1! . .. :.
.thvshatt .

e
u :

 

 

.flnfiufl? .
{flaflnnfiuwi

.1
3|!
.1311...) .

 

 

 

. .3 . ‘44...."

 

 

v1.13? £1.41. 2.1.1.... «I: ... ..

 

 

THESIS LIBRARY
3 Michigan State

2 00‘l University

 

This is to certify that the
dissertation entitled

Analysis of Ribose Dynamics in RNA Molecules Utilizing

13C NMR Spin Relaxation Techniques Determined with
Novel Specific Isotope Labeling Scheme

presented by

James Edward Johnson, Jr.

has been accepted towards fulfillment
of the requirements for the

PhD, degree in Biochemistry & Molecular Biology

 

 

(Hakim

Major Professor’s Signature

NW [1’2068

Date

 

MSU is an Affirmative Action/Equal Opportunity Employer

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K'IPrqlAchresIClRC/DateDue.indd

 

 

Analysis of Ribose Dynamics in RNA Molecules Utilizing 13c NMR Spin
Relaxation Techniques Determined with Novel Specific Isotope Labeling Scheme

By

James Edward Johnson, Jr.

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY
Biochemistry and Molecular Biology

2008

ABSTRACT

Analysis of Ribose Dynamics in RNA Molecules Utilizing 130 NMR Spin
Relaxation Techniques Determined with Specific Isotope Labeling Scheme

By
James Edward Johnson, Jr.

With the increased recognition of the varied roles that RNA molecules can
perform in biology, there has come a larger interest in the study of RNA structure
and function. In many cases, the function of structured RNA molecules such as
ribozymes depends to a great extent on conformational dynamics on various time

scales. While conformational exchange on the us-ms timescale has been
studied in nucleic acid side-chain bases using sophisticated 13C NMR spin

relaxation experiments, the ribose ring of RNA is poorly suited for these

experiments due to magnetic interference arising from 130—13C interactions in
uniform 130 labeled samples. To alleviate these errors, a novel alternate-site
13 . . 1 13 . . .

C labellng scheme was developed to Isolate H- C spln systems Within the

ribose ring suitable for 13C NMR studies.

Using Escherichia coli strains deficient in the oxidative portion of the

pentose phosphate pathway, 2' and 4' 1H-13C spin systems have been isolated

rendering these ribose sugars suitable for 13C NMR studies. Using 13C spin

relaxation measurements for rAMP solvated in Da-glycerol samples to simulate

various correlation times, it was demonstrated that magnetic interactions from

. 13 . .
adjacent C nuclei can be a source of error In R1 measurements for molecules

. . . . 13 .
Wlth long correlation tlmes and In R1p measurements where coupled C nuclei

have similar effective fields. The alternate-site labeling scheme is a practical
approach to removing these errors. Comprehensive analysis of ribose carbons
for the tetraloop region of the GCAA RNA hairpin has been determined via the

incorporation of ribonucleotides using the alternate-site labeling scheme. From

the combined analysis of CPMG and R1p data measurements, timescales of

ribose pucker transitions were observed for the dynamic tetraloop region.
Exchange lifetimes for ribose carbons within the tetraloop were observed to be
similar within error, indicative of concerted motions for multiple residues. Also,
temperature dependent studies, activation energies for ribose pucker transitions
were determined.

Wlth the validation of the alternate site labeling scheme, preliminary
studies have been performed for more biologically relevant RNA ribozymes,
particularly the hairpin ribozyme. The methods reported in herein will open a
new window into the analysis of conformational dynamics in RNA molecules and
advance efforts to understand the correlation between dynamics and function in

ribozymes and other RNA systems.

To my mother and father for many years of sacrifice so that I could have the
opportunities I do now.

To my wife for her love and support so I could be the man I am today.

ACKNOWLEDGMENTS

I’ve never been eloquent with words, so the words here are insufficient to
express my deep gratitude to so many people who have helped me get here. I
want to thank Dr. Charles Hoogstraten for letting me join his lab and mentoring
me all these years, even though I had no experience in the fields of NMR or
RNA. His guidance has helped shape me into a budding scientist. I’m thankful
for him seeing potential in me, even when I couldn’t see it myself. would like to
thank my committee members Drs. John Wang, Bob Hausinger, Honggao Yan,
and David Weliky for their critical analyses of my work so I could become a better
scientist.

I would like to thank all past and present members of the Hoogstraten lab
for a friendly and wonderful environment in which to work. I appreciate Mina
trying to teach me kinetic principles while I tried to teach her NMR methods. I
want to thank Kristy for not only being a fellow co-worker but a wonderful friend.
I want to thank all of the friends I’ve made since joining the biochemistry
department.

I want to thank my wife and family for all their love and support as l
pursued my dreams, especially Prof., Papa, and Mother who passed on before

they could see me accomplish this milestone.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ ix
LIST OF FIGURES .............................................................................................. xi
ABBREVIATIONS .............................................................................................. xv
CHAPTER 1: Introduction ..................................................................................... 1
Discovery of Ribozymes .................................................................................... 2
General Mechanism of Catalysis ....................................................................... 2
Evidence of Dynamics in RNA Systems ............................................................ 5
Experimental Methods to Probe Dynamics ........................................................ 7
NMR Dynamic Studies ..................................................................................... 10
Model-Free Fomlalism ..................................................................................... 13
Relaxation Dispersion ...................................................................................... 15
NMR Dynamic Studies of RNA Molecules ....................................................... 17
Limitation of NMR Studies in RNA Molecules .................................................. 19
REFERENCES .................................................................................................... 23
CHAPTER 2: The Development and Characterization of a Novel Alternating
Isotope13C Labeling Scheme for 13 C NMR Dynamic Studies In RNA ............... 35
INTRODUCTION ................................................................................................. 36
MATERIALS and METHODS ............................................................................. 40
Resuscitation and Storage of E. coli strains ..................................................... 40
Harvest of rNMPs from E. coli cells .................................................................. 41
NMR Spectroscopy .......................................................................................... 42
Data Analysis ................................................................................................... 43
RESULTS ........................................................................................................... 44
Labeling scheme .............................................................................................. 44
lsotopomer Content Analysis from Glycerol Carbon Sources .......................... 51
Analysis of lsotopomers from Specific-Labeled Glucose ................................. 62
Measurement of C Relaxation Rates ............................................................ 64
DISCUSSION ...................................................................................................... 72
ACKNOWLEDGMENTS ..................................................................................... 80
REFERENCES .................................................................................................... 81
1C 3l-IAPTER 3: Analysis of Ribose Dynamics In the GCAA RNA Hairpin Studied by
3C NMR Spin Relaxation Experiments .............................................................. 88
INTRODUCTION ................................................................................................. 89
MATERIALS and METHODS ............................................................................. 98
Preparation of Labeled 13C Ribonucleotides ................................................... 98
Transcription of 13C enriched GCAA RNA hairpin ........................................... 98

vi

NMR Data Setup, Acquisition, and Processing ................................................ 99

Data Analysis ................................................................................................. 102
RESULTS ......................................................................................................... 104
Verification of Chemical Shifts ....................................................................... 104
Measurement of 13C Relaxation Rates .......................................................... 107
Model-free Analysis ....................................................................................... 110
Measurement of Relaxation Dispersion Curves ............................................. 114
13C Hahn Echo Measurements ..................................................................... 115
Measurement of us to ms Motions ................................................................. 116
Single Exchange Process for Ribose Sugar Pucker ...................................... 125
Analysis of Base Dynamics ............................................................................ 131
Temperature Dependence of kex ................................................................... 133
Activation Energy Determination .................................................................... 141
DISCUSSION .................................................................................................... 145
ACKNOWLEDGMENTS ................................................................................... 155
REFERENCES .................................................................................................. 157
CHAPTER 4: Analysis of Ribose Dynamics at the Cleavage Site of the Lead-
dependent Ribozyme Using 13C NMR Spin Relaxation Experiments ............... 164
INTRODUCTION ............................................................................................... 165
MATERIALS and METHODS ........................................................................... 168
Preparation of 13C Cytidine Ribonucleotides ................................................. 168
Transcription of the 13C Lead-dependent Ribozyme ..................................... 169
NMR Data Setup, Acquisition, and Processing .............................................. 170
Data Analysis ................................................................................................. 171
RESULTS ......................................................................................................... 173
Analysis of us-ms Motions ............................................................................. 177
DISCUSSION .................................................................................................... 180
REFERENCES .................................................................................................. 185
CHAPTER 5: Assignment of 1H, 13C, and 15N Resonances of Domain A of the
Hairpin Ribozyme .............................................................................................. 189
INTRODUCTION ............................................................................................... 190
MATERIALS and METHODS ........................................................................... 196
Transcription of the 13C, 15N Domain A of the Hairpin Ribozyme ................. 196
NMR Data Setup, Acquisition, and Processing .............................................. 198
RESULTS ......................................................................................................... 201
One-Dimensional Spectroscopy ..................................................................... 201
Two-Dimensional Exchangeable Spectroscopy ............................................. 203
Two-Dimensional Spectroscopy of Non-exchangeable Resonances ............. 214
DISCUSSION .................................................................................................... 227
ACKNOWLEDGMENTS ................................................................................... 232
REFERENCES .................................................................................................. 233

vii

CHAPTER 6: Conclusions and Future Work .................................................... 238

CONCLUSIONS ................................................................................................ 239
FUTURE WORK ............................................................................................... 243
Further Characterization of the GCAA RNA Hairpin ...................................... 244
Investigation of Dynamics for the Lead-dependent Ribozyme ....................... 244
Investigation of Dynamics for the Hairpin Ribozyme ...................................... 245
REFERENCES .................................................................................................. 247

Images in this thesis are presented in color

viii

LIST OF TABLES

Table 2.1: Table 2.1: 13C lsotopomer species obtained in nucleotide
preparations ........................................................................................................ 62

Table 2.2: 13C R1 and R1 p measurements obtained from specific and uniform
labeled rAMP ....................................................................................................... 67

Table 3.1: R1 and R1 p 13C measurement at 600 MHz for C2' and C4' atoms of
the GCAA RNA hairpin ...................................................................................... 110

Table 3.2: Model-free analysis of picosecond-nanosecond motion using 600 MHz
data ................................................................................................................... 112

Table 3.3: Motional parameters derived via fitting of relaxation dispersion data at
both fields for a single atom at 25 °C ................................................................ 121

Table 3.4: Motional parameters derived via fitting of all data for a single atom at
25 °C ................................................................................................................. 124

Table 3.5: Motional parameters derived via fitting C2' and C4' data for a single
residue for a single kex value at 25 °C .............................................................. 128

Table 3.6: Motional parameters derived via fitting 02' and C4' resonance data for
multiple residues for a single kex value at 25 °C ............................................... 130

Table 3.7: Motional parameters derived via fitting of all data for a single atom at
1 5 °C ................................................................................................................. 140

Table 3.8: Motional parameters derived via fitting of all data for a single atom at
20 °C ................................................................................................................. 140

Table 3.9: Motional parameters derived via fitting of all data for A7 C2' or C4' at
35 °C ................................................................................................................. 140

Table 3.10: Activation energies determined from fitting temperature dependent
Rex values to the linear form of the Arrhenius equation ..................................... 145

Table 4.1: Motional parameters derived via fitting C5 and C30 C2' R1 p data at 25
°C ...................................................................................................................... 180

Table 5.1: Wild type domain A 1H and 15N chemical shifts ............................... 212

Table 5.2: Mutant U+2C/C+3U domain A 1H and 15N chemical shifts ............... 213
Table 5.3: Ribose 1H and 13c chemical shifts for wild type domain A .............. 226
Table 5.4: Aromatic 1H and 13C chemical shifts for wild type domain A ........... 227

LIST OF FIGURES

Figure 1.1: Proposed mechanisms for RNA strand scission catalyzed by

ribozymes ............................................................................................................. 4
Figure 1.2: Reaction coordinate illustrating motional timescales best suited for
NMR spin relaxation studies ................................................................................. 9
Figure 1.3: Structure of the nucleic acid base and sugar ring for

ribonucleosides ................................................................................................... 21
Figure 2.1: Schematic representation of the PPP ............................................... 45

Figure 2.2: (A) Theoretical labeling pattern for the oxidative portion of the PPP
and (B) interconversion of xylulose-5-phosphate to R5P via ribulose-5-
phosphate ........................................................................................................... 48

Figure 2.3: Theoretical labeling pattern of carbons for the non-oxidative portion of
the PPP using 1,3-13C2 glycerol as the sole carbon source ............................... 49

Figure 2.4: Theoretical labeling pattern of carbons for the non-oxidative portion of
the PPP using 2-1 C glycerol as the sole carbon source .................................... 50

Figure 2.5: 1H-130 HSQC spectrum of 13C rNMPs purified from E. coli DL323
. 13
usmg 1,3— C2 glycerol as the carbon source ..................................................... 53

Figure 2.6: 1H-13C HSQC spectrum of fully labeled 13cm rAMP obtained
commercially ....................................................................................................... 54

Figure 2.7: 1H-13C HSQC spectrum of 13c rNMPs purified from E. coli DL323
using 2-13C glycerol as the carbon source .......................................................... 55

Figure 2.8: 1H-13C HSQC spectrum of 13C rNMPs purified from E. coli K10-15-

1 6 utilizing 2-13C glycerol as the carbon source ................................................. 57
Figure 2.9: Fraction of 130 label at individual sites ............................................. 59
. 13 . 1 13
Figure 2.10: C shoes from H- C HSQC spectra ........................................... 61
Figure 2.11: Fraction of 13c label at C3' of rAMP ............................................... 64

xi

Figure 2.12: Longitudinal 13C relaxation of rAMP in glycerol .............................. 69

Figure 2.13: Transverse 13C relaxation of rAMP in glycerol ................................ 71

Figure 3.1: The GCAA RNA hairpin .................................................................... 90

Figure 3.2: Diagram of sugar pucker conformations adopted by A-form RNA
molecules ............................................................................................................ 91

Figure 3.3: Functional forms of the dependence of exchange contributions to

relaxation (Rex) for R p and CPMG experiments on the effective 7B1 field ......... 96

Figure 3.4: 1H-13C HSQC spectrum of a specifically-labeled GCAA RNA

hairpin ............................................................................................................... 106
. . 13 obs

Figure 3.5: Representatlve C (a) R1 and (b) R1 p curves .......................... 109

Figure 3.6: Analysis of relaxation data at 600 MHz using the model-free
formalism at 25 °C ............................................................................................. 113

Figure 3.7: 130 R2 measurements from Hahn spin-echo experiments for (a) C2'
and (b) C4' atoms at 25 °C ................................................................................ 116

Figure 3.8: Relaxation dispersion curves for tetraloop atoms in the GCAA RNA
hairpin ............................................................................................................... 119

Figure 3.9: Representative relaxation dispersion curves for aromatic atoms for
the GCAA RNA hairpin ...................................................................................... 132

Figure 3.10: Relaxation dispersion curves in the GCAA tetraloop at 15 °C ....... 135
Figure 3.11: Relaxation dispersion curves in the GCAA tetraloop at 20 °C ....... 137

f: igure 3.12: Relaxation dispersion curves in the GCAA tetraloop at 35 °C ....... 139
I:igure 3.13: Temperature dependence of A7 C4' ............................................. 142

Figure 3.14: Arrhenius-type plots of the exchange rate constant kex ................ 144
Figure 3.15: Representation of the tetraloop region of GUAA RNA hairpin ....... 153

Figure 4.1: Secondary structure of the lead-dependent ribozyme used in this
work .................................................................................................................. 166

xii

Figure 4.2: 1H-13C HSQC spectra of the cytidine-only specifically-labeled lead-
dependent ribozyme corresponding to C2' (top) and C4' (bottom) resonances at

25 °C ................................................................................................................. 176
Figure 4.3: 13C R2 measurements from Hahn echo experiments for cytidine C2'
(grey) and (b) C4' (black) resonances at 25 °C ................................................. 177
Fig. 4.4: CPMG and R1 p data for C5 (a) 02' and (b) C4' of the lead-dependent
ribozyme ........................................................................................................... 179
Figure 5.1: Diagram of the hairpin ribozyme utilizing a four-way junction ......... 191

Figure 5.2: Secondary structure of the (a) wild type and (b) U+2CIC+3U mutant
domain A ........................................................................................................... 195

Figure 5.3: One-dimensional comparison of the imino proton region for the (top)
mutant U+2C/C+3U and (bottom) wild type isolated domain A at 15 °C ............ 203

Figure 5.4: 1H-15N HSQC spectrum of the imino proton region for (a) wild type
and (b) mutant U+2C/C+3U domain A of the hairpin ribozyme at 15 °C ............ 204

Figure 5.5: 1H-15N HSQC spectrum of the amino proton region for (a) wild type
and (b) mutant U+2CIC+3U domain A of the hairpin ribozyme at 15 °C ............ 207

Figure 5.6: 1H-1H NOESY-HSQC sub-spectra of the wild type hairpin domain A
at 15 °c ............................................................................................................. 210

Figure 5.7: 1H-1H NOESY sub-spectra of the mutant U+2CIC+3U hairpin domain
A at 15 °C .......................................................................................................... 211

Figure 5.8: 1H-13C HSQC spectrum of the non-exchangeable resonances of the
Wild type domain A of the hairpin ribozyme at 25 °C acquired at 600 MHz ....... 215

Figure 5.9: 1H-1H HCCH-COSY sub-spectra of the wild type domain A at 25 °C
at 600 MHz ........................................................................................................ 218

Figure 5.10: 1H-1H HCCH-TOCSY sub-spectra of the wild type domain A at 25
°C at 600 MHz ................................................................................................... 220

xiii

Figure 5.11: 1H-1H 13C-edited NOESY-HSQC sub-spectra of the wild type
hairpin loop A at 25 °C at 600 MHz ................................................................... 223

Figure 5.12: 1H-1H NOESY of the wild type hairpin loop A at 25 °C with a 150 ms
mixing time at 900 MHz ..................................................................................... 225

xiv

 

4WJ
BBrG
BPTI
COSY
CPMG
CSA
DSS
E. coli
FRET
hNOE
HSQC
LB
LNA
NMR
NOE
NOESY

PPP

ABBREVIAITONS

four-way junction

8-bromo-guanosine

basic pancreatic trypsin inhibitor

correlated spectroscopy
Carr-Purcell-Meiboom-Gill

chemical shift anisotropy

sodium 2,2 dimethyl-2-siIapentane-5-sulfonate
Escherichia coli

fluorescence resonance energy transfer
heteronuclear nuclear Overhauser enhancement
heteronuclear single quantum coherence
Luria-Bertani

locked nucleic acid

nuclear magnetic resonance

nuclear Overhauser enhancement

nuclear Overhasuer enhancement spectroscopy

pentose phosphate pathway

longitudinal relaxation rate

rotating-frame transverse relaxation rate

transverse relaxation rate

XV

R5P
rAMP
rcCPMG
RF

RNA
rNMPs
rNTPs
S/N

sshNOE

T1

TOCSY

ribose-5-phosphate

adenosine monophasphate

relaxation compensated Carr-Purcell-Meiboom-Gill
radio frequency

ribonucleic acid

ribonucleotide monophosphates

ribonucleotide triphosphates

signal to noise

steady-state heteronuclear nuclear Overhauser enhancement

longitudinal relaxation time

transverse relaxation time

total correlated spectroscopy

Chapter 1

Introduction

Discovery of Ribozymes. RNA molecules take part in essential roles in many
cellular processes. Previously, RNA was thought to have a secondary role in the
cell: a genetic translator in the form of messenger RNA, an amino acid
chaperone in the form of transfer RNA, and a structural support for
macromolecular complexes in the form of ribosomal RNA (1). However, the
scientific community was forever changed in the early 19803 when the labs of
Drs. Thomas Cech and Sydney Altman discovered the existence of catalytic
RNAs, or ribozymes. Both labs were able to demonstrate that RNA alone
performs chemistry without the aid of amino acid side chains from proteins (2,3).
These findings have changed the way RNA is viewed in the cell and have
sparked a revolution of RNA discoveries and uses. Different RNA systems have
since been discovered to control gene expression including RNA riboswitches,
microRNAs, and RNA interference in addition to ribozymes (4-7), demonstrating
the diversity of RNA functions in the cell.

General Mechanism of Catalysis. Ribozymes have been found in various
cellular functions such as viral replication, splicing of intronic genes, maturation
of tRNA molecules (8), and there is evidence that the ribosome is a ribozyme (9).
Examples of naturally occurring ribozymes are the hammerhead (10), hairpin
( 11), Hepatitis Delta virus (12), Neurospora Varkud satellite (13), group I (2) and
II intron (14), and the RNA subunit of ribonuclease P (3). Most ribozymes
catalyze phosphotransferase reactions along the phosphate backbone with
mechanisms similar to ribonucleases (15,16) (Fig. 1.1). The active site

architecture of ribozymes are positioned to deprotonate the nucleophile, stabilize

the charge build-up of the transition state, and protonate the leaving group.
Metal ions have been implicated to participate in the catalytic mechanism similar
to the amino acid side chain imidazoles in ribonuclease A (9). This is similar for
ribozymes where metal ions have been implicated in assisting the catalytic
mechanism in most cases. In special cases, such as the hairpin ribozyme, metal
ions are not observed in the active site leading to the theory that the nucleobases
take part as the primary role in catalysis (17,18). This theory is further supported
from the crystal structure of the full-length hammerhead ribozyme where no

metals were observed in the active site (19).

 

 

 

J ” .ro/ \o
5 o\9$92 V" 3 33592 0-1
\0( 25+ {0251 “its 06...,
3m— 30.22 w l...
33 3H ll: 5.1+ 3),,_g,(
L p 5.11

Figure 1.1: Proposed mechanisms for RNA strand scission catalyzed by
ribozymes.

Evidence of Dynamics in RNA Systems. Conformational dynamics are well
known to play an important role in the function of enzymes. Similarly, there is
increasing evidence that conformational changes are crucial to ribozyme
function. For example, active site architecture has been studied in minimal
sequence hammerhead ribozyme through several crystal structures. Pley et al.
(20) crystallized the hammerhead using a DNA inhibitor strand, while Scott et al.
(21) crystallized the hammerhead using a 2'-O-methyl group in place of the
reactive 2'-OH. The reactive 2' oxygen was not observed in either structure
within proper distance of the scissile phosphate for in-line nucleophilic attack.
However, Klug and coworkers (22) crystallized unmodified hammerhead
molecules with and without metal ions, using flash-freezing to capture the
intermediate structure. Also, Dunham et al. (23) crystallized a hammerhead
sequence with a phosphodiester linkage between stems l and II to mimic the pre-
catalytic state. In this structure, the nucleophile was positioned very close to
proper orientation; it was theorized that a sugar pucker transition could complete
the alignment necessary for in-line nucleophilic attack. Comparison of all of the
crystal structures suggests a model where the hammerhead undergoes a
conformational change to position the reactive groups congruent with in-line
nucleophilic attack. Recently, the crystal structure of the full length 63 nucleotide
hammerhead ribozyme solved by Maltick and Scott (19) with a 2'-O-methyl group
revealed tertiary interactions that induce structural changes aligning the reactive

ribose ring very close to the proper geometry for in-line nucleophilic attack.

Taken together, these results suggest that investigation of dynamics within the
active site of ribozymes can be crucial to understand the catalytic mechanism.
Another ribozyme system where conformational change has been shown
to contribute greatly to the catalytic mechanism is the hairpin ribozyme. The
hairpin ribozyme is a RNA four-way helix junction (4WJ) where two helices A and
B must interact for catalysis. While metal ions have been shown to facilitate
proper RNA folding, they have not been directly linked to the catalytic
mechanism. Ferre-D’Amare and co-workers (18) have crystallized several 4WJ

hairpin constructs to investigate the catalytic site of the hairpin ribozyme. Using

a 2'-O-methyl group in place of the reactive 2'-OH for A4, extensive

conformational changes were observed, including the formation of a cross-stem

Watson-Crick base pair for 6+1 and C25. The formation of this base pair

constrains A-1 and G+1 into C2'-endo conformation aligning the proposed

nucleophile and leaving group. The significance of sugar pucker was further
elucidated in the comparison of transition state mimic hairpin crystals as well as
wild type crystals (24). Superposition of the precursor, transition state mimic,

and product constructs revealed a rigid active site were motion was confined to

the scissile phosphate and ribose ring of A1, where a change in the sugar
pucker was observed. Salter et al. (25) crystallized multiple analogs for G3 using
a 2'-O-methyl group in place of the reactive 2'-OH for A4. Removal of functional

groups for GB was shown, in some cases, to alter the sugar pucker of A1. Along

with the hammerhead ribozyme, conformational changes appear to be critical to
the reaction mechanism.

Experimental Methods to Probe Dynamics. Several methods have been used
to study conformational changes of ribozymes. Turner and coworkers (26) used
fluorescence detected stopped-flow methods to investigate binding of RNA
oligomers to Tetrahymena themiophila ribozyme. Also, Burke and coworkers
(27) have used fluorescence resonance energy transfer to study conformational
changes such as loop-loop interaction for the hairpin ribozyme. One drawback to
these methods is that investigation of the fluorophore can be insensitive to
localized conformational changes that do not alter the global structure. To focus
on more localized motions, incorporation of fluorescent nucleotide analogs can
elucidate internal motions in nucleic acids. For example, the use of 2-
aminopurine analogs has been used to probe structural changes through base
extrusions for trans-activator responsive region hairpin of human
immunodeficiency virus type 1 binding to ligands (28).

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method to
measure localized conformational changes. Using NMR techniques, each
residue can be investigated, which can reveal localized motions throughout the
molecule in a site-specific fashion. NMR methods can be used to investigate
dynamics across a broad range of timescales. For ribozymes, different
timescales may be important for catalysis. A possible model for the reaction
mechanism of some ribozymes is presented in figure 1.2, where the ground state

(E-S), which is not suitable for catalysis, proceeds to a pre-catalytic state (E-S*)

that more closely resembles the transition state before products (E-P) are
formed. NMR spin relaxation studies can measure “fast” dynamics on the
picosecond to nanosecond timescale, such as segmental motions. In addition,
“slow” dynamics can be measured on the microsecond to millisecond timescale
to investigate conformational changes between states. The use of NMR dynamic

studies can be useful to help reveal how motions participate in catalysis.

TS

llS-mS

 

{-—

 

 

 

 

E-P

Figure 1.2: Reaction coordinate illustrating motional timescales best suited for
NMR spin relaxation studies. E-S represents enzyme-substrate ground state
complex, E-S* represent pre-catalytic state, TS represents transition state, and
E-P represents enzyme-product complex. Figure was adapted from Hoogstraten
and Sumita, “Structure-function relationships in RNA and RNP enzymes: recent
advances” published in Biopolymers (2007) (29).

NMR Dynamic Studies. NMR spin relaxation experiments have been used to

investigate dynamics of macromolecules, particularly longitudinal (T1) and

transverse (T2) relaxation as well as heteronuclear nuclear Overhauser
enhancement (hNOE) measurements. In an early study, Gurd and co-workers
(30) employed natural abundance 13C NMR T1 measurements to investigate

motions within ribonuclease A. Interestingly, multiple dynamics were observed

for B- and e-carbons. Glushko et al. (31) further analyzed segmental motions of
ribonuclease A through pH studies. Investigating 13C T1 measurements with a

pH range of 6.55 to 1.46, they were able to analyze not only segmental motions,
but conformational changes due to protein denaturation. Roberts and coworkers

(32) were perhaps one of the first groups to measure rotation correlation times
from 15N T1 and hNOE measurements in not only proteins, but tRNA as well.
For amide resonances of lysozyme, determination of motional parameters for

. . . . 13
amldes of lysme groups was In excellent agreement Wlth C T1 measurements

of e-carbons of lysine side chains. These results were significant because they

demonstrate that NMR methods can be used to investigate fast internal motions

. . 1 15 . . . .
Wlthln macromolecules. Also, 3C and N dynamic studles can report on Slmllar

motional modes. For example, Hawkes et al. (33) measured 13C and 15N T1

measurements for gramicidin S. In short, a method was outlined to study internal

dynamics of macromolecules that can differentiate local fluctuations from global

10

motions. Several groups used this approach to extract dynamic properties using

13C and 15N NMR spin relaxation measurements (34-38).

. 13 15 . .
Whlle early C and N spin relaxatlon measurements were valuable for

determining local motional modes within macromolecules, these experiments

suffered from limitations. All the previously mentioned works were obtained

using one-dimensional direct ion detect spectroscopy of natural abundance 13C

and/or 15N. 13C has a natural abundance of 1% while 15N has a natural

abundance of just 0.4%. To offset this limitation, groups have used substantial
sample concentrations, in excess of 20 mM for solution state NMR. This also
leads to issues with sample solubility, which can severely limit studies of

macromolecules. Another issue that hampered data analysis is spectral overlap.

In most cases, one-dimensional 13C and 15N spectra had very few residues that

were resolved from other residues. For example, Gurd and co-workers (30,31)
measured effective correlation times for a-carbon envelopes as opposed to
individual residues. In this case, individual residues with different rotational
correlation times can be masked by global motions.

To offset this, heteronuclear two-dimensional spectroscopy experiments
were developed (39,40). These pulse sequences are important because they
provide enhanced sensitivity to nuclei with low gyromagnetic ratios as well as

providing a second dimension of chemical shifts for decreased spectral overlap.

Several groups have used these pulse sequences to measure T1 values in

11

proteins (41-43). Nirmala and Wagner (43) developed a two-dimensional pulse
sequence to measure 13C T1 values for nearly all a-carbons in basic pancreatic
trypsin inhibitor (BPTI) at natural abundance. This result is significant because

they were able to measure individual 13C T1 values for or-carbons as opposed to

a bulk T1 values. Indeed, 13C T1 measurements in the BPTI showed a high

degree of variability. With the advances in spectrometer field strength combined
with two-dimensional pulse sequences, the measurement of dynamics in

macromolecules became more practical.

. 13 15 . . . . .
Relaxation of C and N nuclei in solution predominately arises from

dipolar interaction from covalently bound protons. T1, T2, and hNOE depend on

spectral density functions, which are Fourier transformations of the auto-

correlation function for molecular motion (44). Richarz et al. (34) used 13C T1

and hNOE values to determine internal dynamics for 13C carbons of BPTI. In

this work, internal dynamics were interpreted using the wobbling in a cone model
proposed by Kinosita et al. (45). From this model, information such as rotation
about single bonds and diffusion of a rotation axis inside the cone can be
determined. Other models can be used to determine internal dynamics within
macromolecules (46,47). Likewise, Lipari and Szabo (48) were able to extract
similar internal dynamic parameters for nucleic acid fragments using different
models of internal motions. The models corresponded to different physical

representations, but yielded the same numerical value of the order parameter.

12

Model-Free Fonnalism. Lipari and Szabo (49,50) developed a model-
independent formalism to correlate dynamic properties to measured relaxation

rates. In this “model—free" approach, fast internal motions can be described by a

generalized order parameter, 82, which is the measure of the degree of spatial

restriction of the motion, and an effective correlation time, 13, which is a measure

of the rate of the motion for a given nucleus. This formalism removes the
ambiguity of internal dynamics interpretation. The model-free formalism has
been validated over a wide range of motions, including for anisotropic rotations
where the overall motion cannot be described by a single correlation time.
Because motional parameters describing fast motion can be determined in
an independent fashion, the model free approach has been widely accepted to
investigate internal dynamics using NMR. Early use of the model-free approach

was done by Bax and co-workers (51), where internal dynamics for amides of

15M uniformly enriched staphylococcal nuclease were investigated at several
field strengths using 15N T1, T2 and hNOE measurements. Pulse sequences
were developed to accurately measure T1, T2, and hNOE data. Staphylococcal
nuclease exhibited a uniform structure with similar 82 for nearly all amide

2 .
resonances measured, an average value of 0.86 for S . Some N- and C-terminal

residues were shown to have significant disorder as well as effective correlation

times. In particular, the C-terminal residue Gly-148 was reported to have a 82

and re of 0.23 and 120 ps, respectively (51). In addition to interpreting fast

13

motions, investigations of slow motions were performed. Transverse relaxation
measurements are sensitive chemical or conformational exchange on the

microsecond to millisecond timescale. In staphylococcal nuclease, Glu-52 and
Lys-53 exhibited 1/T2 values 2.5 times larger than the average 1/T2 values (51),
indicative of conformational motions on the 11s to ms timescale. This is plausible
because these residues are located within a flexible region of the molecule (52).

Interestingly, T1 values measured for these residues are similar to T1 values

measured for nearly all residues. Taken together, these results were significant
to the field of NMR spectroscopy because the validity of using NMR spin
relaxation studies to probe internal motions across a broad range of timescales
were demonstrated.

Torchia and co-workers (53) extended interpretation of internal dynamics

to 13C methyl groups of leucine residues. In this approach, internal dynamics

were determined from an extension to the Lipari-Szabo formalism by Clore et al.
(54). The approach by Clore et al. was developed to include “slow” motions that
are an order of magnitude larger than effective correlation times, yet still faster

than the overall isotropic correlation time. Using this extended model free

approach, Torchia et al. were able to separate order parameters of leucine 13C
methyl carbons into flexible and rigid groups (53). The rigid 13C carbons 825
(see next paragraph) values were found to be similar to 82 values for backbone

15N amide resonances using the standard model free approach. This result is

14

useful because it demonstrates the presence of dynamics of side chains in
addition to backbone nuclei.

Previously, exchange contributions were determined from comparisons of

R2 (1/T2) values. Wright and co-workers integrated exchange contributions on

the us to ms timescale that contribute to the overall transverse relaxation
mechanism into the model-free analysis using the term Rex, the contribution to

the observed transverse relaxation rate due to exchange (55,56). Analysis of
internal motions using the extended model-free formalism can be daunting due to
the numerous motional parameters. Palmer and co-workers (57) have
developed the user-friendly ModelFree computer program that aids in the
selection of motional parameters that best represents the data. Using this
program, five different conditions of molecular motions were utilized to best fit the
experimental data, which includes isotropic and anisotropic conditions. Palmer
and co-workers (57) were able to investigate dynamics across a broad range of
motional timescales. For example, complex dynamics were discovered within a
flexible region of E. coli ribonuclease HI where internal motions of nuclei in this
region could be described using all conditions possible (57).

Relaxation Dispersion. The analysis of fast dynamics has been well
documented using the model-free formalism. However, dynamics on the us-ms
timescale are not well determined using this approach. Many biological
processes such as catalysis, protein folding, and allosteric transitions occur on
these timescales (58). Using the model-free approach, chemical or

conformational changes that affect the measured chemical shift are described

15

with the term, Rex. A more definitive description of the Rex term can done by

investigating the transverse relaxation rate as a function of effective field
strength. In early work of this type, Luz and Meiboom previously utilized spin
echo delays to determine proton transfer between trimethylammonium ion and
trimethylamine in solution using Carr-Purcell-Meiboom-Gill (CPMG) pulse
scheme (59). In this work, it was derived that transverse relaxation rates in the
presence of chemical or conformational exchange depend on the average
lifetime of exchange for a nuclei, pulse repetition rate, fractional populations of

exchanging states, and chemical shift difference between states. Similar
derivations were made for the rotating frame R1 p pulse scheme (60,61) where
magnetization is spin-locked in the rotating frame by the application of a radio

frequency field. Again, transverse R1 p measurements in the presence of

exchange varies as a function of the spin-lock power deposition, lifetime of

exchange for the nuclei, fractional population of n sites, and chemical shift

difference between sites. Arseniev and co-workers (62) applied a 15N spin-echo

modulated CPMG pulse sequence to investigate us-ms motions for amides in
bacterioopsin. In this work, all exchange lifetimes for backbone amides
excluding N and C termini were determined to within error of each other, leading

to a model where cooperative motion was occurring for the a-helices. It was

interpreted that the Rex contribution was due to helix-helix interaction whereby

the helices exhibit slight shifting along each other, changing the tilt or bend.

16

Exchange contributions have also been determined using power-dependent R1 p

experiments. Using spin-echo modulated CPMG and/or power-dependent R1 p

experiments, dynamics on the us-ms timescale can be probed.

NMR Dynamic Studies of RNA Molecules. Dynamics of proteins have been
studied extensively using the model-free formalism (55,56,63-72). By contrast,
the investigation of dynamics of nucleic acids using the model free approach has
only been done for a few cases (73-81). Spielmann (73) investigated natural
abundance aromatic and deoxyribose dynamics in a DNA oligomer and DNA
oligomer complexed with a dye. Using the extended model free analysis,

extensive dynamics were observed in the un-Iiganded DNA oligomer including

average 82 values of 0.79 and several residues exhibiting Rex contributions.

Upon binding the dye, previously observed dynamics were quenched or reduced
indicative of the DNA oligomer becoming more structured. Hall and Tang (75)
investigated temperature-dependent dynamics for purine bases in the iron
responsive element RNA hairpin also using the model-free approach. At 20 °C,
order parameters obtained varied little among resonances throughout the
molecule, supporting the NMR structure where loop residues were stacked and
structured similar to stem residues. As the temperature increased, a reduction in
structure was observed for the loop residues while the stem residues maintained
their structure similar to that observed at 20 °C. From these works as well as
others, internal dynamics can be investigated in nucleic acids to probe motional

modes of nucleic acids.

17

Spin-echo modulated CPMG (82-91) and power-dependent R1 p

experiments (92-98) have been implemented even less in nucleic acids (76,99-

102). Hoogstraten et al. (99) investigated 13C aromatic dynamics in the lead-

dependent ribozyme. Using power-dependent R1 p experiments, several

exchange lifetimes on the microsecond regime were observed, including

exchange lifetimes for C2 and CB nuclei of A25. The exchange lifetimes
determined from R1 p experiments were very similar to exchange lifetimes for

protonation of A25 N1 determined by line-shape analysis (103). These results

show the usefulness of obtaining motional parameters in RNA molecules. A25 is

cross-strand from the catalytic residue, C5 (see chapter 4). It was hypothesized

that dynamics observed at this residue was indicative of base flipping for C5.

Blad et al. (100) also obtained motional parameters from power-dependent R1 p

experiments for base as well as C1' sugar atoms in the U6 RNA intramolecular
stem—loop (see figure 1.3 for numbering). This work was unique in the
measurement of exchange lifetimes for the sugar atom C1'. By obtaining multiple

motional parameters for the base and sugar moiety of a residue, correlated

motions can be investigated. Indeed, Blad et al. (100) were able to fit R1 p data to

a single nucleotide exchange lifetime for C1' and base atoms of four residues in
the RNA oligomer. Interestingly, all exchange lifetimes measured were similar

within error. These residues were found throughout the oligomer and not

18

localized to a particular region. It was hypothesized that these residues were
reporting on a single global conformational rearrangement within the U6 RNA by
determining motional parameters using a global exchange lifetime of 84 us (100).
Taken together, us to ms motions can be determined in nucleic acids to probe

not only localized motions, but global conformational fluctuations as well.
Limitation of NMR Studies in RNA Molecules. Both 130 and 15N nuclei have
been used to analyze motions in proteins. Nucleic acids have relatively few

protonated 15M nuclei for analysis similar to proteins (104), since 15M nuclei can

only be found in the aromatic bases of nucleic acids. Because of this, 15N

dynamic studies are restricted to the aromatic ring in RNA molecules. However,
there are protonated carbons located in both the aromatic base and significantly

in the ribose ring (Fig. 1.3). Unfortunately, the structure of the ribose ring leads

to 13C-13C interactions, which can lead to difficulty in unambiguously

understanding relaxation mechanisms in uniformly 30 enriched nucleotides

(105). To offset this limitation, groups employed several methods to obtain

. 1 . . . . .
isolated 1H- 3C spin systems for accurate measurements including acqumng

relaxation measurements at natural isotopic abundance (73,106) and using low

levels of partial 13C fractional enrichment (74,107). While these experiments
13 13 . . . . .
remove unwanted C- C interaction, they suffer severe sensrtlwty loss. Other

ways to highly enrich 13C sites are the use of chemical synthesis to specifically

19

prepare the ribose ring of nucleic acids (108-110). However, this approach can

be laborious and time consuming. While information about RNA dynamics has

been obtained for 13C base resonances (75,111), little information has been

. 13 . . .
obtained for C ribose resonances. In cases where ribose dynamics has been

obtained, analysis has been limited to C1' and/or C5' resonances

(76,79,100,108). As seen earlier, measured 15N amide backbone dynamics of

BPTI showed a high degree of similarity with several 13C methyl groups while

some differed, revealing a case where dynamics are highly localized (53). It is
possible that nucleic acids could display a similar trend where significant base
dynamics can be observed while little to no dynamics are discerned for the ribose
ring, or vice versa. To circumvent these issues, extension of NMR dynamics

studies to the ribose ring is needed.

20

 

\3/ 4 H
HO\ ,H
:5"
H/ 4' 1'
133' 078.
on b.
NH2 0 o

H’N/o

Figure 1.3: Structure of the nucleic acid base and sugar ring for ribonucleosides.
Adenosine, Cytidine, Guanosine, and Uridine ribonucleosides are shown where
carbon nuclei accessible to NMR relaxation studies are highlighted in red.

21

The goal of this thesis is to investigate dynamics in RNA systems using

sophisticated 13C NMR spin relaxation experiments. In chapter 2, methods are
presented to specifically 13C label the ribose ring of ribonucleotides, including an

alternating 13C-12C isotope labeling scheme, rendering the ribose ring suitable

for 13C NMR spin relaxation studies. R1 and R1 p data acquired for specific

labeled ribose is compared to uniformly labeled ribose to investigate the effect of

13C-13C interactions. In chapter 3, extensive analyses of ribose dynamics are

presented for the GCAA RNA hairpin using the specific labeling scheme.

Analyses of multiple timescales are included through the use of the model-free
formalism and combined spin-echo modulated CPMG and R1 p measurements.

In chapter 4, preliminary analysis of ribose dynamics within the active site of the

lead-dependent ribozyme is presented with 13C specific labeled cytidine

incorporation. In chapter 5, preliminary 1H, 13C, and 15N chemical shift

assignments are reported for wild type and mutant domain A of the hairpin
ribozyme. In chapter 6, general conclusions are made and possible directions for

future research are outlined.

22

REFERENCES

10.

Bloomfield, V.A., Crothers, D.M. and Tinoco, l., Jr. (2000) Nucleic Acids:
Structures, Properties, and Functions. University Science Books,
Sausalito, California, USA.

Kruger, K., Grabowski, P.J., Zaug, A.J., Sands, J., Gottschling, DE. and
Cech, TR. (1982) Self-splicing RNA: autoexcision and autocyclization of
the ribosomal RNA intervening sequence of Tetrahymena. Cell, 31, 147-
157.

Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. and Altman, S.
(1983) The RNA moiety of ribonuclease P is the catalytic subunit of the
enzyme. Cell, 35, 849-857.

Montange, R.K. and Batey, RT. (2008) Riboswitches: emerging themes in
RNA structure and function. Annu Rev Biophys, 37, 117-133.

Dykxhoorn, 0M. and Lieberman, J. (2005) The silent revolution: RNA
interference as basic biology, research tool, and therapeutic. Annu Rev
Med, 56, 401-423.

Bushati, N. and Cohen, SM. (2007) microRNA functions. Annu Rev Cell
Dev Biol, 23, 175-205.

Khan, AU. and Lal, SK. (2003) Ribozymes: a modern tool in medicine. J
Biomed Sci, 10, 457-467.

Kuimelis, RS. and McLaughlin, L.W. (1998) Mechanisms of ribozyme—
mediated RNA cleavage. Chem Rev, 98, 1027-1044.

DeRose, V.J. (2002) Two decades of RNA catalysis. Chem Biol, 9, 961-
969.

Symons, RH. (1997) Plant pathogenic RNAs and RNA catalysis. Nucleic
Acids Res, 25, 2683-2689.

23

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Hampel, A. and Tritz, R. (1989) RNA catalytic properties of the minimum
(-)sTRSV sequence. Biochemistry, 28, 4929-4933.

Thill, G., Vasseur, M. and Tanner, MK. (1993) Structural and sequence
elements required for the self-cleaving activity of the hepatitis delta virus
ribozyme. Biochemistry, 32, 4254-4262.

Saville, B.J. and Collins, RA. (1990) A site-specific self-cleavage reaction
performed by a novel RNA in Neurospora mitochondria. Cell, 61, 685-696.

Peebles, C.L., Perlman, P.S., Mecklenburg, K.L., Petrillo, M.L., Tabor,
J.H., Jarrell, K.A. and Cheng, H.L. (1986) A self-splicing RNA excises an
intron lariat. Cell, 44, 213-223.

Roberts, G.C., Dennis, E.A., Meadows, D.H., Cohen, JS. and Jardetzky,
O. (1969) The mechanism of action of ribonuclease. Proc Natl Acad Sci U
SA, 62, 1151-1158.

Doudna, J.A. and Lorsch, JR. (2005) Ribozyme catalysis: not different,
just worse. Nat Struct Mol Biol, 12, 395-402.

Donahue, C.P., Yadava, R.S., Nesbitt, SM. and Fedor, M.J. (2000) The
kinetic mechanism of the hairpin ribozyme in vivo: influence of RNA helix
stability on intracellular cleavage kinetics. J Mol Biol, 295, 693-707.

Rupert, PB. and Ferre-D'Amare, AR. (2001) Crystal structure of a hairpin
ribozyme-inhibitor complex with implications for catalysis. Nature, 410,
780-786.

Martick, M. and Scott, W.G. (2006) Tertiary contacts distant from the
active site prime a ribozyme for catalysis. Cell, 126, 309-320.

Pley, H.W., Flaheity, KM. and McKay, DB. (1994) Three-dimensional
structure of a hammerhead ribozyme. Nature, 372, 68-74.

Scott, W.G., Finch, J.T. and Klug, A. (1995) The crystal structure of an all-
RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic
cleavage. Cell, 81, 991-1002.

24

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

Scott, W.G., Murray, J.B., Arnold, J.R., Stoddard, BL. and Klug, A. (1996)
Capturing the structure of a catalytic RNA intermediate: the hammerhead
ribozyme. Science, 274, 2065-2069.

Dunham, C.M., Murray, J8. and Scott, W.G. (2003) A helical twist-
induced conformational switch activates cleavage in the hammerhead
ribozyme. J Mol Biol, 332, 327-336.

Rupert, P.B., Massey, A.P., Sigurdsson, ST. and Ferre-D'Amare, AR.
(2002) Transition state stabilization by a catalytic RNA. Science, 298,
1421-1424.

Salter, J., Krucinska, J., Alam, S., Grum-Tokars, V. and Wedekind, J.E.
(2006) Water in the active site of an all-RNA hairpin ribozyme and effects
of Gua8 base variants on the geometry of phosphoryl transfer.
Biochemistry, 45, 686-700.

Bevilacqua, P.C., Kierzek, R., Johnson, K.A. and Turner, DH. (1992)
Dynamics of ribozyme binding of substrate revealed by fluorescence-
detected stopped-flow methods. Science, 258, 1355-1358.

Walter, N.G., Hampel, K.J., Brown, KM. and Burke, J.M. (1998) Tertiary
structure formation in the hairpin ribozyme monitored by fluorescence
resonance energy transfer. EMBO J, 17, 2378-2391.

Bradrick, TD. and Marino, JP. (2004) Ligand-induced changes in .2-
aminopurine fluorescence as a probe for small molecule binding to HIV-1
TAR RNA. RNA, 10, 1459-1468.

Hoogstraten, CG. and Sumita, M. (2007) Structure-function relationships
in RNA and RNP enzymes: recent advances. Biopolymers, 87, 317-328.

Allerhand, A., Doddrell, D., Glushko, V., Cochran, D.W., Wenkert, E.,
Lawson, P.J. and Gurd, F.R.N. (1971) Conformation and segmental
motion of native and denatured ribonuclease A in solution. Application of
natural-abundance carbon-13 partially relaxed Fourier transform nuclear
magnetic resonance. J Am Chem Soc, 93, 544-546.

Glushko, V., Lawson, P.J. and Gurd, FR. (1972) Conformational states of
bovine pancreatic ribonuclease A observed by normal and paltially

25

32.

33.

34.

35.

36.

37.

38.

39.

40.

relaxed carbon 13 nuclear magnetic resonance. J Biol Chem, 247, 3176-
3185.

Gust, D., Moon, RB. and Roberts, JD. (1975) Applications of natural-
abundance nitrogen-15 nuclear magnetic resonance to large
biochemically important molecules. Proc Natl Acad Sci U S A, 72, 4696-
4700.

Hawkes, G.E., Randall, E.W. and Bradley, CH. (1975) Theory and

practice for studies for peptides by 15M nuclear magnetic resonance at
natural abundance: gramicidin S. Nature, 257, 767-772.

Richarz, R., Nagayama, K. and Wuthrich, K. (1980) Carbon-13 nuclear
magnetic resonance relaxation studies ofinternal mobility of the
polypeptide chain in basic pancreatic trypsininhibitor and a selectively
reduced analogue Biochemistry, 19, 5189-5196.

Fuson, MM. and Prestegard, J.H. (1983) Dynamics of an interfacial
methylene in dimyristoylphosphatidylcholine vesicles using carbon-13 spin
relaxation. Biochemistry, 22, 1311-1316.

Henry, G.D., Weiner, J.H. and Sykes, ED. (1986) Backbone dynamics of

a model membrane protein: 13C NMR spectroscopy of alanine methyl
groups in detergent-solubilized M13 coat protein. Biochemistry, 25, 590-
598.

Smith, G.M., Yu, LP. and Domingues, DJ. (1987) Directly observed 15N
NMR spectra of uniformly enriched proteins. Biochemistry, 26, 2202-2207.

Dellwo, M.J. and Wand, A.J. (1989) Model-independent and model-
dependent analysis of the global and internal dynamics of cyclosporin A. J
Am Chem Soc, 111, 4571 -4578.

Morris, G. and Freeman, R. (1979) Enhancement of nuclear magnetic
resonance signals by polarization transfer. J Am Chem Soc, 101, 760-762.

Freeman, R., Mareci, TH. and Morris, GA. (1981) Weak satellite signals
in high-resolution NMR spectra: Separating the wheat from the chaff. J
Magn Reson, 42, 341-345.

26

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

Kay, L.E., Jue, T.L., Bangerter, B. and Demou, PC. (1987) Sensitivity

enhancement of 130 T1 measurements via polarization transfer. J Magn
Reson, 73, 558-564.

Sklenar, V., Torchia, DA. and Bax, A. (1987) Measurement of carbon-13
longitudinal relaxation using 1H detection. J Magn Reson, 73, 375-379.

Nirmala, NR. and Wagner, G. (1988) Measurement of 13C relaxation

times in proteins by two-dimensional heteronuclear 1H--13C correlation
spectroscopy. J Am Chem Soc, 110, 7557-7558.

Abragam, A. (1961) Principles of Nuclear Magnetism. Oxford University
Press, USA.

Kinosita, K., Jr., Kawato, S. and Ikegami, A. (1977) A theory of
fluorescence polarization decay in membranes Biophys. J., 20, 289-305.

Wittebort, R.J. and Szabo, A. (1978) Theory of NMR relaxation in
macromolecules: Restricted diffusion and jump models for multiple internal
rotations in amino acid side chains. J Chem Phys, 69, 1722-1736

Woessner, DE. (1962) Spin relaxation processes in a two-proton system
undergoing anisotropic reorientation J Chem Phys, 36, 1-4.

Lipari, G. and Szabo, A. (1981) Nuclear magnetic resonance relaxation in
nucleic acid fragments: models for internal motion. Biochemistry, 20,
6250—6256.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 1. theory
and range of validity. J Am Chem Soc, 104, 4546-4559.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 2. analysis
of experimental results. J Am Chem Soc, 104, 4559-4570.

27

51.

52.

53.

54.

55.

56.

57.

58.

Kay, L.E., Torchia, DA. and Bax, A. (1989) Backbone dynamics of

proteins as studied by 15N inverse detected heteronuclear NMR

spectroscopy: application to staphylococcal nuclease. Biochemistry, 28,
8972-8979.

Torchia, D.A., Sparks, SW. and Bax, A. (1989) Staphylococcal nuclease:
sequential assignments and solution structure. Biochemistry, 28, 5509-
5524.

Nicholson, L.K., Kay, L.E., Baldisseri, D.M., Arango, J., Young, P.E., Bax,
A. and Torchia, DA. (1992) Dynamics of methyl groups in proteins as

studied by proton-detected C NMR spectroscopy. Application to the
leucine residues of staphylococcal nuclease. Biochemistry, 31, 5253-
5263.

CIore, G.M., Szabo, A., Bax, A., Kay, L.E., Driscoll, PC. and Gronenborn,
AM. (1990) Deviations from the simple two-parameter model-free
approach to the interpretation of nitrogen-15 nuclear magnetic relaxation
of proteins. J Am Chem Soc, 112.

Palmer, A.G., 3rd, Rance, M. and Wright, PE. (1991) lntramolecular
motions of a zinc finger DNA-binding domain from Xfin characterized by
proton-detected natural abundance carbon-13 heteronuclear NMR
spectroscopy. J Am Chem Soc, 113, 4371-4380.

Stone, M.J., Fairbrother, W.J., Palmer, A.G., 3rd, Reizer, J., Saier, M.H.,
Jr. and Wright, RE. (1992) Backbone dynamics of the Bacillus subtilis

glucose permease "A domain determined from 15N NMR relaxation
measurements. Biochemistry, 31, 4394-4406.

Mandel, A.M., Akke, M. and Palmer, A.G., 3rd. (1995) Backbone dynamics
of Escherichia coli ribonuclease HI: correlations with structure and function
in an active enzyme. J Mol Biol, 246, 144-163.

Palmer, A.G., 3rd, Kroenke, CD. and Loria, JP. (2001) Nuclear magnetic
resonance methods for quantifying microsecond-to-millisecond motions in
biological macromolecules. Methods Enzymol, 339, 204-238.

28

59.

60.

61.

62.

63.

64.

65.

66.

67.

Luz, Z. and Meiboom, S. (1963) Nuclear magnetic resonance study of the
protolysis of trimethylammonium ion in aqueous solution - order of the
reacton with respect to solvent. J Chem Phys, 39, 366-370.

Jones, G.P., Douglass, DC. and McCall, D.W. (1965) Apparatus for the
detection of ultraslow atomic motion by magnetic resonance techniques
Rev Sci Instrum, 36, 1460-1465.

Douglass, DC. and Jones, GP. (1966) Nuclear magnetic relaxation of n-
alkanes in the rotating frame. J Chem Phys, 45, 956-963

Orekhov, V., Pervushin, K.V. and Arseniev, AS. (1994? Bgckbone

dynamics of (1-71)bacterioopsin studied by two-dimensional H- N NMR
spectroscopy. Eur J Biochem, 219, 887-896.

Akke, M., Skelton, N.J., Kordel, J., Palmer, A.G., 3rd and Chazin, W.J.
(1993) Effects of ion binding on the backbone dynamics of calbindin D9k

determined by 15N NMR relaxation. Biochemistry, 32, 9832-9844.

Powers, R., CIore, G.M., Stahl, S.J., Wingfield, PT. and Gronenborn, A.
(1992) Analysis of the backbone dynamics of the ribonuclease H domain

. . . . . 15
of the human immunodefiCIency VII‘US reverse transcriptase usmg N
relaxation measurements. Biochemistry, 31, 9150-9157.

Kordel, J., Skelton, N.J., Akke, M., Palmer, A.G., 3rd and Chazin, W.J.
(1992) Backbone dynamics of calcium-loaded calbindin D9k studied by

two-dimensional proton-detected 15N NMR spectroscopy. Biochemistry,
31, 4856-4866.

Palmer, A.G., 3rd, Hochstrasser, R.A., Millar, D.P., Rance, M. and Wright,
RE. (1993) Characterization of amino acid side chain dynamics in a zinc-
finger peptide using carbon-13 NMR spectroscopy and time-resolved
fluorescence spectroscopy. J Am Chem Soc, 115, 6333-6345.

Stone, M.J., Chandrasekhar, K., Holmgren, A., Wright, RE. and Dyson,
H.J. (1993) Comparison of backbone and tryptophan side-chain dynamics

of reduced and oxidized Escherichia coli thioredoxin using 15N NMR
relaxation measurements. Biochemistry, 32, 426-435.

29

68.

69.

70.

71.

72.

73.

74.

75.

76.

Mandel, A.M., Akke, M. and Palmer, A.G., 3rd. (1996) Dynamics of
ribonuclease H: temperature dependence of motions on multiple time
scales. Biochemistry, 35, 16009-16023.

Wikstrom, M., Forsen, S. and Drakenberg, T. (1996) Backbone dynamics
of a domain of protein L which binds to immunoglobulin light chains. EurJ
Biochem, 235, 543-548.

Zhou, H., McEvoy, M.M., Lowry, D.F., Swanson, R.V., Simon, ML and
Dahlquist, F.W. (1996) Phosphotransfer and CheY-binding domains of the
histidine autokinase CheA are joined by a flexible linker. Biochemistry, 35,
433-443.

Palmer, A.G., 3rd, Williams, J. and McDermott, A. (1996) Nuclear
magnetic resonance studies of biopolymer dynamics. J Phys Chem, 100,
13293-13310.

Vugmeyster, L., Trott, O., McKnight, C.J., Raleigh, DP. and Palmer, A.G.,
3rd. (2002) Temperature-dependent dynamics of the villin headpiece
helical subdomain, an unusually small thermostable protein. J Mol Biol,
320, 841-854.

Spielmann, HP. (1998) Dynamics of a bis-intercalator DNA complex by

1H-detected natural abundance 1 C NMR spectroscopy. Biochemistry, 37,
16863-16876.

Kojima, C., Ono, A., Kainosho, M. and James, TL. (1998) DNA du lex

dynamics: NMR relaxation studies of a decamer with uniformly 3C-
labeled purine nucleotides. J Magn Reson, 135, 310-333.

Hall, KB. and Tang, C. (1998) 13C relaxation and dynamics of the purine
bases in the iron responsive element RNA hairpin. Biochemistry, 37,
9323-9332.

Shajani, Z. and Varani, G. (2005) 13C NMR relaxation studies of RNA
base and ribose nuclei reveal a complex pattern of motions in the RNA
binding site for human U1A protein. J Mol Biol, 349, 699-715.

30

77.

78.

79.

80.

81.

82.

83.

84.

85.

Shajani, Z., Drobny, G. and Varani, G. (2007) Binding of U1A protein

changes RNA dynamics as observed by 13C NMR relaxation studies.
Biochemistry, 46, 5875-5883.

Duchardt, E. and Schwalbe, H. (2005) Residue specific ribose and

nucleobase dynamics of the cUUCGg RNA tetraloop motif by NMR 13C
relaxation. J Biomol NMR, 32, 295-308.

Trantirek, L., Caha, E., Kaderavek, P. and Fiala, R. (2007) NMR 13C-

relaxation study of base and sugar dynamics in GCAA RNA hairpin
tetraloop. J Biomol Struct Dyn, 25, 243-252.

Hansen, AL. and AI-Hashimi, HM. (2007) Dynamics of large elongated
RNA by NMR carbon relaxation. J Am Chem Soc, 129, 16072-16082.

Ferner, J., Villa, A., Duchardt, E., Widjajakusuma, E., Wohnert, J., Stock,
G. and Schwalbe, H. (2008) NMR and MD studies of the temperature-
dependent dynamics of RNA YNMG-tetraloops. Nucleic Acids Res, 36,
1928-1940.

Loria, J.P., Rance, M. and Palmer, AG. (1999) A relaxation-compensated
Carr-PurcelI-Meiboom-Gill sequence for characterizing chemical exchange
by NMR spectroscopy. J Am Chem Soc, 121, 2331-2332.

Millet, O., Loria, J.P., Kroenke, C.D., Pons, M. and Palmer, A.G., 3rd.
(2000) The static magnetic field dependence of chemical exchange
Iinebroadening defines the NMR chemical shift time scale. J Am Chem
Soc, 122, 2867-2877.

Mulder, F.A., Hon, B., Muhandiram, D.R., Dahlquist, F.W. and Kay, LE.
(2000) Flexibility and ligand exchange in a buried cavity mutant of T4
lysozyme studied by multinuclear NMR. Biochemistry, 39, 12614-12622.

Mulder, F.A., Mittermaier, A., Hon, 8, Dahlquist, F.W. and Kay, LE.
(2001) Studying excited states of proteins by NMR spectroscopy. Nat
Struct Biol, 8, 932-935.

31

86.

87.

88.

89.

90.

91.

92.

93.

94.

Tollinger, M., Skrynnikov, N.R., Mulder, F.A., Forman-Kay, JD. and Kay,
LE. (2001) Slow dynamics in folded and unfolded states of an SH3
domain. JAm Chem Soc, 123, 11341-11352.

Cole, R. and Loria, JP. (2002) Evidence for flexibility in the function of
ribonuclease A. Biochemistry, 41, 6072-6081.

Kovrigin, E.L., Cole, R. and Loria, JP. (2003) Temperature dependence of
the backbone dynamics of ribonuclease A in the ground state and bound
to the inhibitor 5'-phosphothymidine (3'-5')pyrophosphate adenosine 3'-
phosphate. Biochemistry, 42, 5279-5291.

Korzhnev, D.M., Bezsonova, l., Evanics, F., Taulier, N., Zhou, Z., Bai, Y.,
Chalikian, T.V., Prosser, RS. and Kay, LE. (2006) Probing the transition
state ensemble of a protein folding reaction by pressure-dependent NMR
relaxation dispersion. J Am Chem Soc, 128, 5262-5269.

Korzhnev, D.M., Salvatella, X., Vendruscolo, M., Di Nardo, A.A.,
Davidson, A.R., Dobson, CM. and Kay, LE. (2004) Low-populated folding
intermediates of Fyn SH3 characterized by relaxation dispersion NMR.
Nature, 430, 586-590.

Butterwick, J.A., Patrick Loria, J., Astrof, N.S., Kroenke, C.D., Cole, R.,
Rance, M. and Palmer, A.G., 3rd. (2004) Multiple time scale backbone
dynamics of homologous thermophilic and mesophilic ribonuclease HI
enzymes. J Mol Biol, 339, 855-871.

Vugmeyster, L., Kroenke, C.D., Picart, F., Palmer, A.G., 3rd and Raleigh,

DP. (2000) 15N R1 p measurements allow the determination of ultrafast
protein folding rates. J Am Chem Soc, 122, 5387-5388.

Korzhnev, D.M., Orekhov, V.Y., Dahlquist, F.W. and Kay, LE. (2003) Off-

resonance R1 p relaxation outside of the fast exchange limit: an
experimental study of a cavity mutant of T4 lysozyme. J Biomol NMR, 26,
39-48.

Kim, S. and Baum, J. (2004) An on/off resonance rotating frame relaxation
experiment to monitor millisecond to microsecond timescale dynamics. J
Biomol NMR, 30, 195-204.

32

95.

96.

97.

98.

99.

100.

101.

102.

103.

Massi, F., Johnson, B, Wang, C., Rance, M. and Palmer, A.G., 3rd.

(2004) NMR R1p rotating—frame relaxation with weak radio frequency
fields. J Am Chem Soc, 126, 2247-2256.

Massi, F., Grey, M.J. and Palmer, A.G., 3rd. (2005) Microsecond
timescale backbone conformational dynamics in ubiquitin studied with
NMR R1rho relaxation experiments. Protein Sci, 14, 735-742.

Grey, M.J., Tang, Y., Alexov, E., McKnight, C.J., Raleigh, DP. and
Palmer, A.G., 3rd. (2006) Characterizing a partially folded intermediate of
the villin headpiece domain under non-denaturing conditions: contribution
of His41 to the pH-dependent stability of the N-terminal subdomain. J Mol
Biol, 355, 1078-1094.

Butterwick, J.A. and Palmer, A.G., 3rd. (2006) An inserted Gly residue fine
tunes dynamics between mesophilic and thermophilic ribonucleases H.
Protein Sci, 15, 2697-2707.

Hoogstraten, C.G., Wank, JR. and Pardi, A. (2000) Active site dynamics
in the lead-dependent ribozyme. Biochemistry, 39, 9951-9958.

Blad, H., Reiter, N.J., Abildgaard, F., Markley, J.L. and Butcher, SE.
(2005) Dynamics and metal ion binding in the U6 RNA intramolecular
stem-loop as analyzed by NMR. J Mol Biol, 353, 540-555.

Shajani, Z. and Varani, G. (2008) 13C relaxation studies of the DNA target
sequence for hhai methyltransferase reveal unique motional properties.
Biochemistry, 47, 7617-7625.

Maltseva, T.V., Foldesi, A., Ossipov, D. and Chattopadhyaya, J. (2000)
Comparative 13C and 2H relaxation study of microsecond dynamics of the

AT tract of selectively 130le double-labelled DNA duplexes. Magn Reson
Chem, 38, 403-414.

Legault, P. and Pardi, A. (1997) Unusual dynamics and pK,.,I shift at the
active site of a lead-dependent ribozyme. J Am Chem Soc, 119, 6621-
6628.

33

104.

105.

106.

107.

108.

109.

110.

111.

Akke, M., Fiala, R., Jiang, F., Patel, D. and Palmer, A.G., 3rd. (1997) Base

dynamics in a UUCG tetraloop RNA hairpin characterized by 15N spin
relaxation: correlations with structure and stability. RNA, 3, 702-709.

Batey, R.T., Battiste, J.L. and Williamson, JR. (1995) Preparation of
isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol,
261, 300-322.

Borer, P.N., LaPlante, S.R., Kumar, A., Zanatta, N., Martin, A., Hakkinen,

A. and Levy, cc. (1994) 13c-NMR relaxation in three DNA

oligonucleotide duplexes: model-free analysis of internal and overall
motion. Biochemistry, 33, 2441-2450.

Boisbouvier, J., Brutscher, B., Simorre, JP. and Marion, D. (1999) 130
spin relaxation measurements in RNA: Sensitivity and resolution

improvement using spin-state selective correlation experiments. J Biomol
NMR, 14, 241-252.

Gaudin, F., Chanteloup, L., Thuong, NT. and Lancelot, G. (1997)

Selectively 13C-enriched DNA: dynamics of the C1'H1' and C5'H5' or
CS'H5" vectors in d(CGCAAA'ITI'GCG)2. Magn Reson Chem, 35, 561-
565.

Kline, PC. and Serianni, AS. (1990) 13C-enriched ribonucleosides:

synthesis and application of 13C-1H and 13C-13C spin-coupling constants
to assess furanose and N-glycoside bond conformations. J Am Chem Soc,
112, 7373-7381.

SantaLucia, J., Jr., Shen, L.X., Cal, 2., Lewis, H. and Tinoco, l., Jr. (1995)
Synthesis and NMR of RNA with selective isotopic enrichment in the
bases. Nucleic Acids Res, 23, 4913-4921.

Dayle, K.T., Brodsky, AS. and Williamson, JR. (2002) Base flexibility in

HIV-2 TAR RNA mapped by solution 15N, 13c NMR relaxation. J Mol Biol,
317, 263-278.

34

Chapter 2

The Development and Characterization of a Novel Alternating Isotope 130
Labeling Scheme for 13C NMR Dynamic Studies in RNA

Portions of this chapter were derived from “Alternate-site isotopic labeling of
ribonucleotides for NMR studies of ribose conformational dynamics in RNA”
published in the Journal of Biomolecular NMR (2006), written by Dr. Charles G.
Hoogstraten and myself.

35

INTRODUCTION

Heteronuclear NMR spin relaxation is an effective experimental technique
to investigate conformational dynamics in biological molecules. Studies have
been conducted on timescales that cover the picosecond to millisecond regime
(1-5). These studies were made possible by the model-free formalism developed
by Lipari and Szabo (6,7) that examine picosecond to nanosecond timescales,
and relaxation dispersion curves (8) that probe microsecond to millisecond
timescales. Using these approaches, motional parameters can be determined.

NMR relaxation is the process by which the magnetization returns to
equilibrium. There are two types of NMR relaxation: longitudinal (z-axis) and
transverse (x-y plane). Longitudinal relaxation is the process by which the bulk
magnetization reappears along the z-axis, or static field, according to its
Boltzmann distribution. Transverse relaxation is the process by which
magnetization perpendicular to the static field disappears due to coherence loss.
Nuclear spins within a molecule are sensitive to other spins within their
environment leading to unique local fields for each nuclear spin. Dipolar coupling
is the interaction of nuclear spins through space, which depends on the nuclear
spin’s orientation to the static magnetic field. Chemical shift anisotropy (CSA) is
the orientation dependence of the nuclei with the induced field of the electrons
surrounding the nuclei. As a molecule tumbles in solution, the orientation of the
nuclei changes and the dipolar coupling and CSA change the localized field at

the nucleus which determines the relaxation. The overall molecular tumbling is

defined as the overall correlation time 1c, which is the time it takes for the

36

molecule to rotate by one radian. Measurements of longitudinal, R1, and/or

transverse, R2 (R1p), relaxation rates can yield valuable information.

Another experiment that can affect longitudinal magnetization is steady
state heteronuclear Overhauser effect (sshNOE). During this process,
application of a weak radio frequency (RF) field at the frequency of one spin for a
sufficiently long time can affect non-irradiated spins, enhancing the longitudinal
magnetization for the non-irradiated spin. The NOE enhancement depends on
the correlation time, and thus can be used to probe internal motions in
macromolecules.

As mentioned in chapter one, the model-free approach uses spectral

density functions for R1, R2 and sshNOE in order to probe motions of nuclei

. . . . 2
Within a molecule to determme motional parameters: an order parameter, S

which is a measure of spatial restriction, an effective correlation time, 1., which is

the timescale of internal motion, and Rex. which is the contribution to the

observed transverse relaxation rate due to exchange on the us to ms timescale.
It should be noted that CSA relaxation can be neglected at lower to medium field
strengths for liquids; however, at higher field strengths, CSA contributions can
equal dipole—dipole contributions (4).

The application of the model-free formalism was pioneered by Kay et al.

. . 15 .
(9) where motions for amide N nuclei of staphylococcal nuclease were

investigated. From R1, R2, and hNOE data, Kay et al. (9) were able to obtain an

37

average value for the order parameter, 82, of 0.86 for all measured amide 15N

nuclei of the protein excluding three residues which were located in the termini.

These techniques have been applied to other proteins (10-15) as well as nucleic

acids (16-21). To interpret Rex contributions more definitively, the use of

relaxation dispersive curves have been used to determine chemical shift
perturbations for an atom through chemical or conformational changes. In this

approach, spin-echo modulated Carr-Purcell-Meiboom-Gill (CPMG) and/or

power-dependent spin-locked rotating frame (R1p) relaxation rates are measured

as a function of effective field strength. Millet et al. (22) measured exchange
contributions for 15N amide resonances in basic pancreatic trypsin inhibitor. In

this approach, exchange contributions were observed for several residues near

the region of the Cys14-Cys38 disulfide bond where isomerization occurs.

. . . . 15 . 13
Determination of motional timescales for backbone N amldes as well as C

sidechain atoms using relaxation dispersion curves in proteins have been utilized
(23-32) while applications to 13C atoms in nucleic acids have been sparsely done
(18,33-36).

NMR spin relaxation experiments have been aided greatly by the use of

isotopic enriched media to incorporate magnetically active nuclei into molecules

of interest. Prior to use of isotope labeling, large amounts of sample had to be

prepared to compensate for weak signal due to small gyromagnetic ratios for 13C

and 15N. In addition, early NMR relaxation experiments were limited to one-

38

dimensional direct detection (37-39), for which relaxation rates were limited to an
ensemble of resonances or isolated resonances. The use of two-dimensional
correlated spectroscopy has eliminated this limitation by reducing spectral

overlap, thereby greatly aiding dynamic studies within macromolecules.

. . 13 . . . . . .
In nucleic aCldS, C relaxation experiments have been llmlted primarily to

the purine and pyrimidines bases (5). Complete 13C labeling of carbons of the

ribose ring using standard uniform enrichment complicates data analysis due to

13 13 . . .
C- C scalar coupling which can cause spectral overlap and/or lnaccurate data

measurement, as well as dipolar coupling which leads to additional relaxation

contributions that can complicate the analysis of spin relaxation experiments. To

. . . . 13 . . .
overcome this limitation, C relaxation experiments have been carried out on

RNA molecules with natural abundance of 13C isotope (20,40-42) or partial 13C

labeling (43,44). Both of these experimental setups are still limited by signal to

noise, which can be offset with significant NMR acquisition times and/or the use

of a cryogenic-chilled probe. Another way to overcome 13C-13C magnetic

interference is the use of specific labeling of 13C resonances. This has been

previously done using chemical synthesis (21 ,45,46), which entails multiple steps
not commonly found outside of a synthetic chemistry lab.
Another method to specifically label RNA can be done using bacterial

metabolic pathways. Previously, LeMaster and Cronan (47) engineered bacterial

strains to specifically 13C label the side chains of amino acids. By deleting

39

certain enzymes of the citric acid cycle, glycolytic and citric acid cycle
intermediates do not interchange carbons. Because of this, labeled precursors
can be introduced that give rise to predictable, desirable patterns. LeMaster and
Kushlan (48) used one of these Escherichia coli strains, DL323, to investigate
side chain motions for E. coli thioredoxin. From this analysis, the authors were
able to observe decreased stability for side chain atoms when compared to
backbone atoms of the same residue. It was theorized that this metabolic
labeling scheme could be utilized for the ribose of nucleic acids.

Conformational dynamics have been shown to play a critical role in the

function of ribozymes, where the ribose ring is often central to the catalytic

mechanism. In this chapter, a description of 130 isotopic patterns of
ribonucleotides obtained from various E. coli harvests is presented. Comparison

of R1 and R1 p values obtained from alternate-site and uniformly labeled samples
. . 13 13 . . . .
is made to examine the effects of C- C magnetic interactions for the ribose

ring. With the removal of 13C interactions, accurate motional parameters can be

obtained through the use of NMR spin relaxation experiments.

MATERIALS and METHODS

Resuscitation and Storage of E. coli strains. E. coli strains DL323, K10-15-
16, and DF2001 were obtained from the E. coli Genetic Stock Center. Samples
arrived as lyophylisates on circular pieces of cellulose. The cellulose slips were

deposited in a culture tube with 5 mL of enriched Luria-Bertani (LB) media. After

40

the cellulose was dissolved with mild shaking, 1 mL of each solution was
transferred to a culture tube containing 3 mL of enriched LB media, which was
incubated overnight at 37 °C. After overnight growth, 1 mL was transferred to 99
mL of LB media. The 100 mL sample was incubated at 37 °C and growth was
monitored by measuring the absorbance, A600, to follow the growth curve. When
the growth curve indicated stationary growth phase, E. coli cell culture was
prepared for long-term storage. 500 uL aliquots of E. coli cell culture were
prepared by making a 1:1 ratio of sample:60% glycerol. The aliquots were frozen
in a dry ice and ethanol solution for five mins. The aliquots were then labeled

and stored at -80 °C.

Harvest of rNMPs from E. coli cells. To obtain 13C labeled ribonucleotide

monophosphates (rNMPs) for NMR analysis, frozen E. coli cells were
resuscitated from long term storage. After minor thawing, a sterile toothpick was
used to transfer E. coli cells to a culture tube containing 5 mL of LB media and

incubated overnight at 37 °C. The overnight growth was then transferred to a

flask containing 995 mL of sterile M9 minimal media and 20% 130 specifically

labeled glycerol (Cambridge Isotope Laboratories, Inc.) as the sole carbon
source. The liter growth of E. coli cells was incubated at 37 °C until cell growth

reached an A500 of 1. Cells were then harvested, Iysed, and total nucleic acids

were collected, which were subsequently hydrolyzed (49). 13C rAMP was

separated from other ribonucleotides using an Akta Basic FPLC (GE Healthcare)

using a POROS Pl/20 column (20 um, 4.6 mm x 100 mm) with a linear gradient

41

from 50 mM NH4COOH, pH 3.0 to 500 mM NH4COOH, pH 2.5 over 3.5 mins

with a flow rate of 5 mUmin collecting 10 mL fractions. rNMP elution was
monitored by UV absorbance, and fractions were pooled and stored at -20 °C.
NMR Spectroscopy. To analyze samples by NMR spectroscopy, all rNMPs

were concentrated to 1 to 2 mM in 600 uL with a buffer concentration of 10 mM

NaH2PO4, pH 7.5, and 60 uL of 10 mM sodium 2,2 dimethyl-2-silapentane-5-

sulfonate for reference. H2O was exchanged with 99.9% D20 with repeated

lyophilization. The final resuspension was done with 99.96% D2O. For 13C

relaxation rates, specifically and uniformly labeled rAMP were prepared to 1 and
0.8 mM concentrations, respectively, as mentioned above and solvated to a final

volume of 600 uL in Dg-glycerol. To achieve spectral lock, a special capillary
insert filled with D20 was placed inside the NMR tube.

All spectra were obtained on a 600 MHz Varian Unitylnova spectrometer.

rNMP mixtures were characterized using one-dimensional 13C decoupled and

. . 1 13 .
non-decoupled spectra and two-d1mensronal H- C heteronuclear Single

quantum correlation (HSQC) spectra. Relaxation rates were determined using
slightly modified, nonconstant-time versions of published sequences as

described previously (13). Due to the limited number of resonances and good

resolution in the 1H dimension, relaxation spectra were obtained without

incrementing t1. R1 experiments at 15 °C, 20 °C, 25 °C, and 30 °C with were

42

acquired with delay times ranging from 100 ms to 3000 ms. R1 p experiments for
the 2' 13C were also acquired at 15 °C, 20 °C, 25 °C, and 30 °C, while R1p

experiments for the 4' 13C was acquired at 20 °C, 25 °C, and 30 °C at

approximately 3 kHz spin lock field strength. Data for the 4' 13C was not
collected at 15 °C due to spectral overlap with glycerol satellite peaks. For R1 p

data for 13C C2' and C4' were collected separately with the 130 carrier placed on

resonance, respectively, at 1550 and 2980 Hz spin-lock fields. Delay periods
ranged from 4 to 76 ms.

Data Analysis. All NMR data were processed using FELIX 2002 (Felix NMR,

Inc.). Prior to Fourier transformation in the t2 dimension, a 20% DC offset was

applied, data were zero filled, and a 3 Hz exponential line broadening function

was applied. For two-dimensional experiments, a cosine-squared apodization

function was applied prior to Fourier transformation of the t1 dimension. To

achieve near uniform baseline, the baseline of all one-dimensional spectra were
corrected using a baseline correction algorithm. Peak intensities were exported

to Igor Pro 4.0 (WaveMetrics). Error bars on intensity measurements were

determined by repeating a single delay time three times. 13C relaxation rate

constants were determined by nonlinear fitting of the intensity data to a single
exponential using Igor Pro 4.0. Errors in rates are those derived from the

covariation matrix as reported by the nonlinear fitting routine.

43

Using the ratio of T1 to T1 p for 2' 13C of the specifically labeled rAMP, it

was estimated that the four temperatures of 15, 20, 25, and 30 °C corresponded
to rotational correlation times of 16.9, 11.8, 7.8, and 5.4 ns, respectively (50).

Assuming a linear scaling of correlation time with molecular weight and taking

6.0—6.4 ns as the correlation time for a 30-nucleotide RNA stem loop in D2O at

25 °C (33), the 2' 13C resonances in our samples exhibit relaxation behavior

roughly characteristic of RNA oligomers of 82, 57, 38, and 26 nucleotides,

respectively, at the four temperatures used.

RESULTS

Labeling scheme. A goal of this work was to develop a biosynthetic isotope

labeling pattern that eliminates 13C-13C interaction within the ribose ring. In E.

coli, ribose-5-phosphate (R5P) used for rNMPs is made via the oxidative and
non-oxidative portions of the pentose phosphate pathways (PPP) (Fig 2.1). If
glycerol is used as a sole carbon source for bacterial cell cultures, it can enter
the metabolic flux of E. coli cells via phosphorylation by glycerol kinase. The
phosphorylated glycerol-3-phosphate is converted to dihydroxyacetone-3-
phosphate by glycerol phosphate dehydrogenase. lsomerization is carried out
by triosephosphate isomerase to interconvert dihydroxyacetone-3-phosphate and
glyceraIdehyde-3-phosphate. GlyceraIdehyde-3-phosphate can enter PPP
where shown or be converted to other metabolites that enter PPP through

gluconeogenisis enzymes.

44

NADPH+H* + C"(3’0 O. NADPHtCOz

W H “‘90“ mow Ct'20“
.. “WM bag—ii. °\ WW __.U tits.
6?“

 

 

 

 

 

 

 

H C-0— H— -OH
C—d,’ 6ducono- H" ’OH 5- H—C—OH
(silicone-5- W GQPfiosphO- Rbulose-o-
Cle-I
c=o H‘c’o
Hoe-H translietolase l H-c-OH
H H—C-OH H—c-OH +——
Q‘C’ H—C-OH H-é-oH WW
H-c-OH l-l-c-Ol-i 63W m
cm} Ribose-5- I
am mm 2““
7-Phosphde I
not}: J/
H—C-OH ‘==-—-——
transaldolase étl20F’03"" duress-sumac
Cle-I
fro l 9‘20“
Ho-c—H “0’” i=0
l-l—c-OH H—c-OH WWW“ K H
H- -OH l-l- —0H ”'00“ C,
H—é—OH H-i-OH
HzoPos“ H20P032°
Fructooo-O- rose-4-
Fnictoee-G-

Figure 2.1: Schematic representation of the PPP. The number of arrows
represents the number of times the reaction occurs. Atoms are highlighted in
color to help illustrate the transfer of atoms and enzymes are italicized. The
desired metabolite, R5P, is emphasized. Figure was adapted from Voet, Voet,
and Pratt (51).

45

For the oxidative portion of the PPP, glucose-6-phosphate is the initial

metabolite. If glycerol were 13C labeled at the 1' and 3' carbon positions, then

the glucose-6-phosphate made via gluconeogenesis would be labeled at the 1',
3', 4’, and 6' carbon positions. Alternatively, if the glycerol is labeled only at the 2'
carbon position, then the glucose-6-phosphate molecules would be labeled at the
2' and 5' carbon positions. In order to make R5P using the oxidative portion of

the PPP, glucose-6-phosphate molecule would yield a ribulose-5-phosphate

molecule that was labeled at the 2', 3', and 5' carbon positions for 1.3-13C2

glycerol and 1' and 4' carbon positions for 2-13C glycerol, which can be

converted to R5P with similar patterns (Fig. 2.2A).

When the cell needs ribose for nucleotide synthesis, the non-oxidative
portion of the PPP is utilized. In this pathway, the enzyme transketolase
transfers the first two carbons from fructose-6-phosphate to glyceraldehyde-S-
phophate to make xylulose-5-phosphate and erythrose-4-phosphate. In the next
step, transaldolase transfers the first three carbons from another fructose-6-
phosphate molecule to erythrose-4-phosphate yielding glyceraldehyde-3-
phosphate and sedohepuIose-7-phosphate. Transketolase then transfers the

first two carbons from sedohepquse-7-phosphate onto glyceraldehyde-3-

phosphate to make xylulose-5-phosphate. Using 1,3-13C2 glycerol as the sole

carbon source, xylulose-5-phosphate would be obtained with a labeling pattern of
1', 3', 5' at the carbon positions while ribose-5-phosphate is made with a labeling

isotope at the 1', 2', 3', and 5' carbon positions for the non-oxidative portion of the

46

PPP (Fig. 2.3). Using 2-13C glycerol as the sole carbon source, xylulose-5-

phosphate would be obtained with a labeling pattern of 2', 4' at the carbon
positions while R5P is made with a labeling isotope at the 4' carbon position for
the non-oxidative portion of the PPP (Fig. 2.4). For purposes of ribonucleotide

synthesis, xylulose-5-phosphate can be isomerized to R5P (Fig. 2.2B).

47

A OscO'

CHzogoaH CH2°P°3 H-C-OH CH20H
lc/C \OH C/B— HO" "C-H =
H H; -> H H, \C=0 —> b —> _¢_
0 C—C’ RoC—(Izl H .‘OH H . 0”
H20P03 20P03

6-phosphoglucono-delta-lactone

 

 

G6P 6-phosphogluconate RU5P
CHZOPO 30’0-
C—0 3 3301303 H—C—Ol—l CH20H
H \OH H O | i
('g/ ' , '/ \ —> HO-C-H —> 9’0
i H H (I: C H H 0:0 I H-C-OH
H 0“ CH20P03 CH20P03
O H
CH20H C|3H2OH \c/
c=o (I): H—C—OH
HO—C—H ———‘__ H—(l3—OH H—C—OH
H—C—OH H—CI:_OH H—C—OH
CH20P03 CH20P03 CH20P03
XUSP RU5P R5P

Figure 2. 2: (A) Theoretical labeling pattern for the oxidative portion of the PPP.
The metabolites would be derived using (top) 1,3-13 C2 glycerol and (bottom) 2-

13C glycerol as the sole carbon source. Carbons expected to be C are labeled
red. (B) lnterconversion of xylulose-5-phosphate to R5P via ribulose-5-
phosphate.

48

CH20H

é: CH H
O\C/H | 20
H— —OH + H_C_°H Ho— —H
H—C—OH H2°P°3 H——c—0H
sap
H20P03 20PO3
Fsp Xu5P
CHZOH
C: O H CHon
\c/ |_o
o H Ho— —H J: -—
\C/ H— —OH
I H— —OH <— | HO— —H
—— — —— —— +
H C OH + H_ —OH H c OH H_ —OH
H20P03 H—CIJ—OH H20P03 H—C—OH
esp E4P
H20P03 H20F’03
S7P FBP
H o
ICHZOH \C/
0=0 H—C—OH
HO— —H H—— —OH
H—C—OH H—C—OH
H20P03 H20P03
Xu5P R5P

Figure 2.3: Theoretical labeling pattern of carbons for the non-oxidative portion

of the PPP using 1,3- C2 glycerol as the sole carbon source. Expected C
carbons are labeled in red.

49

_O . CH H
O§C/H 2°
HO—f—H é “—0
H—C—OH , H— I —OH Ho—c—H
H— —OH CH2°P°3 H-— —OH
G3P
CH20P03 CH20PO3
FGP Xu5P
CHZOH
l _
H — — | 0
O\C/ H O (I: H H—‘C—OH
J; H—C—OH ‘— H OH HO—C—H
CH20P03 CH20PO3 — _
G3P H— —OH 54p H I OH
CH20PO3 CH20P03
STP FGP
H O
CHZOH \c/
|
‘0 H—C—OH
H0—C—H * H—C—OH
H— —OH H— —OH
CH20P03 CH20PO3
XuSP R5P

Figure 2.4: Theoretical labeling pattern of carbons for the non-oxidative portion

of the PPP using 2-1 C glycerol as the sole carbon source. Expected 13C
carbons are labeled in red.

50

lsotopomer Content Analysis from Glycerol Carbon Sources. LeMaster and

Kushlan (48) previously demonstrated the ability to specifically 130 label the side

chains of amino acids using E. coli strain DL323 with 13C labeled glycerol as the

carbon source. E. coli strain DL323 has deletions to enzymes of the citric acid
cycle, preventing metabolic scrambling of carbons in precursors for amino acid

side chains. Based on these results, E. coli strain DL323 from the E. coli Genetic

Stock center was used to specifically 130 label the ribose ring of rNMPs. The

1H-mC HSQC spectrum of rNMPs harvested from E. coli strain DL323 grown

with 1,3-1302 glycerol as the sole carbon source are presented in Figure 2.5.

Four crosspeaks corresponding to the four ribonucleotides can be observed in

each spectral region of ribose carbons except for the 4' region. This result

confirms the predicted labeling pattern where no 130 labeling is observed for the

4' atoms. Also presented is the 1H-13C HSQC spectrum of uniformly 13C labeled

adenosine monophosphate (rAMP) for comparison (Fig. 2.6). From this

spectrum, the splitting of crosspeaks due to 1Jcc scalar coupling can be
observed. rNMPs synthesized using 2-130 glycerol as the sole carbon source

were also analyzed. Similar to the 1,3-1302 glycerol prep, cross peaks were

observed only for those predicted to be labeled. From the HSQC spectrum (Fig
2.7), cross peaks can be observed for the 1', 2', and 4' spectral regions.

However, no crosspeaks are detected in the 3' and 5' spectral regions. While

51

specific carbons were 13C enriched using E. coli strain DL323, the desired

alternating pattern was not obtained. To achieve the alternating 13C-12C pattern,

additional E. coli strains were used.

52

 

 

 

 

 

 

l‘
P70
- 6"
3 o .
'75
2° C“:
. E
Q.
F803
0
52
l
'85
_,
4r
1' —" ~
0 ° '90
o .
0
,....,..-.,....1.--.,....,
6.5 6.0 5.5 5.0 4.5 4.0

1H (ppm)

Figure 2.5: 1H-130 HSQC spectrum of 13C rNMPs purified from E. coli DL323
using 1.3-1302 glycerol as the carbon source.

53

 

 

 

 

5': ,
l
-70
3mg:- .
F75
E
2"} 3
- 0
CO
-80"
-85
l
4n} .
1' .
2 ~90
.,....,.-..,....,.-..,.
6.0 5.5 1 5.0 4.5 4.0
H(ppm)

Figure 2.6: 1H-13C HSQC spectrum of fully labeled 130“) rAMP obtained
commercially.

54

 

 

5U

 

 

 

 

 

 

 

 

 

 

L-70
3' '
-75
i
2' _' ’CE:
. 3
-800
52
--85
L
4. .3
1' .
° —90
...,. .,....,...,...
6.0 5.51 5.0 4.5
H(ppm)

Figure 2.7: 1H-13C HSQC spectrum of 13c rNMPs purified from E. coli DL323
using 2-130 glycerol as the carbon source.

55

From the PPP, one can discern that an alternating 120-130-12C labeling

pattern could be obtained if R5P was biosynthetically synthesized via the non-
oxidative enzymes exclusively. Deletion of the glucose-6-phosphate
dehydrogenase gene should shut down the oxidative reactions and shunt R5P
production exclusively to the non-oxidative portion of the PPP. The E. coli Stock
center was queried for E. coli strains that were deficient in glucose-6-phosphate

dehydrogenase gene, zwf. Out of eight possible cell lines, two cell lines were
obtained, K10-15-16 and DF2001 (52). The 1H-13C HSQC spectrum of rNMPs

harvested from E. coli K10-15-16 cultures shows crosspeaks for the 2' and 4'

regions with no detectable crosspeaks for the other ribose spectral regions (Fig.

2.8). The 1H-130 HSQC spectrum of DF2001 is indistinguishable from the
spectrum of K10-15-16 (data not shown). Hence, rNMPs with the ribose ring 13C

labeled in an alternating 120-130 fashion can be obtained using zwf mutants.

56

 

5'Db

-70

2'. ” t

 

 

 

° ° E
Q.
53:
-sog9
~85
4':
1' °
l
...,....,....,....,....,
6.0 5.5 5.0 4.5 4.0
1H(ppm)

Figure 2.8: 1H-13C HSQC spectrum of 13C rNMPs purified from E. coli K10-15-
.. . 13
16 uttltzmg 2- C glycerol as the carbon source.

57

To further characterize the specific labeling pattern obtained for each cell

culture protocol, the percentage of 13C labeling at a given resonance was
determined. This was done by comparison of one-dimensional 13C decoupled

and non-decoupled 1H spectra and evaluation of 13C scalar couplings from 130

slices of HSQC spectra. The ribose protons have a narrow chemical shift range,

causing data analysis of one-dimensional 1H spectra for rNMP mixtures to be

ambiguous due to spectral overlap. For unambiguous isotopomer content

analysis, rAMP was isolated from E. coli strains DL323 and K10-15-16. One-

dimensional 1H spectra were obtained at 5°C for commercial uniformly labeled
rAMP and specifically labeled rAMP using 2-13C glycerol sole carbon sources for
DL323 and K10-15-16 E. coli strains (Fig. 2.9). The effect of 130 interaction can

be observed in the uniformly 130 labeled rAMP spectra as ribose 1H singlet

peaks in the decoupled spectrum are completely split into the muliplet structure

in the non-decoupled spectra. It can be concluded that the C4' atom in both E.

coli strains DL323 and K10-15-16 non—decoupled spectra is completely 13C

labeled due to the absence of a singlet peak, which is consistent with the model

previously mentioned for R5P obtained from the non-oxidative portion of the PPP

using 2-13C glycerol. Also supporting the model is the absence of multiplet

structures for H3' and H5' peaks in the non-decoupled spectra. In the DL323 and

58

K10-15-16 spectra, both multiplet and singlet peaks can be observed for H1' and
H2', which is indicative of partial 130 enrichment at those sites.

H1' H2 H3' H" ”5

 

 

-
r-1nr-‘lnr-‘ -‘J‘

    

 

sIo' ' ' 'sfs' ' ' 's'c' ' ' '415' 4'.o'
1H proton chemical shift ppm

Figure 2.9: Fraction of 130 label at individual sites. One-dimensional 1H NMR

spectra of rAMP acquired at 5 °C. The top spectrum is a 13C-decoupled
spectrum of uniformly labeled material shown for reference; the remaining

spectra were acquired without 13C decoupling. From top to bottom, uniformly
labeled rAMP (decoupled); uniformly labeled rAMP (not decoupled); rAMP
isolated from E. coli DL323; and rAMP isolated from E. coli K10-1516. Both

bacterial strains were grown on 2 13C glycerol. Colored lines are added to help
guide the eye.

While analysis of one-dimensional 1H spectroscopy can ascertain the
level of 130 enrichment for a given site, little information can be obtained for

neighboring 130130 interaction. This is vital to determine whether a 130 atom is

59

indeed isolated from other 130 neighbors. To probe neighboring 130
interactions, one-dimensional 13C slices from HSQC experiments were analyzed
for each carbon atom of the ribose ring. One-dimensional 13C slices at the C4'
show no detectable 1JCC scalar coupling (Fig 2.10). From these results, one can
conclude the 4' 1H-13C spin systems is essentially isolated using 2-13C glycerol
in DL323 and zwf mutants. For the DL323 prep, 13C labeling can be detected for
both the 1' and 2' resonances, whereas 13C labeling is observed only for the 2'

resonance of K10-15-16. Consistent with this result is the absence of 1Joe

scalar coupling for the C2' of the K10-15-16 spectrum, unlike the DL323
spectrum. Combining the two analyses, percentages of isotopomers have been

obtained for the different strains (Table 2.1). Taken together, these results

demonstrate the ability to obtain 22421302 rNMPs using E. coli strains that
exclusively use the non-oxidative portion of the PPP with 2-130 glycerol as the

sole carbon. For the purposes of 13C NMR spin relaxation studies,

ribonucleotides obtained from zwf strains have the most useful isotopic labeling

pattern.

60

(a) (b) A

 

 

 

 

 

 

 

 

 

 

L L

LNA
L,

 

 

 

 

 

150 100 50 -50 -100 -150 HZ 150100 50 0 -50 -100-150 HZ

Figure 2.10: 1300 slices from 1H-130 HSQC spectra. Sections parallel to the

13C frequency axis through the (a) C2' and (b) C4' resonance of rAMP. rAMP
was obtained from (top) commercial uniformly labeled rAMP; (middle) E. coli
strain DL323; and (bottom) E. coli K10- 15- 16. Both bacterial strains were grown

with 2-130 glycerol as the sole carbon source.

61

Table 2.1: 13C isotopomer species obtained in nucleotide preparations

 

E. coli
strain

DL323 1,3-13C2 glycerol 1',3',5'-13C3 ribose 25
232521303 ribose 55
1',2',3',5'-13c4 ribose 10
31521302 ribose 5
Various 1301 ribose < 5

Carbon Source lsotopomer Yield (%)

DL323 2-13c glycerol 13421302 ribose 55
2',4'-1302 ribose 30

1222421303 ribose 5

4'-13C1 ribose 10

Various 13C1 ribose < 5
K10-15-16 2-130 glycerol 2.421302 ribose 30
421301 ribose 5
12421302 ribose 5
, , , 13 . 5
1 ,2 ,4- C3 rlbose
Various 1301 ribose < 5

 

Analysis of lsotopomers from Specifically-Labeled Glucose. The above

procedures can provide spectroscopically useful methods to isolate the 2', 4', and

5' 130 resonances. The 3' 13C resonance, however, has been predicted to

undergo the greatest chemical shift change upon C3'-endo to CZ'-endo
interconversion (53,54), and thus may be the most sensitive spin relaxation

probe of conformational exchange processes. Therefore procedures were

62

explored which would give rise to a fully isolated 3' 1H-130 spin system. Based

on previous results for 13C enrichment, it was determined that R5P can be

obtained from the oxidative portion of the PPP. Because the oxidative reactions

remove the first carbon, 13C enrichment of 03' could potentially be obtained with

glucose enriched at the fourth carbon atom. This prediction is verified by the

one-dimensional 1H NMR spectrum of rAMP (Fig 2.11) using wild type E. coli

cells with 4-130 glucose as the sole carbon source. In the non-decoupled

spectrum of this preparation, the H3' peak is mostly split into a doublet.

Consistent with the model, no observable multiplet peaks are observed for the

remaining sugar protons. This procedure provides the means to 13C enrich the

C3' atom of R5P at a high percentage and excellent specificity.

63

H5'

H2' H3' H4'

L

. .fi,....,....,...r
6.0 5.5 5.0 4.5 4.0

 

 

 

 

 

 

A-

 

1H proton chemical shift (ppm)
Figure 2.11: Fraction of 13C label at C3' of rAMP. 13C-decoupled (top) and

undecoupled (bottom) NMR spectra of rAMP prepared from wild type E. coli
grown on 4 C glucose. Lines were added to help guide the eye.

13

Measurement of 136 Relaxation Rates. 0130 dipolar and scalar

contributions to the relaxation mechanism have limited extensive analysis for the

ribose ring of RNA molecules. An alternate-site labeling pattern has been

developed that isolates 1H-1BC spin systems within the ribose ring of

ribonucleotides which can remove these contributions allowing for accurate

. . . 13 1 . .
analySIs ln RNA molecules. To examlne the effects of C- 3C lnteractlons on
. . . 13 .
the measured relaxatlon rate, a preVIous procedure measurlng C relaxatlon

rates in side chain amino acid atoms (13) has been‘ adapted to compare 130

64

relaxation rates of 02' and C4' atoms for specifically and uniformly labeled
samples. rAMP NMR samples were solvated in 98% Dg-glycerol. The

temperature dependent viscosity of glycerol allows for the control of the

correlation time for global tumbling, 1c. Acquiring the data at multiple

temperatures, different To values can be simulated with a single sample to
13 13 . . . . 13 .

analyze C- C lnteractlons for speCIfically and unlformly C relaxatlon rates.

Because 1,; increases with molecular size, it can be interpreted that multiple

molecular weights, and thus different size molecules, were also simulated. Since

the data were collected in similar fashion for both samples, any differences

detected can be attributed to 130-130 interactions.

Because the H2' and H4' peaks are well resolved from other peaks in 13C-

decoupled spectra (Fig. 2.9), the two-dimensional pulse sequence was reduced

to a one-dimensional pulse sequence by not incrementing t1 to frequency label

130 carbons, allowing for shorter acquisition times. Table 2.2 compares the

effect of varying the temperature, and thus the correlation time, on longitudinal

relaxation rates in specifically labeled (22421302) rAMP, derived from E. coli '
strain K10-1516 grown on 2-130 glycerol, and uniformly 130 labeled rAMP. For
02', measured R1 values are similar at 30 and 25 °C for both specifically and

uniformly labeled samples. However, the measured R1 values are different at 20

65

and 15 °C, which can be attributed to 13C-130 interactions at higher molecular
weight molecules (Fig. 2.12). This observation is in agreement with the results of
Bax and coworkers (19), who showed that the contributions due to 13C—13C
interactions are negligible for correlation times of less than 4 ns. For C4', the

measured R1 values differ at all measured temperatures (data not shown).

These results reveal the contribution of the 13C-130 interaction to the measured

relaxation rate at C4' and the removal of this interaction with the specific labeling
scheme. Taken together, these results demonstrate the usefulness of the
specific labeling scheme to measure accurate relaxation rates for ribose carbon

atoms in RNA molecules.

66

Table 2.2: 13C R1 and R1 p measurements obtained from specifically and
uniformly labeled rAMP.

13C R1 Measurements (3'1)

 

22421302 Uniform 130
T (°C) 02' C4' 02' C4'
30 1.48 :l: 0.05 1.13 :l: 0.02 1.59 :l: 0.07 1.29 :l: 0.05
25 1.1 i 0.1 0.90 d: 0.08 1.12 :l: 0.01 1.07 d: 0.04
20 0.95 1: 0.06 0.67 :l: 0.02 0.79 i 0.02 0.84 :t 0.04
15 0.9 i 0.2 1.9 :l: 0.1 0.4 i 0.2 0.9 :l: 0.1

13c R1 p Measurements (3'1) at yB1I2n=2980 Hz

 

2',4'-13c2 Uniform 13c
T (°C) 02' C4' 02' C4'
30 25 : 1 37.0 : 0.9 25.8 : 0.7 47 : 3
25 38:4 53:3 29:4 57:2
20 70:10 140:20 67:3 120:20
15 150 :l: 40 nda 90 1:10 nda

13C R1,, Measurements (3'1) at yB1I2n=1550 Hz

 

2421302 Uniform 13c
T (°C) 02' C4' 02' C4'
30 22:2 36:2 23:1 38:2

 

aRates could not be measured due to interference from sidebands from the
strong residual glycerol signal.

67

Figure 2.12: Longitudinal 13C relaxation of rAMP in glycerol. R1 curves for 02' of

(solid line) uniformly labeled and (dashed line) 2', 4' 1302 labeled rAMP at (a) 30
°C, (b) 20 °C, and (c) 15 °C.

68

0.

l

 

 

- — _
8. 6. A l
0 0 0 0

£23.: 85.2.52

 

 

- d u 4
0. 3 6. 4. 2
.I. 0 0 0 0

 

>333:— 13:5:qu

 

 

 

--
4.2 4.2.0
ll 00

1.0

36:85 35.-«5.5 Z

3.0

For R1 p values, the largest source of error arising from 13C—130

interaction is magnetization transfer via the Hartmann—Hahn effect during the

spin-lock period (19). The Hartmann-Hahn effect can be observed for the 02'

resonance (Table 2.2). At 30 °C using a spin-lock field of almost 3 kHz, R1,,

values are similar between the specifically and uniformly labeled rAMP.

However, as the temperature decreases, the measured relaxation rates become

dissimilar (Fig. 2.13a). Measured R1 p for the C4' resonance are similar within
error for both 25 and 20 °C (Fig. 2.13b). The R1 p was not similar for the C4'

resonance at 30 °C (see Discussion). R1 p measurements were not obtained at

15 °C due to spectral overlap arising from sidebands of the residual labeled

glycerol. In the absence of exchange on the us to ms timescale, R1 p values are

determined to be independent of applied yB1 field strength (8). Thus, relaxation
rates obtained at 3 kHz spin lock power should be identical to the relaxation rates

obtained at a second spin lock power. To confirm this, R1 p values were

measured using a spin-lock field of 1.6 kHz at 30 °C. Measured R1 p for 02' and

C4' are similar for both field strengths, which is consistent with exchange models.

In short, these results demonstrate the effectiveness of the specific labeling

scheme to remove unwanted 13C-13C effects and permit accurate NMR spin

relaxation analysis in RNA molecules.

70

Normalized Intensity

 

 

 

Time (ms)

Normalized Intensity

 

 

 

Time (ms)

Figure 2.13: Transverse 13C relaxation of rAMP in glycerol. On resonance R1 p

curves for (solid line) uniformly labeled and (dashed line) 22451302 labeled rAMP
at 25 °C @ 2980 Hz spin-lock power for (a) C2' and (b) C4'.

71

DISCUSSION

In this work, methods have been described and characterized to efficiently

130 enrich specific carbons of the ribose ring of RNA molecules for 13C NMR
. . . . . . 13 13
spln relaxatlon experlments (50). Also, quantitatlve analyses problng C- C

interactions for R1 and R1 p values have been compared between specifically and

uniformly labeled rAMP samples.

The E. coli strain DL323 was designed to prevent metabolic scrambling of

13C labels in the side chains of amino acids derived from the citric acid cycle

intermediates (47,48). For ribonucleotides, the distribution of 13C carbons within

the sugar ring would be dependent on the enzymes of the PPP used for R5P

production. When grown on 1,3-1302 glycerol, E. coli strain DL323 yields a
' r i r13 r r :13 - .

mlxture of 1,3,5- C3 and 2,3,5- C3 lsotopomers, Wlth small amounts of
. . . . , , , , 13 . 13

mlnor specres includlng 1 ,2,3,5,- C4 lsotopomers. Due to the lack of C

enrichment at C4', C5' is isolated from other 130 carbons, thus removing any

13 13 13

C-13C interactions. Unfortunately, there are still considerable C— C

interactions for 03'. When grown on 2-13C glycerol, rNMPs obtained from DL323
. . , , 13 , , 13 .

yield a mlxture of the 1 ,4- C2 and 2 ,4- 02 lsotopomers of R5P, plus small

amounts of minor species including 1222421303. Thus, C4' is isolated from other

130 carbons similar to 05' using 1,3-13C2 glycerol as a carbon source.

72

Unfortunately, 02' is not completely isolated due to interference from neighboring
C1' in a subset of isotopomers. The most likely source for 130 scrambling arises
due to repetitive iterations of the complete (oxidative and non-oxidative) PPP.

130 scrambling could also emerge due to intermediates bleeding off the PPP into

other pathways and re-entering the PPP. Finally, the level of 13C incorporation

at 02' is low, which could potentially lead to problems with data analysis due to
low sensitivity. Based on all these observations using E. coli strain DL323, other
methods were explored to highly enrich 02' in ribonucleotides.

The results obtained indicate that growth of zwf E. coli strains (K10-15-16

and DF2001) on specific 13C glycerol synthesize essentially all of their R5P, and

consequently their rNMPs, from the non-oxidative portion of the PPP. When 2-

13 . . .
C glycerol was used as a carbon source, spectroscoplcally useful Isolation of

the 2' 1H-130 and 4' 1H-13C spin systems was obtained, with the desired
carbons labeled at 85% and >95%, respectively. It should be noted that, in this
preparation, the ratio of 2',4'-13C2 R5P to 42130 R5P is considerably higher than

the 2:1 value expected. This effect can be attributed to the depletion of the

intermediate erythrose-1-phosphate for use in anabolic reactions including amino

acid synthesis (55). The exact source for the small amount of 13C enrichment at

C1' is not clear at this time. A hypothetical reason could be due to 13C

scrambling in pathways not considered in this work.

73

The 13C labeling pattern for the ribose ring using zwf E. coli strains is

superior to the pattern obtained from E. coli DL323 for NMR spin relaxation

studies because the alternate-site labeling pattern removes 130130 interactions
as well as yields a substantially higher fraction of C2' 130 enrichment, increasing
the signal-to-noise ratio, while the 1',2',4'-13C3 isotopomer is a minor contributor
, 13 . . 13 13 . .
to the total 2 C atom populatlon (z5%). Because of this, C- C lnteractlons
are deemed to be negligible. Therefore, the 224-1302 labeling scheme obtained
from 2-13C glycerol preparations of E. coli strains K10-15-16 or DF2001 should
allow for the analysis of the 2' and 4' 13C atoms at nearly full or full occupancy,

respectively, yielding a near ideal case for 130 NMR spin relaxation studies of

these two carbons.

Another advantage of the alternate-site labeling scheme is the collapse of

ribose peak triplets (for 2’, 3', and 4' 13C resonances) into singlets, which can

help alleviate spectral overlap in multidimensional NMR spectra. This effect is of
particular importance in the crowded ribose spectral region of HSQC spectra. In
uniformly labeled samples, multiplet collapse is typically achieved via the use of
constant-time spectroscopy (13), but at the cost of a substantial reduction in
experimental sensitivity.

Based on calculations that predicted large chemical shift changes for C3'

upon a change in ribose conformations, methods were explored to reliably 130

74

enrich C3'. This was done with wild type E. coli cultures using 4-130 glucose as
the sole carbon source. The results show outstanding effectiveness in 13C

enrichment of C3'. Glucose labeled with 130 at each individual carbon atom is
commercially available, albeit at considerable financial expense. For example, 4-

13C glucose was listed at $1600/g from Cambridge Isotope Laboratories, Inc.

compared to $449/g for 2-130 glycerol and $699/g for 1,3-1302 glycerol. Hence

this route provides a robust and selective, albeit costly, method to label a desired

individual ribose carbon. Even though this method costs more than specifically

or uniformly labeled methods, it can be beneficial for RNA 130 NMR spin

relaxation studies because of the large expected chemical shift change for the

03', due to sugar re-puckering, which will lead to a greater Rex dependence in

dispersion experiments (8) (see Chapter 3). This can potentially offset the need

to use higher magnetic fields.

In the standard model-free analysis in biomolecules, R1, R2 (R1p), and

heteronuclear NOE values are interpreted as arising from 1H—X (X = 13C or 15N)

dipole—dipole and X chemical-shift anisotropy (CSA) relaxation mechanisms,

along with possible contributions of us-ms timescale conformational exchange to

R2 (R1p) (6). In the case of 13C, however, the presence of scalar and dipolar

couplings between the resonance of interest and directly bonded 130 atoms

gives rise to additional terms in the relaxation equations that substantially

75

complicate the collection and/or interpretation of results (13). The alternate-site

labeling scheme was developed to remove such effects. Therefore, the effects of

neighboring 13C atoms on measured R1 and R1 p relaxation rates in specifically
and uniformly 130 labeled rAMP have been examined (50).
13 . . 13
For C R1 values in the presence of directly bonded C carbons, the
longitudinal relaxation rates will be affected by 13C-13C dipolar interactions as
1 13 . 13 . . . .

well as H- C dipolar and C CSA contributions. B0isbouwer and coworkers
13 13 . . . . .

(19) demonstrated that C- C dipolar relaxation can be negligible in the

analysis of longitudinal relaxation rates. The TC for the DNA oligomer used was
. . . 13 13 .

estimated to be 3.35 ns. However it was predicted that C- C dipolar

contributions would become more significant as the re value increased, and

therefore not negligible.

By adjusting the temperature, different Tc values could be simulated

ranging from 5.4 to 16.9 ns (30 °C to 15 °C) using a single NMR sample. Due to

sample setup, any differences observed in the relaxation rates for the alternate-

site labeling sample and uniformly labeled sample can be attributed to 130-13C
interactions. Such interactions were detected in both R1 and R1 p values. At

higher temperatures corresponding to short correlational times, R1 values for 02'

were similar for the alternate-site and uniformly labeled samples, while R1 values

76

differed at higher temperatures. This is consistent with predictions Bax and co-

workers (19) made. C4' R1 values are different at all temperatures. A possible

reason for this could be due to the CSA for C4'. Bax and coworkers (56)
measured chemical shift tensors for ribose carbons in helix-35 of 238 rRNA.
From this work, the CSAs for 03' and C4' were determined to be greater than
C1', C2', and C5'. Because the measured CSA of ribose C4' is almost three
times larger than ribose 02', it can be theorized that the CSA of C4' is not
negligible at short correlation times.

For the measurement of transverse relaxation rates, two distinct

measurement schemes are possible. R2 measurements based on the Carr-

Purcell-Meiboom-Gill (CPMG) pulse scheme are completely unsuitable in the

presence of 130—130 couplings due to oscillatory behavior arising from echo
modulation (13). We did not obtain R2 rates using CPMG pulse sequences.
However, due to the removal of 1Jcc: scalar coupling, R2 measurements could
be reliably obtained using the 13C specific labeling pattern. It should be noted
that in the 13C specific labeling pattern, the ZJCC scalar coupling between C2'
and C4' is still present and, in principle, could affect R2 measurements.
However, the ZJCC coupling has been measured to 0.9 Hz in adenosine (57) as

compared to 40 Hz for 1Jcc (58). Due to this low value, the 2JCC scalar coupling

can be neglected.

77

Another method to measure transverse magnetization is using a spin-

locked rotating-frame (R1p) pulse sequence. In this setup, the greatest source of

. 13
error arises from homonuclear Hartmann—Hahn transfer to covalently bound C

carbons during the spin-lock period (13,59). This effect is negligible if magnitude

of the scalar coupling is small relative to the difference in effective field strength

between the two nuclei, which is the resonance offset. In uniformly 13C labeled

DNA, this effect is slight due to the wide separation of chemical shifts for carbons

at different sites. For example, in the dodecamer studied by the Bax group (19),

the closest approach is 8 ppm, between C3' and C4', and accurate R1 p

measurements could be obtained. In RNA, by contrast, the possibilities for
Hartmann—Hahn transfer are substantially greater. In the lead-dependent
ribozyme, the median separation between C2' and C3' shifts is 2.6 ppm, and the
ranges of the two carbon types actually overlap (60). in rAMP, the measured
chemical shift difference between the 02' and C3' resonances is 4 ppm, while the
measured chemical shift difference between the C3' and C4' resonances is 12

ppm. One would presume Hartmann-Hahn transfer to be more likely between

02' and 03' as opposed to C3' and C4'. 13C R1 pvalues are dissimilar among the
alternate-site and uniformly C2' at temperatures below 30 °C whereas 13C

R1 pvalues are similar for C4' at all temperatures.

78

R1 p measurements are sensitive to motions on the ps-ms regime. In the
absence of motion, R1 p values are independent of RF field strength (2,33). R1p

values were obtained at 30 °C at 1.5 and 3 kHz. R1 p for C2' and C4' are similar

within error at both fields measured. This result was expected because
ribonucleotide monomers are not predicted to have sugar pucker conformation
transitions. However, the Hartmann—Hahn effect could be particularly
troublesome in the use of relaxation dispersion studies to study ps-ms timescale

exchange in RNA molecules, as magnetization could vary with spin-lock strength

and therefore could possibly yield an artifactual dependence of R1p relaxation

rate on wait that would be erroneously interpreted as conformational exchange.

To analyze unambiguously interconversion of C3'-endo and C2'-endo

conformations at the active sites of ribozymes using CPMG and R1 p relaxation

methods, the use of alternate-site labeling or related schemes is thus an absolute
necessity (see Chapter 3).

Here, a method has been presented to use a convenient biosynthetic

procedure to prepare rNMPs with favorable 13C labeling patterns that isolate 1H-
13 . . . . . 13 . . .
C spin systems Within the ribose ring for C NMR spin relaxatlon studles.

Using 13C relaxation measurements for rAMP solvated in Dg-glycerol samples to

simulate various correlation times, it was demonstrated that magnetic

. . . 13 . .
interactions from adjacent C nuclel can be a source of error in R1

79

measurements for molecules with long correlation times and in R1p

13 . . . .
measurements where coupled C nuclei have Similar effective fields. The

alternate-site labeling scheme is a practical approach to removing these errors
(50). The methods reported in herein will open a new window into the analysis of
conformational dynamics in RNA molecules and advance efforts to understand
the correlation between dynamics and function in ribozymes and other RNA

systems.

ACKNOWLEDGMENTS

I would like to thank Kristine Julien for growth and isolation of rNMPs using 2-13C

glycerol for the DL323 strain.

80

REFERENCES

10.

Dayle, K.T., Wagner, G. and Lefevre, J.F. (1996) Theory and practice of
nuclear spin relaxation in proteins. Annu Rev Phys Chem, 47, 243-282.

Palmer, A.G., 3rd. (2004) NMR characterization of the dynamics of
biomacromolecules. Chem Rev, 104, 3623-3640.

Palmer, A.G., 3rd. (1997) Probing molecular motion by NMR. Curr Opin
Struct Biol, 7, 732-737.

lshima, R. and Torchia, DA. (2000) Protein dynamics from NMR. Nat
Struct Biol, 7, 740-743.

Shajani, Z. and Varani, G. (2007) NMR studies of dynamics in RNA and
DNA by 13C relaxation. Biopolymers, 86, 348-359.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 1. theory
and range of validity. J Am Chem Soc, 104, 4546-4559.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 2. analysis
of experimental results. J Am Chem Soc, 104, 4559-4570.

Palmer, A.G., 3rd, Kroenke, CD. and Loria, J.F. (2001) Nuclear magnetic
resonance methods for quantifying microsecond-to-millisecond motions in
biological macromolecules. Methods Enzymol, 339, 204-238.

Kay, L.E., Torchia, DA. and Bax, A. (1989) Backbone dynamics of

proteins as studied by 15N inverse detected heteronuclear NMR

spectroscopy: application to staphylococcal nuclease. Biochemistry, 28,
8972-8979.

Zhou, H., McEvoy, M.M., Lowry, D.F., Swanson, R.V., Simon, MI. and
Dahlquist, F .W. (1996) Phosphotransfer and CheY-binding domains of the

81

11.

12.

13.

14.

15.

16.

17.

18.

histidine autokinase CheA are joined by a flexible linker. Biochemistry, 35,
433-443.

Mandel, A.M., Akke, M. and Palmer, A.G., 3rd. (1995) Backbone dynamics
of Escherichia coli ribonuclease HI: correlations with structure and function
in an active enzyme. J Mol Biol, 246, 144-163.

Mandel, A.M., Akke, M. and Palmer, A.G., 3rd. (1996) Dynamics of
ribonuclease H: temperature dependence of motions on multiple time
scales. Biochemistry, 35, 16009-16023.

Yamazaki, T., Muhandiram, R. and Kay, L. (1994) NMR experiments for
the measurement of carbon relaxation properties in highly enriched,

uniformly 13C, 15N-labeled proteins: application to Ca carbons. J Am
Chem Soc, 116, 8266-8278.

Wikstrom, M., Forsen, S. and Drakenberg, T. (1996) Backbone dynamics
of a domain of protein L which binds to immunoglobulin light chains. EurJ
Biochem, 235, 543-548.

Lienin, S.F., Bremi, T., Brutscher, B., Bruschweiler, R. and Ernst, RR.
(1998) Anisotropic intramolecular backbone dynamics of ubiquitin
characterized by NMR relaxation and MD computer simulation. J Am
Chem Soc, 120, 9870-9879.

Duchardt, E. and Schwalbe, H. (2005) Residue specific ribose and

nucleobase dynamics of the cUUCGg RNA tetraloop motif by NMR 130
relaxation. J Biomol NMR, 32, 295-308.

Hall, KB. and Tang, C. (1998) 13C relaxation and dynamics of the purine
bases in the iron responsive element RNA hairpin. Biochemistry, 37,
9323-9332.

Shajani, Z. and Varani, G. (2005) 13C NMR relaxation studies of RNA
base and ribose nuclei reveal a complex pattern of motions in the RNA
binding site for human U1A protein. J Mol Biol, 349, 699-715.

82

19.

20.

21.

22.

23.

24.

25.

26.

27.

Boisbouvier, J., Wu, Z., Ono, A., Kainosho, M. and Bax, A. (2003)

Rotational diffusion tensor of nucleic acids from 3C NMR relaxation. J
Biomol NMR, 27, 133-142.

Borer, P.N., LaPlante, S.R., Kumar, A., Zanatta, N., Martin, A., Hakkinen,

A. and Levy, GO. (1994) 13C-NMR relaxation in three DNA
oligonucleotide duplexes: model-free analysis of internal and overall
motion. Biochemistry, 33, 2441-2450.

Gaudin, F., Chanteloup, L., Thuong, NT. and Lancelot, G. (1997)

Selectively 13C-enriched DNA: dynamics of the C1'H1' and C5'H5' or
C5'H5" vectors in d(CGCAAATTTGCG)2. Magn Reson Chem, 35, 561-
565.

Millet, O., Loria, J.P., Kroenke, C.D., Pons, M. and Palmer, A.G., 3rd.
(2000) The static magnetic field dependence of chemical exchange
Iinebroadening defines the NMR chemical shift time scale. J Am Chem
Soc, 122, 2867-2877.

Loria, J.P., Rance, M. and Palmer, AG. (1999) A relaxation-compensated
Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange
by NMR spectroscopy. J Am Chem Soc, 121, 2331-2332.

Mulder, F.A., Mittermaier, A., Hon, 3., Dahlquist, F.W. and Kay, LE.
(2001) Studying excited states of proteins by NMR spectroscopy. Nat
Struct Biol, 8, 932-935.

Tollinger, M., Skrynnikov, N.R., Mulder, F.A., Forman-Kay, JD. and Kay,
LE. (2001) Slow dynamics in folded and unfolded states of an SH3
domain. JAm Chem Soc, 123, 11341-11352.

Cole, R. and Loria, JP. (2002) Evidence for flexibility in the function of
ribonuclease A. Biochemistry, 41, 6072-6081.

Kovrigin, E.L., Cole, R. and Loria, JP. (2003) Temperature dependence of
the backbone dynamics of ribonuclease A in the ground state and bound
to the inhibitor 5'-phosphothymidine (3'-5')pyrophosphate adenosine 3'-
phosphate. Biochemistry, 42, 5279-5291.

83

28.

29.

30.

31.

32.

33.

34.

35.

36.

Korzhnev, D.M., Bezsonova, |., Evanics, F., Taulier, N., Zhou, Z., Bai, Y.,
Chalikian, T.V., Prosser, RS. and Kay, LE. (2006) Probing the transition
state ensemble of a protein folding reaction by pressure-dependent NMR
relaxation dispersion. J Am Chem Soc, 128, 5262-5269.

Kim, S. and Baum, J. (2004) An on/off resonance rotating frame relaxation
experiment to monitor millisecond to microsecond timescale dynamics. J
Biomol NMR, 30, 195-204.

Massi, F., Johnson, B, Wang, C., Rance, M. and Palmer, A.G., 3rd.

(2004) NMR R1 p rotating-frame relaxation with weak radio frequency
fields. J Am Chem Soc, 126, 2247-2256.

Massi, F., Grey, M.J. and Palmer, A.G., 3rd. (2005) Microsecond
timescale backbone conformational dynamics in ubiquitin studied with
NMR R1 rho relaxation experiments. Protein Sci, 14, 735-742.

Grey, M.J., Tang, Y., Alexov, E., McKnight, C.J., Raleigh, DP. and
Palmer, A.G., 3rd. (2006) Characterizing a partially folded intermediate of
the villin headpiece domain under non-denaturing conditions: contribution
of His41 to the pH-dependent stability of the N-terminal subdomain. J Mol
Biol, 355, 1078-1094.

Hoogstraten, C.G., Wank, JR. and Pardi, A. (2000) Active site dynamics
in the lead-dependent ribozyme. Biochemistry, 39, 9951-9958.

Blad, H., Reiter, N.J., Abildgaard, F., Markley, J.L. and Butcher, SE.
(2005) Dynamics and metal ion binding in the U6 RNA intramolecular
stem—loop as analyzed by NMR. J Mol Biol, 353, 540-555.

Shajani, Z. and Varani, G. (2008) 13C relaxation studies of the DNA target
sequence for hhai methyltransferase reveal unique motional properties.
Biochemistry, 47, 7617-7625.

Maltseva, T.V., Foldeséi, A., Ossipov, D. and Chattopadhyaya, J. (2000)
Comparative 13C and H relaxation study of microsecond dynamics of the

AT tract of selectively 13C/2 H double-labelled DNA duplexes. Magn Reson
Chem, 38, 403-414.

84

37.

38.

39.

40.

41.

42.

43.

44.

45.

Fuson, MM. and Prestegard, J.H. (1983) Dynamics of an interfacial
methylene in dimyristoylphosphatidylcholine vesicles using carbon-13 spin
relaxation. Biochemistry, 22, 131 1-1316.

Henry, G.D., Weiner, J.H. and Sykes, 8D. (1986) Backbone dynamics of

a model membrane protein: C NMR spectroscopy of alanine methyl
groups in detergent-solubilized M13 coat protein. Biochemistry, 25, 590-
598.

Dellwo, M.J. and Wand, A.J. (1989) Model-independent and model-
dependent analysis of the global and internal dynamics of cyclosporin A. J
Am Chem Soc, 111, 4571-4578.

lsaacs, R.J., Rayens, WS. and Spielmann, HP. (2002) Structural
differences in the NOE—derived structure of G-T mismatched DNA relative

to normal DNA are correlated with differences in 130 relaxation-based
internal dynamics. J Mol Biol, 319, 191-207.

Spielmann, HP. (1998) Dynamics in psoralen-damaged DNA by 1H-

detected natural abundance 13C NMR spectroscopy. Biochemistry, 37,
5426-5438.

Spielmann, HP. (1998) Dynamics of a bis-intercalator DNA complex by

1H-detected natural abundance 130 NMR spectroscopy. Biochemistry, 37,
16863-16876.

Kishore, A.l., Mayer, MR. and Prestegard, J.H. (2005) Partial 130 isotopic
enrichment of nucleoside monophosphates: useful reporters for NMR
structural studies. Nucleic Acids Res, 33, e164.

Kojima, C., Ono, A., Kainosho, M. and James, TL. (1998) DNA du lex

dynamics: NMR relaxation studies of a decamer with uniformly 3C-
labeled purine nucleotides. J Magn Reson, 135, 310-333.

SantaLucia, J., Jr., Shen, L.X., Cal, 2., Lewis, H. and Tinoco, |., Jr. (1995)
Synthesis and NMR of RNA with selective isotopic enrichment in the
bases. Nucleic Acids Res, 23, 4913-4921.

85

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

Hatala, P.J., Kallmerten, J. and 1Borer, PN. (2001) Regioselective

synthesis of 1,3,5-13C3 and 2,4-1 302- labeled 2-deoxyribonolactones.
Nucleosides Nucleotides Nucleic Acids, 20, 1961- 1973.

L3eMaster, D. M and Cronan, J. E., Jr. (1982) Biosynthetic production of

3-C labeled amino acids with site-specific enrichment. J Biol Chem, 257,
1224- 1230.

LeMaster, D. M. and Kushlan, D. M. (1996) Dynamical mapping of E. coli

thioredoxin via13C NMR relaxation analysis. J Am Chem Soc, 118, 9255-
9264.

Batey, R.T., Battiste, J.L. and Williamson, JR. (1995) Preparation of
isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol,
261, 300-322.

Johnson, J.E., Jr., Julien, K.R. and Hoogstraten, CG. (2006) Alternate-
site isotopic labeling of ribonucleotides for NMR studies of ribose
conformational dynamics in RNA. J Biomol NMR, 35, 261-274.

Voet, D., Voet, J. and Pratt, CW. (2002) Fundamentals of Biochemistry.
John Wiley & Sons, Inc, Hoboken, New Jersey, USA.

Fraenkel, D.G. (1968) Selection of Escherichia coli mutants lacking
glucose-6-phosphate dehydrogenase or gluconate-6-phosphate
dehydrogenase. J Bacteriol, 95, 1267-1271.

Ebrahimi, M., Rossi, P, Rogers, C. and Harbison, GS. (2001)

Dependence of 13C NMR chemical shifts on conformations of RNA
nucleosides and nucleotides. J Magn Reson, 150, 1-9.

Rossi, P. and Harbison, GS. (2001) Calculation of 13C chemical shifts in

RNA nucleosides: structure-13C chemical shift relationships. J Magn
Reson, 151, 1-8.

Hoogstraten, CG. and Johnson, J.E., Jr. (2007) Metabolic Labeling:
Taking advantage of bacterial pathways to prepare spectroscopically

86

56.

57.

58.

59.

60.

useful isotope patterns in proteins and nucleic acids. Concepts Magn
Reson Part A, 32A, 35-55.

Bryce, D.L., Grishaev, A. and Bax, A. (2005) Measurement of ribose
carbon chemical shift tensors for A-fonn RNA by liquid crystal NMR
spectroscopy. J Am Chem Soc, 127, 7387-7396.

Kline, PC. and Serianni, AS. (1990) 13C-enriched ribonucleosides:

synthesis and application of 13C-1H and 13C-13C spin-coupling constants
to assess furanose and N-glycoside bond conformations. J Am Chem Soc,
112, 7373-7381.

Wijmenga, S. and van Buuren, B. (1998) The use of NMR methods for
conformational studies of nucleic acids. Prog Nucl Magn Reson
Spectrosc, 32, 287-387.

Hartmann, SR. and Hahn, EL. (1962) Nuclear double resonance in the
rotating frame. Phys Rev, 128, 2042.

Legault, P., Hoogstraten, C.G., Metlitzky, E. and Pardi, A. (1998) Order,
dynamics and metal-binding in the lead-dependent ribozyme. J Mol Biol,
284, 325-335.

87

Chapter 3

Analysis of Ribose Dynamics in the GCAA RNA Hairpin Studied by 130
NMR Spin Relaxation Experiments

Portions of this chapter were derived from “Extensive backbone dynamics in the

GCAA RNA tetraloop analyzed using 13C NMR spin relaxation and specific
isotope labeling” to be published by the Journal of the American Chemical
Society (2008), written by Dr. Charles G. Hoogstraten and myself.

88

INTRODUCTION

Conformational dynamics play an important role in the function of
enzymes. For RNA molecules, conformational dynamics play an equally
important role in events such as RNA catalysis and ligand recognition (1-4). A
common feature found in RNA tertiary structures are tetraloop sequences, which
can be found at the capping ends of hairpin stems (5), particularly GNRA
sequences where N stands for any nucleotide and R stands for either guanosine
or adenine. Studies have revealed that tetraloop sequences are highly stable (6-
8). In addition to stabilizing hairpin structures, GNRA tetraloop sequences have
been shown to mediate tertiary RNA-RNA (9,10) and RNA-protein interactions
(11,12).

NMR solution and X-ray crystal structures of GNRA RNA hairpins have
been solved (13-19). The structures of GNRA RNA hairpins determined from
NMR structures solved by Jucker et al. (14) were found to be A-form RNA with
anti conformations for all glycosidic bond angles with C3'-endo sugar pucker

conformations for nearly all residues. Within the tetraloop, only the ribose ring of

G5 was determined to have C3'-endo conformation while the remaining tetraloop

sugar pucker conformations were determined to be in equilibrium. The GNRA

tetraloop has a major change in the direction of the phosphate backbone

between the first and the second loop nucleotide (Gs-Ne) forming an asymmetric

loop in which the opening base stacks on the 5' side of the helix while the

remaining bases stack on the 3' side, and G5 and A3 form a sheared anti-anti

89

base pair (Fig. 3.1a). In two of the ten structures of the NMR ensemble for the

GCAA RNA hairpin (pdb code 1zih), the base of 03 was exposed to solvent and

not stacked upon A7 (Fig. 3.1b), in contrast to A5 of GAGA and GAAA RNA

hairpins (14). This is consistent with results from molecular dynamic simulations

of the GCAA RNA hairpin where the ribose ring of Ca was observed to undergo a

change in sugar conformation from C3'-endo to C2'-endo (Fig. 3.2) in a

correlated fashion with base extrusion (20).

 

Figure 3.1: The GCAA RNA hairpin. (a) Secondary structure of the RNA
construct used in these studies. Tetraloop residues are displayed in red and the
unstructured 3' tail is displayed in blue. (b) Superimposition of the ten NMR
structures (pdb code 1zih) for the tetraloop residues 5-8 of the GCAA RNA
hairpin (14).

90

R1O3°O

 

Figure 3.2: Diagram of sugar pucker conformations adopted by A-form RNA
molecules.

NMR spin relaxation techniques can be used to study molecular motions
on several timescales (21-30). A conformational exchange process that alters
the local magnetic environment for a given nucleus will contribute to the
dephasing of the transverse coherences (26). For the simplest case of a two-site

exchange model of A reversibly interconverting with B, the exchange rate

constant, kex, is the sum of the fonivard and reverse rate constants, k1 and k.1.

kex = k1 + k.1 [3.1]

The populated states can be observed in the NMR spectra as two resolved
resonances in the slow exchange regime or as a single population-weighted

average resonance in the fast exchange regime. Determination of exchange

regimes depends on the relation between kex and Art), the chemical shift

difference between the populated states. In the fast exchange regime, the

exchange rate is much greater than the chemical shift difference. Carr-Purcell-

Meiboom-Gill (CPMG) (31-33) and rotating-frame (R1p) (34) transverse

relaxation experiments are sensitive to motional processes that are on the us to

91

ms timescale. These motional processes can contribute to the overall transverse

relaxation rate as an additional term, Rex:
32°"s = R20 + Rex [3.21

b . . .
where R20 5 is the measured transverse relaxation rate and R20 is the

transverse relaxation rate in the absence of exchange.

CPMG experiments measure the transverse relaxation rate as a function

of successive spin echo time periods, ‘rCP, between 180° pulses. Conformational

changes that affect the frequency of precession lead to imperfect refocusing of

transverse magnetization during the spin-echo period. An expression for

transverse relaxation rate dependence on ‘tCP in the fast exchange regime is
given by:
R2(1/TCP )=R2(1/’CCP—*°°) + pApB(Aw)2/kex*(1-((tanh(kex/(4*tcP)))*4*ICP/kex))
[3.3]
where R2(1/th—-°°) is the transverse relaxation rate in the absence of
exchange, pp, and p53 are the exchangeable populations where the sum of pp, and

p3 are equal to one, Aw is the chemical shift difference in radians between the

populated states A and B, kex is the exchange rate constant, and 2101: is the

delay time between 180° pulses.
CPMG experiments have been used to measure exchange on the slow us

to ms timescale. For short us timescales, measurements require rapid spin echo

92

periods which can damage the NMR sample and/or probe from excessive

heating. Also, accurate measurement of relaxation rates is limited by the

evolution of heteronuclear scalar coupling during th, where in-phase and anti-

phase magnetization interconvert. In the absence of exchange, R20 is expressed
as:

R20 = eRzm + (1 — emf“ [3.4]

' a t' . .
where Rzm and R2 m are the transverse relaxation rate constants for in-phase

and anti-phase coherences averaged over the populations of each

conformational state. 0 s e s 1 reflects the averaging between in-phase and anti-

phase coherences due to evolution under the scalar coupling during ‘CCP.

Accurate motional parameters can be determined if e z 1, which corresponds to

rep less than 1/(4JXH). Loria et al. (35) have developed a relaxation

compensated CPMG pulse sequence that explicitly averages in-phase and anti-

phase magnetization such that E = 0.5. Using this pulse sequence, TCP values

as large as 2/JNH have been used for CPMG experimentation for basic
pancreatic trypsin inhibitor.

R1 p experiments measure the transverse relaxation rate as a function of
the effective field, a)eff, under spin-locked conditions. The effective field is the
vector sum of the applied (01 radio frequency (RF) field and the resonance offset,

0. The effective field, weff, in radians per second can be expressed as:

93

2 2 1/
weff= (601 + Q ) 2 [3-5]
In the fast exchange regime, the transverse relaxation rate measured as a

function of (Def-f is:
2 ,, 2 2
R1 p (weft) = R1p (weft —+°°) + pAPB(Aw) kex / (weir + kex ) [35]
where R1 p(a)eff—>°°) is the transverse relaxation rate in the absence of exchange

and Arc is the chemical shift difference in radians between pp, and p3. During

spin-locked conditions, magnetization is rotated from the z-axis to its resultant

effective field at a tilt angle given by tan6 = (01/0 and magnetization precesses

around the resultant effective field. Conformational changes which alter the

resonance offset lead to a loss in transverse magnetization.

R1 p experimentation also suffers from limitation of timescale

measurements. Yamazaki and co-workers (36) have designed an on-resonance

R1 p pulse sequence for accurate measurement of transverse relaxation rates
with tilt angles > 70°. To satisfy this condition, transverse relaxation rates are
measured at various (01 field strengths with constant resonant offset. However,
at low an field strengths, R1 p measurements become spurious due to oscillatory

behavior of the magnetization (25). Alternatively, one can use an adiabatic pulse
sequence (off-resonance) to align magnetization to effective fields with tilt angles

< 70° (37).

94

Generally, only one type of experimentation is used depending on the
timescale of the conformational exchange process. While advances have been

made such that individual techniques can cover broader timescales, relatively

few studies have combined CPMG and R1 p data for analysis (38,39). Such

limitation depends on the timescale of conformational exchange. This is

represented by simulated CPMG and R1 p relaxation dispersion curves in figure

3.3. For exchange rates >104 s4, R1 p experiments are better suited to

determine exchange parameters, while CPMG experiments contribute very little

to the overall curve. Conversely, CPMG experiments determine the shape of the

relaxation dispersion curve while R1 p experiments yield little information for

exchange rates < 103 s'1. Alternatively, the Palmer group has designed a R1 p

pulse sequence to measure transverse relaxation rates at effective fields as low
as 150 Hz (40). It is feasible that exchange rates that fall in between these

ranges would be best analyzed using both experimentations.

95

 

Rex (s'1)

 

 

 

ns,/2: (kHz)

  
   
 

A
O

lllllllljlllllJl

oo

-1
Rex (5 )
o:

 

h

 

  

 

 

781/27t (kHz)

Figure 3.3: Functional forms of the dependence of exchange contributions to
relaxation (Rex) for Rip (solid lines) and CPMG (dashed lines) experiments on
the effective yB1 field are shown based on equations 3.3 and 3.6. Simulations
are for 13C nuclei at 600 MHz (150 MHz 13C) and used Aw = 1 ppm, pA = 0.95,

and kex = 60,000 31 (top) or 4000 6'1 (bottom). Approximate experimentally-
accessible ranges for the three major types of relaxation dispersion experiments
are shown with horizontal lines, with “on” and “off" denoting on-resonance and

off-resonance R p experiments, respectively.

96

As mentioned previously, conformational dynamics play an important role
in the catalytic mechanism of ribozymes. A change in sugar pucker conformation
was observed for the adenosine residue adjacent to the cleavage site in the
active site of the hairpin ribozyme as determined from crystal structures (41).

Analysis of exchange rates for this sugar pucker transition could aid in the

understanding of the catalytic mechanism. While standard uniform 13C

enrichment renders the sugar ring of nucleotides unsuitable for NMR 13C spin

relaxation studies, the alternate-site labeling scheme can be used to probe sugar
dynamics (see chapter 2). The tetraloop region of the GCAA RNA hairpin
represents an excellent test-case for measurements of exchange rates for ribose
pucker transitions. With the validation of the alternate site labeling scheme as a
method to probe ribose dynamics in RNA molecules, analysis can be performed
for biologically relevant RNA systems, such as the lead-dependent ribozyme or
the hairpin ribozyme.

In this chapter, ribose dynamics across a broad timescale have been
probed for the GCAA RNA hairpin using the alternate-site labeling scheme. Fast

motions on the ps to ns timescales were analyzed by the model-free formalism of

Lipari and Szabo (42-44). By analyzing combined CPMG and R1 p relaxation
dispersion curves at multiple field strengths, novel 02' and C4' ribose dynamics
were observed within the tetraloop. From the similarity of measured exchange

rates for 02' and C4' atoms, the existence of concerted residue dynamics has

been proposed. Relaxation dispersion curves measured at multiple

97

temperatures has allowed for the determination of activation energies for ribose

pucker transitions.

MATERIALS and METHODS

Preparation of Labeled 13c Ribonucleotides. 2342130 rNTPs for the RNA

GCAA hairpin were prepared as described earlier (45,46). The phosphorylation
reaction was monitored by an Akta Basic FPLC (GE Healthcare) with a Vydac

302lC4.6 Ion-Chromatography column (10 pm, 4.6mm x 250 mm) using a linear

gradient from 25 mM 1:1 NaH2PO4zNa2HPO4, pH 2.8 to 125 mM 1:1
NaH2PO4zNa2HPO4, pH 2.9 over 34 min with a flow rate of 1 mL/min monitored

by UV absorbance. 13C rNTPs were purified from proteins using centrifugation,

concentrated using a Rotovapor RE 120 (Buchi) under vacuum at 40 °C to a final
volume of 1 mL, and purified using a DEAE Sephadex column with a 25 mL step

gradient from 50 mM to 1M triethylamine bicarbonate, pH 7.5 at a flow rate of 2

mL/min with 4 mL fractions. 13C rNTP fractions with an A250 above 0.1 were
pooled and lyophilized repeatedly to remove excess salt. Uniformly labeled 99%
13C, 15N rNTPs were purchased commercially (Spectra Stable Isotopes).

Transcription of 13C enriched GCAA RNA hairpin. 22421302 and uniform 130

rNTPs were incorporated into the fifteen nucleotide GCAA RNA hairpin by in vitro
transcription using recombinant T7 RNA polymerase and a synthetic DNA

template (47,48). Transcribed RNA was ethanol precipitated and purified using

98

denaturing polyacrylamide gel electrophoresis on a Bio-Rad model 491
preparative cell (49) with a flow rate of 1 mUmin with 4 mL fractions. RNA
fractions corresponding to GCAA RNA hairpin as determined by gel
electrophoresis were pooled, condensed as above to 1 mL, and desalted using a

G-25 Sephadex column collecting 1 mL fractions. RNA was dried, exchanged

into 99.9% D20 with repeated lyophilization, and resuspended to a final
concentration of 1.2 mM (2',4'-13c2) and 1.0 mM (uniform 13C) in 280 pL 99.96%
D20, 10 mM sodium phosphate pH 6.8, 100 mM NaCI, and 200 pM EDTA in an

advanced microtube matched with D20 (Shigemi, lnc.).

NMR Data Setup, Acquisition, and Processing. All NMR data were acquired

on Varian UnitleOVA 600 MHz (130 150 MHz) or Bruker Avance 900 MHz (130

225 MHz) spectrometers. The 900 MHz spectrometer was equipped with a

cryogenically cooled probe (Bruker TCI) in most cases. At 25 °C, 1H-13C

hetereonuclear single quantum coherence (HSQC) spectra were acquired to

verify chemical shift assignments previously obtained (14). The 1H-130 HSQC
spectrum at 600 MHz was acquired with 1024 x 512 complex points in the t2 and

t1 dimensions, respectively, with corresponding spectral widths of 6000 and 7540

Hz, and a 1 s recycle delay. The proton RF carrier was centered on the residual

HDO signal, and the 13C carrier frequency was set at 85 ppm to allow differential

folding of the ribose and base 13C resonances. The 1H-13C HSQC spectrum at

99

900 MHz was acquired with 512 x 64 complex points in the t2 and t1 dimensions,

respectively, with corresponding spectral widths of 9920 and 15848 Hz, and 1 s

recycle delay. The proton RF carrier was centered on the residual HDO signal,

and the 13C carrier frequency was centered at 75 ppm.
For 13C relaxation experiments, all two-dimensional spectra acquired on
the 600 MHz spectrometer were obtained with 1024 x 96 complex points in the t2

and t1 dimensions, respectively, with corresponding spectral widths of 6000 and

1800 Hz, a 2 s recycle delay, and 15 min of steady-state scans to allow for

temperature equilibrium. The proton RF carrier was centered on the residual

HDO signal, and the 13C carrier frequency was set at 78.3 ppm. For
experiments focusing on the aromatic ring, the 130 carrier frequency was

positioned at 138 ppm for C8 and C6 resonances and 153 ppm for C2

resonances. R1, R1 p, and 1H-13C heteronuclear NOE (hNOE) data were
acquired using minor variations of published pulse sequences (36). R1 delay

times ranged from 100 ms to 2000 ms collected in a random format. For R1 p,

delay times ranged from 10 ms to 120 ms in a random format acquired at 2980
Hz spin-lock field. For saturated heteronuclear NOE experiments, protons were
irradiated for 3 s with a total recycle delay of 7 s. For non-saturated
heteronuclear NOE experiments, protons were not irradiated with a recycle delay

of 7 s. Dispersion data were acquired using relaxation compensated CPMG

100

(35,50), on-resonance R1 p (36), off-resonance R1 p (37,51), and Hahn echo (52)

pulse sequences. CPMG experiments were acquired as a function of inter-pulse

spacing 210p, where TCP values ranged from 180 us to 2500 us, corresponding

to effective spin-lock powers 1781/21: [VCP=1/(4TCP)] of ca. 1400 Hz to 80 Hz. On
resonance R1 p experiments were acquired as a function of the applied (01 RF,
where 01/21: ranged from 1.5 to 6 kHz. Off resonance R1 p experiments were

acquired as a function of Q at a (01/27: of 3 kHz, where (2/21: ranged from 3.5 to

10 kHz. Magnetization was rotated to its effective field using adiabatic half

passages of 4 ms. Hahn echo experiments were acquired with top values
ranging from 25 to 62.5 ms. R1, R1 p, 1H-13C hNOE, Hahn Echo, CPMG, on-
and off-resonance data were acquired at 25 °C, and 35 °C. Off-resonance R1 p
was omitted at 15 °C. R1 and on-resonance R1 pwere acquired at 20 and 30 °C.

For 130 experiments acquired on the 900 MHz spectrometer, all two-

dimensional spectra were obtained with 512 x 64 complex points in the t2 and t1

dimensions, respectively, with corresponding spectral widths of 9921 and 5656

Hz, a 1.6 s recycle delay and 256 steady-state scans. The proton RF carrier was

centered on the residual HDO signal, and the 13C carrier frequency was set at 77

ppm. R1, R1 p, and heteronuclear NOE were acquired as described above. R1

101

delay times used ranged from 0 s to 1.705 s, while R1 p delay times ranged from
10 ms to 100 ms collected in a random format acquired at yB1/21t of 1 kHz.
CPMG and on-resonance R1 p data were acquired as described above. On-
resonance R1 p experiments were acquired with a room temperature probe

(Bruker TXI), and the 130 RF carrier was set in the middle of the C2' and C4'

regions, respectively. All data were acquired at 25 °C.

All NMR data were processed using FELIX 2002 (Felix NMR, Inc.). Prior

to Fourier transformation in the t2 dimension, a 20% DC offset was applied, data

were zero filled, and a 3 Hz exponential line broadening function was applied.

Prior to Fourier transformation in the t1 dimension, t1 was extended by 20% using

linear prediction and a cosine-squared apodization function was applied.
Data Analysis. Resolved crosspeak intensities were integrated using FELIX

2002 and exported to Igor Pro 5.0.4 (WaveMetrics). In all experiments, one data

. . . . b .
pomt was acqmred three times to determine error. R1 and R1 p0 s relaxation

rates were extracted by fitting the integrated peaks to a single exponential decay.

The 1H-130 hNOE was determined from the ratio of the saturated and non-
saturated spectra. For dispersion curves, R2 and R1 pObs rates were calculated

using the equation R2/R1pObs = 1fT * In (l(T)/Io), where T is the relaxation delay

time, I is the measured intensity, and lo is the measured intensity with no

102

relaxation delay. The IC was acquired twice to ensure reproducibility. R1 p rates
obs . . obs 2 . 2
were calculated from R1 p usmg the equation R1 p = R1 cos 6+ R1 p Sin (9,

where 8 = tan.1 ((01 IQ) is the tilt angle of the spin lock axis from the z axis, (01 is

the spin lock power in Hz and Q is the offset in Hz. Errors were propagated from

a single coeff or vcp repeated three times. Minimum thresholds of errors were set
0 _ 2 o o
to 2/o. kex and (Dex, where (Dex - pApB(Aw ), R2 , and R1 p values were

extracted by simultaneously fitting dispersion curves to CPMG and R1 p equations

(26) using Igor Pro 5 Global Analysis at a single field. To determine whether the
timescale of exchange is in the fast, intermediate, or slow regime, a scaling

factor, a, was determined using the equation (52):

<1 = (32 + Bl) / (32 - Bl) * (Rexz - Rex1) / (Rex2 + Rexl) [37]
where B1 and B2 are the magnetic field strengths in Tesla for the measured
values of Rex1 and Rexz, and Rex = pApBAw2 / kex.

Exchange parameters were determined from CPMG and R1 p dispersion
curves at 600 MHz and 900 MHz where (Dex at 900 MHz was imposed to be 2.25
times (Dex at 600 MHz. A kex was determined for each C2' and C4' atoms. In
cases were kex values coincided in error for 02' and C4' atoms of a single

residue, kex for that residue was refit to a single value. In cases where kex

103

values overlapped for several residues, multiple residues were refit to an overall

kex value. F-statistic critical values (or) were calculated to justify fitting dispersion

curves to motional dependent equations 3.3 and 3.6 as opposed to a horizontal

line. F-values for 02' and C4' resonances were obtained by comparing X2 values

obtained by fitting data sets to a horizontal line versus the dispersion equations.
F-statistic critical values were obtained using the website:
http://www.stattrek.com/tables/f.aspx. Confidence level was set to an a value of

>0.95.

RESULTS

A method was previously reported to specifically 13C label the ribose ring

of ribonucleotides in an alternate-site fashion (46). Using these rNTPs, it was

demonstrated that 13C-13C interactions were removed for R1 and R1 p

measurements using the alternate-site labeling scheme when compared to
uniform 13C labeling. With this proof of concept, rNTPs utilizing the alternate-site

labeling scheme were incorporated into a well characterized GCAA RNA hairpin
to study ribose dynamics, particularly sugar re-puckering, by measuring motional

timescales of C2' and C4' atoms.

Verification of Chemical Shifts. The 1H-13c HSQC at 600 MHz of the GCAA

RNA was obtained to verify chemical shifts previously reported (14). Figure 3.4

shows the crosspeaks for (top) C2' and (bottom) C4' atoms of the GCAA RNA

104

hairpin. Thirteen 02' and nine C4' resonances were sufficiently resolved for

analysis. All tetraloop resonances were resolved allowing for complete analysis
of the tetraloop. From the 1H-13C HSQC, one can discern the severe
broadening of the A7 02' resonance. At 900 MHz, this resonance was so

broadened that it is not observed in any spectra (data not shown). Due to this,

data analysis for A7 02' was only obtained at 600 MHz, while all other

resonances were analyzed at both fields.

105

 

 

 

75.0

‘67 E
[531;
./ r‘I‘K\
”i «532:: a
‘ - . ' l
I. I :l j.\\ fl‘w 411..
ft" ‘ 'l I’\ 'A’:\\\ ,j r“ 31.5 _,) _ if", l/x ;
ll.“- --'r . :1 ,» . .1 ~.;---~« l (.03
lg, ” . -. 7.3, j, /..-—/‘\v x C; /\. ‘l‘ _.
V’ ”ye/r" W 5 O
V M
{— c
A; \ \ I“ ‘ F
l ‘ .‘l' . 749s ‘ R‘
'1‘; . iii/j. 71.-.1 > ‘..:\\ @‘a 'a, "1,9, (74' ,5) 76 ' o
.,_ r, ('r.“ Itl‘Il I, I“ .,, CC"
561/ 53”- :‘32/ \7 \gj/ v
v gr;.r

I I I I I I I I I I I I I I I I I j I l' I I I f

 

- 77.0

 

4.2

1"
o

 

. I

C4 ,
_— 82.0
- E
- a.
' 3
I o
- 2

I
on
9"
o

 

I I I I I T I I I

 

 

 

I l T T I

4.8

. 34.0
1 4.4
H (9me

5.0

Figure 3.4: 1H-130 HSQC spectrum of a specifically-labeled GCAA RNA hairpin.
02' (top) and C4' (bottom) spectral regions are shown where tetraloop
resonances are labeled.

106

Measurement of 130 Relaxation Rates. Using modifications of published pulse

sequences (36), R1, R1 p, and 1H-13C hNOE measurements were acquired for

02' and C4' atoms of the GCAA RNA hairpin to confirm the presence of

dynamics for ribose ring atoms within the tetraloop. Presented in Figure 3.5 are

13C (a) R1 and (b) R1100bs curves for representative C2' atoms for the stem,
tetraloop, and 3' tail obtained at 25 °C at 600 MHz. Shown in Table 3.1 are R1
and offset-corrected R1 p 130 measurements obtained for 600 MHz data at 25 °C.

The effect of ps to ns dynamics can be revealed by an increase in R1
values as well as a decrease in R1 p values. The R1 for U15 C2' is higher (=17°/o)
than the average value of R1 for helical C2's. Also, the R1 p is diminished

(:18%). This is in good agreement with previous work (53,54). Because U15 is

at the terminus in a single stranded region and presumably the least structured
residue, relaxation rates obtained can be inferred as the maximum of ps to ns

measurable for residues in the GCAA RNA hairpin. Dynamics on the ps to ns

timescale were observed for resonances within the tetraloop. Increased R1
values were observed for G5 and 05 C23. Critical analysis of C4' atoms is less

clear due to the limited number of R1 values.

107

Figure 3.5: Representative 13C (a) R1 and (b) R1 p°bs curves obtained for (red)

A7; (black) G9; and (blue) U15 at 25 °C. Curves represent non-linear least
squares two-parameter fits to a single exponential.

108

 

 

 

   

a 1.0‘l
Q .
in
C
9 .
E
‘8 0.5-
g
(B ..
E
o I
2
0.0 TIIIIIIT‘IIIIIIT
0.0 0.5 1.0 1.5
Time(s)
b
.12:
0)
C
.9
.E
8
.5
To
E
o
2

0 A7 02'
0 G9 02'

     

2.0

B0.
__C

2.5

>
\l

O

"2

G)
0.0
as

 

 

Time (s)

109

 

Table 3.1: R1 and R1 ,3 13C measurement at 600 MHz for C2' and C4' atoms of
the GCAA RNA hairpin.

 

 

R1 Relaxation (5'1) R1 p Relaxation (s4)
62' Region C4' Region 62' Region C4' Region
G1 2410.1 G1 21210.05 G1 10.7103 G1 13310.6
G2 2.13 1 0.05 62 13.2 1 0.4
G3 2.27 1 0.07 G3 13.1 i 0.6

G5 2.49 1 0.06 G5 2.38 1 0.09 G5 16.6 1 0.7 Gs 17.6 1 0.7
C6 2.40 1 0.04 C6 2.44 1 0.08 C6 12.6 1 0.4 Ca 39.3 1 0.9
A7 2.29 1 0.09 A, 2.52 1 0.06 A7 53 1 4 A7 29.1 1 0.6
A8 2.27 1 0.02 As 2.38 1 0.03 A8 19.5 1 0.6 A8 21.5 1 0.8
Gg 2.24 1 0.06 Gg 2.32 1 0.04 Gg 12.9 1 0.6 Gg 13.4 1 0.6
C10 2.151003 C10 2410.1 C10 13.0106 C10 13.2106
2010.1 U12 2310.1 U12 12.4109 15810.9

U12

U12

    

Tetraloop atoms are shaded in grey while 3' single stranded atoms are shaded in
blue.

Model-free Analysis. To further quantify disorder on the ps-ns timescale, the

model-free formalism of Lipari & Szabo (42-44) was performed using R1, R1 p

and 1H-130 hNOE measurements acquired at 600 MHz. Calculations using the

model-free analysis were performed by Dr. Charles G. Hoogstraten. Using the

computer program Modelfree 4.1, motional parameters were determined for

atoms in the GCAA RNA hairpin using R1, R1 p, and hNOE values measured at

600 MHz. Motional parameters determined were 82, re, and Rex. The results of

this analysis are shown in Table 3.2 and Figure 3.6 With the exception of the

110

terminal G12U12 base pair and the unpaired 3'-tail, the order parameter $2, for a

given type of carbon, is high and uniform, indicating a lack of structural disorder
in the helix and tetraloop which is consistent with the well-structured and
thermodynamically stable nature of the system (7). The resulting overall isotropic
correlation time, 1c, for overall molecular tumbling was 3.00 ns, in good

agreement with a value recently reported from analysis of nucleotide aromatic

13C data in a 14-nucleotide GCAA RNA hairpin construct (55). Of the total of 22
resonances analyzed, 11 could be satisfactorily fit using 82 alone, 2 required the
re term in addition to the 82 term, 6 (comprising 6 of the 8 resonances within the

tetraloop) required Rex, and 3 (including one resonance from the tetraloop)

required all three parameters. It should be noted that relative values obtained for
the two carbon types are strongly dependent on the values of the chemical shift
anisotropy parameter (CSA) used (data not shown). The values of CSA used

here, 26.6 ppm for 02' and 91.7 for C4', have strong experimental support for

rigid A-form helices (56). Rigorous interpretation of relative 82 values between

C2' and C4' resonances would depend on careful consideration of the values and
relative orientation of the C—H bond vector and the CSA tensor for various states

of the ribose ring, which is beyond the scope of the present work.

111

Table 3.2: Model-free analysiszof ps — ns motion using 600 MHz data.

 

Resonancea 8 re, ps Rex, s-1
62 (32' 0.808 1 0.044 69.2 1 19.0 2.00 1 0.67
G3 02' 0.973 1 0.023
(35 02'” 0.903 1 0.038 892.4 1 291.2 3.84 1 0.79
C6 (32' 0.901 1 0.028 266.0 1 83.8
A7 C2' 0.984 1 0.027 39.5 1 4.2
A3 C2’ 0.975 1 0.011 6.28 1 0.64
(39 (32' 0.960 1 0.022
010 02' 0.926 1 0.014
U12 (:2 0.892 1 0.042
A14 (32' 0.972 1 0.017
U15 c2! 0.709 1 0.041 459.6 1 108.3
(31 (34' 0.815 1 0.018
(35 C4' 0.903 1 0.036 3.57 1 0.92
CS 04’ 0.926 1 0.030 24.9 1 1.0
A7 (:41 0.956 1 0.023 14.2 1 0.7
A8 (34’ 0.903 1 0.014 7.47 1 0.86
(39 C4' 0.878 1 0.015
C10 041 0.875 1 0.028
U12 04’ 0.792 1 0.049 40.3 1 20.5 3.31 11.19

 

(a) No satisfactory model could be fit for G1 C2', U13 02', and U13 C4' using the

combinations of 82, re, and Rex tested. As these residues are at the 5'-terminus
or in the 3'-unstructured tail, and thus of little relevance to the goals of the study,
further detailed analysis was not attempted.

satisfactorily fit by allowing a re value of ca. 30% of the isotropic correlation time,
in violation of the known limits of validity of the model-free approach. Results

should be interpreted with caution.

112

(b) This residue could only be

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0-3 — d
l I—T—r—T—T—r—T—‘r
G1 62 G3 04 Gs C6 A7 A8 G9 010 C11U12 U13A14 U15

 

a:
O
I

" b

A
O
l
J_—l-—'l
[J
O
[\2

-1
)
on
0

Rex (s
N
O

 

.3
O

lllllllllllllllllll

 

 

 

DI i

l6 l l lfiil I I l l l I l l T l
G1 62 G3 C4 Gs C6 A7 A8 G9 010 C11U12 U13 A14 U15

Residue

0

Figure 3.6: Analysis of relaxation data at 600 MHz usizng the model-free
formalism at 25 °C where (a) generalized order parameter S and (b) exchange

contribution to transverse relaxation Rex are shown. Open bars are C2' atoms
and shaded are C4' atoms.

113

Measurement of Relaxation Dispersion Curves. Motions on the us to ms

timescales can affect the transverse relaxation as a measurable term, Rex.
While model-free analysis can suggest the existence of Rex contributions, 3 more

accurate determination of Rex contributions is the analysis of relaxation

dispersion curves. Relaxation dispersion curves are the dependence of the
measured transverse relaxation rate on the effective field determined by spin-

echo modulated spacing for CPMG based experiments or applied RF field and

the resonance offset for R1 p experiments. While relaxation dispersion curves

have been utilized to investigate conformational exchange processes extensively

in proteins (27-29,50,52,57,58), little analysis has been done for RNA oligomers

(25,59). Because CPMG and R1p experiments are, in principle, similar and both

report on transverse relaxation rates, the combination of these experiments can

allow for complete analysis of effective field-dependence of relaxation rates.

From these curves, one can calculate kex, the timescale of exchange within the
us - ms regime. In general, there are cases where either CMPG or R1 p

experiments were sufficient to obtain kex values of motion. Based on previous
RNA dispersion work (25) as well as using a novel isotope labeling scheme (46),

combinations of CPMG, on-, and off-resonance R1 p were acquired to critically

analyze ribose dynamics within the tetraloop region of the GCAA RNA hairpin.

114

13 . .
C Hahn Echo Measurements. 13C relaxatlon measurements were acquured

using Hahn echo experiments to determine Rex((oeff—>0), which can identify

resonances that are undergoing conformational exchange processes (60).

Shown in Fig. 3.7 are Hahn echo R2 measurements for (a) 02' and (b) C4'
atoms. Increased R2 were observed for both C2' and C4' atoms of A7 and A3,
C2' of G5, C4' of C6, and a possible increase for G5 C4'. This is in good
agreement with R1 p measurements obtained (Table 3.1) as well as the results of
the model-free analysis (Fig 3.5b). However, no Rex contribution was observed
for C5 C2'. It is not immediately obvious why no Rex contribution is detected

given that a significant contribution is observed for C6 C4' as well as Rex

contributions for all other tetraloop C2' and C4' atoms.

115

3 1 :1 C2'
I C4'

1

 

 

 

 

 

 

 

0.“ .JL;

G1G2'G3'C4 G5 cs A7 A8 (3901 11 1 1 A14 1

 

Residue

Figure 3.7: 13C R2 measurements from Hahn echo experiments. Rates are
shown for C2' (open) and C4' (closed) atoms at 25 °C.

Measurement of us to ms Motions. Due to the observation of increased
measured transverse relaxation rates as well as Rex contributions determined

using the model-free analysis, relaxation dispersion curves were acquired to

examine conformational exchange processes within the tetraloop in detail using

relaxation-compensated CPMG (rcCPMG) and on-resonance R1 p experiments

acquired at two field strengths of 600 MHz (130 150 MHz) and 900 MHz (130
225 MHz) at 25 °C. Relaxation dispersion curves at 600 MHz were extended

using off-resonance R1 p experiments. Relaxation dispersion curves were fit to

equations 3.3 and 3.6 to determine kex, R20 or R1 p0, and (Dex, where (Dex =

116

pApB(Aw)2. To validate the use of fast-exchange equations, an a scaling factor

was determined for each residue in the tetraloop using equation 3.7. Exchange

within the fast-exchange regime will have an a scaling factor of 2. a scaling
factors for all 13C carbons in the tetraloop that were able to be measured at both

magnetic fields were calculated to be 1.7 or above. Because of this, exchange
parameters could be determined from equations 3.3 and 3.6, which were derived
for the fast-exchange limit, as opposed to the more general exchange equations
(26,61).

In Figure 3.8, relaxation dispersion curves are shown from simultaneous

fitting to equations 3.3 and 3.6, which gave a statistically significant improvement

versus a description using w1-independent rates (cumulative probability for F-
statistic > 0.99) except for G5 C4' (cumulative probability for F-statistic is 0.91).

Statistically significant dispersive effects for G3 and G9 C2' atoms were

calculated, as well as for the 3' single stranded C2' atoms. Motional parameters

determined by fitting the dispersion curves to equations 3.3 and 3.6 were either

poorly defined or were not consistent with the fast-exchange regime (kex >> Am).

117

Figure 3.8: Relaxation dispersion curves for tetraloop atoms in the GCAA RNA
hairpin. CPMG (open symbols) and on- and off-resonance R1 p (solid symbols)
data are shown for 02' and C4' atoms respectively of (a,b) G5, (c,d) C5, (e,f) A7,
and (g,h) A5. Upper traces represent data at 900 MHz (225 MHz 13C) and lower
at 600 MHz (150 MHz 130) except in the case of A7 C2', for which data at 900

MHz was unavailable due to fast relaxation. For C5 02', a horizontal-line fit to
the data is shown. Otherwise, solid lines denote simultaneous fits of all data for

the corresponding atom to equations 3.3 and 3.6, dashed lines (for G5, A7, and
A5) denote simultaneous fits to C2' and C4' results for a particular residue, and
dotted lines represent global fits of all data for either C5 and A7 (apical residues)

or G5 and A5 (closing base pair). In many cases, dashed and/or dotted lines are
not visible due to coincidence with the solid lines representing resonance-specific
fits.

118

E 011.. EZW
15— F{__ 1

l.
”1.1,...”“5” ...,

mm
0 4000 8000 12000 C) 4000 8000 12000
181/2n(H2) 12 yB1/2n(Hz)

 

 

 

 

d

  

 

 

 

O 4000 8000 12000 4000 8000 12000
181/2n(H2) 7B1/21t(Hz)
01
04
sofl‘om
204
O 4000 8000 12000 0 4000 8000 12000
181/2n(H2) 181/

o 4000 8000 12000 0 4000 8000 12000
731/2n(HZ) 113/MHZ)

119

Table 3.3: Motional parameters derived via fitting of relaxation dispersion data at
both fields for a single atom to equations 3.3 and 3.6 at 25 °C.

120

 

«.1 H 1.2 3 H 1.2 E .1. 1.2 3 .1. 0.2 42 x :3 5 9% A12 x 88 H 1.3 .52
5 .5 1.2 3.1. :2 9o U.. 2.2 3 5 Q2 12 x 86 5 NS m2 10o .4 1.: No?
S .1. 16 0.1 H «.1 3.. H «.2 ma H 1.1 «2 11¢ .1. 08 m2 x 8.2 .1. 1.8 .82
$2 $2 3 .1. o 1.1 .1. N. 2 2- 92 x 66 H 31 M32 x 85 H 1.3 .82
m2 5 1.: 1.1 .1. EN 2.. .11 m. S on .1. Q2 92 x :3 H 18 12 x 38 H N. c .58
3 5 RN 3 .1. mam mo 5 12 ad .1. at 12 x 8.2 H 13 12 x A: H 1.9 .88
o.~ H 1.2 3 5 2: 9o 9. 1.2 no .1. N2 12 x 8.0 H 03 m2 x and H. 1.: .83
3.85121 2-45de 38qu @8de Tm .331 comma 85881

121

Listed in Table 3.3 are parameters for the tetraloop region determined by

simultaneous fitting of the relaxation dispersion curves at 600 MHz and 900 MHz,

where appropriate. (Dex was only reported at 600 MHz because (DeXQOO was

imposed to be 2.25 times that of (Dex600. kex values ranging from 22000 to

40000 s.1 were determined. Because the exchange rate constant, kex, is the

sum of k1 and k.1 for two states A and B, it can be postulated that kex values for
atoms of the ribose ring are reporting on sugar pucker transitions, i.e., C3'-endo

to CZ'-endo interconversions. For A7 C2', non physical R20 and R1 p0 rates were

determined. Although this atom has significant Rex contribution, the data

collected only at 600 MHz are not sufficient to determine accurate exchange

parameters. For this reason, parameters from A7 C2' only should be interpreted

with caution.

Furthermore, estimates for chemical shift changes for C2’ and C4'

resonances were calculated from the (Dex term. Because exchange is within the

fast-exchange regime, a population weighted chemical shift average was

observed. Therefore, estimates for p71, and p5 were made to determine Aw from
the (Dex term. Using a value of 0.5 for pp, and p5 sets a minimum value for the

chemical shift difference, Awmin, between the two populations, C3'-endo and C2'-
endo. Previous work (14) has determined sugar pucker conformations for the

ribose ring for residues in the GAGA RNA hairpin from JH1'-H2'/JH3--H4'

122

measurements. For three out of the four tetraloop residues of GAGA RNA
hairpin, sugar pucker conformations were found to be in equilibrium. Similar
equilibria for sugar pucker conformations were also seen in the GCAA construct.

Based on these equilibrium measurements, estimated chemical shift changes for

sugar pucker transitions, Awest. were determined from using pp, and p5 reflective

of the equilibria.

123

Table 3.4: Motional parameters derived via fitting of all data for a single atom to

equations 3.3 and 3.6 at 25 °C.

(Awmin.

 

Residue Atom kex, 5'1 ppma (:Zfigt’ rex (us)C
G5 c2;1 (2.5 ._. 0.3) x 104 0.90 1 0.08 > 1.26 41 1 5
C4 (24 1 1.3) x 104 0.53 1 0.20 > 0.75 41 1 22
Ce C4' (34 ,_. 54) x 104 2.28 1 0.24 2.63 1 0.28 29 1 4
A7 02' (45 1 56) x 104 3.42 1 0.41 3.73 1 0.44 25 1 4
C4 (35105yxuf 1981021 2151022 2914
A 02' (2.2 1 0.3) x 104 0.91 1 0.07 1.13 1 0.09 46 1 6
8 C4' 4 1.071013 1.341017 40.16

(2.5 1 0.4) x 10

 

(a) Values derived from (Dex = p,1s,p5(Aa))2 using pA = p5 = 0.5. (b) Values
derived using estimates of C3'-endo pucker populations with pA of 0.75 for C5,

0.70 for A7, and 0.8 for A5, based on NMR J-coupling analysis. For G5, J-
coupling values were consistent with a fully C3'-endo population, and a lower

bound for Mo was thus derived using 08, > 0.85. (c) The characteristic time tax
for an exchange process is the reciprocan of kex and is included here to aid

physical insight. (d) Improvements in X2 using Equations 3.3 and 3.6 for this
resonance did not reach statistical significance versus a constant relaxation rate
model (P = 0.09 for standard F-test).

124

Single Exchange Process for Ribose Sugar Pucker. It has been shown
previously that measurement of exchange rates that are similar within error for
multiple residues can suggest a single conformational event. Due to this, a
single exchange rate for multiple atoms can be determined (58). This has also

been demonstrated for C8/C6 and C1' atoms in the U6 RNA ISL (59). From

Table 3.4, kex values for C2' and C4' atoms for a given residue were observed to

be equal within error. Consequently, global fitting was used to test whether the

02' and C4' atoms are reporting on the same conformational event. Fitting the

C2' and C4' atoms to a single kex value does not significantly alter motional
parameters (Table 3.5 and Fig 3.8). Slight alterations of motional parameters
were observed for the residue of A7 compared to those obtained from individual
atom fits. This can be attributed to limited data acquired for A7 C2'. However,

values obtained are well within error compared to individual fit. Because C2' and

C4' atoms were fit to a single kex, it can be interpreted that these atoms are

reporting on the exchange process of ribose pucker interconversion within the

tetraloop region of the GCAA RNA hairpin.

From the residue fit, we also noted a clustering of kex values for multiple
nucleotides. kex values of (2.5 1 0.3) x 104 and (2.3 1 0.2) x 104 s'1 were
measured for G5 and A5, respectively, while (3.7 1 0.4) x 104 5'1 was measured

for A7. These exchange rates suggest that motions of residues are coupled

125

together. Therefore, motional parameters were determined for G5 and A5 using
a single kex value as well as for C5 and A7. A global kex value of (2.4 1 0.2) x
104 s'1 was measured for G5 and A5; and a single kex value of (3.3 1 0.2) x 104
5'1 was determined for C5 and A7 (Table 3.6 and Fig 3.8). These results hint at
correlated motions for G5 and A5, which can be attributed to a dynamic hydrogen
bond network (13,14), while correlated motions for C5 and A7 can be tentatively

attributed to base dynamics for C5 (14,20).

126

Table 3.5: Motional parameters derived via fitting 02' and C4' data for a single
residue to equations 3.3 and 3.6 for a single kex value at 25 °C.

127

 

3 H 1.8 1.. H 1.2 1.0 .1. 1.2 1.. .1. 1.2 12 x Go .1 may .10?
3 5 1.: 3 .H 2.: od 1.. 1.1. we .1. N2 12 x ad .1. 1.5 02 x 8.1 .1. as .82
we .1. 1.. 1.1 H 1.1 .3 H E 2 H E 12 x A: u 1.9 .102
<12 .12 1.1 H 1.1 51 H 1.1- 12 x 11.1 5 we mc2 x 11.0 .1. 1.8 .82
m: .1. gm 1.. H m.- 1.0 5. 1.2 we 5 1.: 12 :11 5 me .1010
ed 5 1.2 1.. .1. 2.1. me u. 1.2 no 5 N2 12 x 8.0 .1. ms 12 x 81 ... 1.: .83
2.181112 311de 2-108.091 30851.1 2-..... .31 comes 811881

128

Table 3.6: Motional parameters derived via fitting C2' and C4' resonance data for
multiple residues to equations 3.3 and 3.6 for a single kex value at 25 °C.

129

 

1.1 .H 1.1 1.1 1 1.1 1.2 1 2. : 1.. 1 1.1 12 x 11.1 1 1.11 .102
<2 <2 1N .1. 1.1 1.1 5 1.1- 12 x AN.1 H 1N1 .82
N1 1 1.1 1.1 1 1.2N 1.. .1. 1.1 1.21 1.1 12 x AN.1 u 1.11 12 x A 2.1 ... N. 1 .1010
5 .1 1.1N 2 .1 1.2 1.1 1 N2 1.. 1 N2 12 X11.1 .1. 1N1 .1012
1.. .1.. 1.: 1.2 1 1.2 1.1 1 1.1. 1.1 1 1.2 12 x11.1 1 NV 1012
1.. .1.. 1.NN 1.21. 1N 1.1 1 1.2 1.1 5 1.: 12 x :N 1 2.1V .1010
1.. 1 1.2 1.. H 1.1. 1.1 1.. 1.2 1.1 1 1.2 12 x 1.1 1 1N1 12 x AN.1 .1. 1. : .N010
2-10.11.11.21 1&8de 2-118.191 3111.12 2-1 211 1211.19 8118111

130

Analysis of Base Dynamics. Timescales for sugar pucker interconversions
within the tetraloop of the GCAA RNA hairpin have been determined. It is
possible that these motions are correlated to the dynamic hydrogen bond

network as well as base motions. To determine if aromatic base and sugar

motions are coupled, analysis has been extended to the aromatic 13C CB, C6,

and C2 atoms in a uniformly labeled 13C, 15N GCAA RNA hairpin sample. This

work was carried out by Jodi Boer, a graduate student doing her rotation in the

Hoogstraten lab. The results from this work are presented in Figure 3.9. From

molecular dynamic simulations, the aromatic ring of A5 was observed to briefly

leave the tetraloop structure (20). From the relaxation dispersion curves, no Rex
contributions indicative of exchange on the us to ms timescale were observed for
A5 C8 or CZ. Because similar kex values were not observed for base and ribose

atoms for the tetraloop, it can be inferred that base motions are not coupled to

ribose dynamics. It should be noted that relaxation dispersion curves were not
obtained for C5 C6 due to ambiguity from 1H only chemical shift assignments

(14).

131

l
l—O—wl
I
11
1—0—

 

1O Ilrlrli11r11t1llrlllIIIII]I[TI

0 1500 3000 4500 6000 7500
181/2n (H2)

Figure 3.9: Representative relaxation dispersion curves for aromatic atoms for
the GCAA RNA hairpin. On-resonance R1,, (solid symbols) data are shown for

C8 resonances of 63 (black diamonds), G5 (red circles), and A5 (blue squares).
Solid lines denote best fit to a horizontal line.

132

Temperature Dependence of kex. Loria and co-workers (57) have obtained

relaxation dispersion curves at multiple temperatures to investigate the activation

energy of us to ms motional process in ribonuclease A. For this purpose, kex

values for C2' and C4' atoms of the GCAA RNA hairpin tetraloop were
determined from relaxation dispersion curves obtained at 15 °C, 20 °C, and 35

°C (Fig. 3.10 to 3.12; Table 3.7 to Table 3.9) to supplement those already

obtained at 25 °C. At 15 °C, kex values were determined for all tetraloop atoms
except C5 C2' that range from 11000 to 20000 s'1. Unlike 25 °C, a single kex
value for 02' and C4' atoms from a single residue fit was determined only for A5.
For G5 and A7, kex values for C2’ and C4' atoms determined were not within
error. At 20 °C, kex values obtained have a high degree of uncertainty due to the

measurement of only on-resonance R1 p data at a single magnetic field. The

acquisition of CPMG data could help define the relaxation dispersion curve for

better analysis as well as acquisition of dispersion curves at multiple fields. At 35

°C, a statistically significant relaxation dispersion curve was obtained for A7 C2'
only. Even though A7 C4' had a cumulative probability for F-statistic of 0.2, both
C2' and C4' atoms of A7 were analyzed to determine a single kex value of (7.0 1

2.3) x 104.

133

Figure 3.10: Relaxation dispersion curves in the GCAA tetraloop at 15 °C.
CPMG (open symbols) and on-resonance R, p (solid symbols) data are shown for

C2' and C4' atoms, respectively, of (a, b) G5, (c) C5, (d, e) A7, and (f, 9) A5.
Solid lines denote simultaneous fits of all data for the corresponding resonance
to equations 3.3 and 3.6.

134

 

 

 

 

 

 

 

 

 

  

 

 

 

 

35 35
a b
A 30 A 30
if; 25 'L’L 25
N
CK
20 [I 20
15l'l'l‘l‘l'l 15|TTTTTITI'I
0 2000 4000 6000 800010000 0 2000 4000 6000 800010000
781/21: (Hz) yB1/21r (HZ)
120 C
100 -‘
A 1
£1 60 4
40 '-
20-'l'l'jle'l
0 2000 4000 6000 800010000
yB1/21t (Hz)
100 1 d 100 e
A 80 1 A 80
'3 60 — I ‘73 60
12 1%.. 12 40
20 '1 20
' I ' I ' I ' I ' I I ' I ' I ' I ' I ' fl
0 2000 4000 6000 800010000 0 2000 4000 6000 800010000
151/211 (Hz) 151/211 (Hz)
40 f 40 9
‘TA 1 ‘TA
3 30 1 3 30 -
N N
m '1 m
20 -1 20 -
.1 .
l l I I I l I l I I "TI‘I'IIIII‘F1I'I'T'I
0 2000 4000 6000 800010000 0 2000 4000 6000 800010000

181/211 (Hz) 181/211 (Hz)

135

Figure 3.11: Relaxation dispersion curves in the GCAA tetraloop at 20 °C. On-
resonance R1,, (solid symbols) data are shown for C2' and C4' resonances,

respectively, of (a, b) G5, (0) C5, (d, e) A7, and (f, 9) A5. Solid lines denote fit of
all data for the corresponding resonance to equations 3.6.

136

30

 

 

 

a
1" 25
'13
1220
15l'lfil'l'l'1
0 2000 4000 6000 800010000
yB1/27r(HZ)
65 d
55
73,45
£135
25
15l'l'l'l’l'l
0 2000 4000 6000 800010000
yB1/27t (Hz)
35 f
A30
'2 25
12‘
20
15I'I'I'I'Irfi
0 2000 4000 6000 800010000
yB1/21t(Hz)

R2 (8‘1)

30 b
.r 25
'12
1“ 20
15

 

I ' I ' I ' | ' I ' I
0 2000 4000 6000 800010000
151/211(Hz)

70 C

 

| ' I ' I ' I ' I ' I
o 2000 4000 6000 800010000
151/211 (Hz)

e

 

| ' I ' I ' I ' I ' I
0 2000 4000 6000 800010000
151/211 (Hz)

9

 

I ' I ' I ' I ' I ' I
0 2000 4000 6000 800010000
151/211 (Hz)

137

Figure 3.12: Relaxation dispersion curves in the GCAA tetraloop at 35 °C.

CPMG (open symbols) and on-resonance R1p (solid symbols) data are shown for

(a) C2' and (b) C4' atoms of A7. Solid lines denote simultaneous fits of all data
for the corresponding resonance to equations 3.3 and 3.6.

138

R2 (8'1)

k)
01

b

R2 (8-1)

N)
01

.5
01
[11'

.h
C)
llel

R) Q) Q)
C) C) 01
LlllllllllllllllllllIlLJJI

.3
UI

 

.3
C)

 

CD

OJ
C)
l

[Illlllllll

Z,

R)
C)

l

_L
C)
I

 

CD

I l I I I I I I I I I I I I I

5000 10000 15000
131/211 (Hz)

 

‘—

I I I I I I I I I I I I f I ‘I

5000 10000 15000
151/211 (Hz)

139

Table 3.7: Motional parameters derived via fitting of all data for a single atom to

equations 3.3 and 3.6 at 15 °C.

 

Resonance (Dex kex. 8-1 R20, S—1 R100, S-1
G5 02' (1.9102) x 105 (1.2101) x 104 14.5108 15.3105
35 C4' (1.6 1 0.2) x 105 (1.6 1 0.2) x 104 20.7 1 0.7 18.9 1 0.6
06 C4’ (8.710.4)x105 (1.110.1)x104 33612.5 19911.0
A7 C2' (8.3 11.8)). 105 (2.0 1 0.4) x 104 7.3 1 3.4 15.4 1 3.9
A7 C4' (7.4 1 0.7) x 105 (1.3 1 0.2) x 104 18.7 1 3.3 19.7 1 1.6
A5 c2' (3.210.3)x105 (1.410.1)x104 142:1.2 14.2110
A5 C4' (3.2 1 0.7) x 105 (1.4 1 0.3) x 104 18.2 1 2.6 18.0 1 1.8

 

Table 3.8: Motional parameters derived via fitting of all data for a single atom to
equation 3.6 at 20 °C.

 

Resonance (Dex kex1 3'1 R100, s-1
G5 02' (1.5 1 0.4)x105 (1.3 1 0.6)x104 15-4 2:12
G5 C4' (1,311.3)1105 (1.911.8)x104 113-813.0
c6 C4' (1.2 1 0.2) x 106 (1.9 1 0.4) x 104 16.4 1 5.5
A7 02' (1.1 1 0.2) x 106 (2.2 1 0.3) x 104 12.2 1 4.2
A7 C4' (6.1 11.5)x 105 (1.7 1 0.5) x 104 19.4 1 3.6
A5 02' (2.5 11.5)x 105 (1.3 11.4)x 104 16.3 1 4.8
A5 C4' (2.3 11.6)1105 (1.5 11.5)). 104 17.8 1 4.4

 

Table 3.9: Motional parameters derived via fitting of all data for A7 C2' or C4' to
equations 3.3 and 3.6 at 35 °C.

 

Resonance (Dex kex. 3'1 R20, 5'1 R100, s-1
A7 C2' (2.1 11.6)x 1.06 (7.0 1 2.4) x 104 N/A 5.3 113.3
A7 C4' (6.5 1 13.3) x 105 (7.3 1 6.5) x 104 8-6 i 10.3 8.7 110.7

 

140

Activation Energy Determination. To determine activation energies for sugar

pucker interconversions, the temperature dependence of kex values was
analyzed. For example, relaxation dispersion curves for A7 C4' are plotted at all
temperatures (Fig. 3.13). The figure demonstrates that Rex contributions are
affected by temperature. The Rex contributions are determined from kex, (Dex,

and R2” terms. Interestingly, the (Dex term is not significantly different for all

measured temperatures for a given atom. From this, it can be suggested that

temperature changes do not significantly affect the populated states, pA and p3,

or the chemical shift difference. kex values were plotted as a function of

temperature (Fig. 3.14) as previously reported (57,58). From these plots,

activation energies were determined by fitting the values of kex to the linear form

of the Arrhenius equation (Table 3.10). Due to high uncertainty, interpretation of

activation energies for G5 and A3 resonances (25% to 120% error) should be

done with caution. Activation energies for the tetraloop atoms ranged from 31 to

79 kJ/mol. Activation energy values determined for G5 and A3 02' and C4'

atoms are similar within error as are Ca and A7 C4' atoms. The results obtained

support the theory that sugar pucker transitions can be coupled together, which
are in good agreement with global fitting data obtained from relaxation dispersion

curves at 25 °C.

141

80

  

 

70
60 —— 15°C
17" -—-— 25°C
52, 50 .
82‘ 35°C
40
30
“r“!
20 "-- ~l---
999 .$‘ .‘i-oo...‘ o-,:.. I.';""} ----------
I I I I l I I I I I I I I I I I l I I I I I I I _l
0 2000 4000 6000 8000 10000 12000
731/2MHZ)

Figure 3.13: Temperature dependence of A7 C4'. CPMG (open symbols) and

R, p (solid symbols) data were acquired at 15 °C, 25 °C, and 35 °C at 600 MHz.
Solid lines denote best fit to equations 3.3 and 3.6 for the individual atom A7 C4'.

142

Figure 3.14: Arrhenius-type plots of the exchange rate constant kex. G5 02'
(black, open circles, short dash), G5 C4' (black, filled circle, solid line), A3 02'
(red, open squares, short dash), and A3 C4' (red, closed squares, solid line) are
presented in panel a, while C5 C4' (red, closed circles, solid line), A7 02' (black,
open squares, short dash), and A7 C4' (black, closed squares, solid line) are

presented in panel b. In all cases, kex values determined from individual
resonance fits were used.

143

 

 

—l

_s

O
I

10.5

10.0

9.5

9.0

IIIkex
IIIIIIIIIIIIIIIIIIIIIII

 

 

 

8.5 I I I I I I I I I I I l' I I I I I I I I I
0.00330 0.00335 0.00340 0.00345 0.00350

Temperature (K'1)

12.5
b

12.0
11.5

11.0

In kex

10.5
10.0
9.5

 

 

9.0 I T fT I l I I l' 1 I I I I I l I I I I I I I I I I I I I I l
0.003200.003250.003300.003350.003400.003450.00350
Temperature (K-1)

144

Table 3.10: Activation energies determined from fitting temperature dependent
kex values to the linear form of the Arrhenius equation.

Resonance Ea, kJ/mol

 

G5 02' 50 :l: 11
G5 C4' 32 :l: 38
C5 C4' 79 :l: 10.
A7 C2' 50 i 12
A7 C4' 72 i 13
A3 C2' 31 :l: 11

A3 C4' 40.118

 

DISCUSSION

In this work, dynamics for 02' and C4' atoms within the tetraloop region of

the GCAA RNA hairpin were analyzed using 130 NMR spin relaxation

experiments. This was carried out by incorporating 2',4'-13C2 rNTPs into the

GCAA RNA hairpin. From the model-free analysis, C2' and C4’ atoms are highly

ordered from 82 values, with little evidence of fast internal motions on the ps to

ns timescale. However, Rex contributions were determined for the tetraloop

atoms. Using relaxation dispersion curves, exchange rates were determined for
02' and C4' atoms in the tetraloop. Coupling of motion for C2' and C4' atoms of

a residue as well as coupling of multiple residues was suggested by

simultaneous fits. By analyzing the temperature dependence of kex values,

activation energies for ribose ring fluctuations were determined.

145

The fifteen nucleotide GCAA RNA hairpin is a small, well studied molecule

suitable to study ribose dynamics using the alternate-site labeling scheme to

extend 13C NMR analysis within the ribose ring as opposed to only the C1'

resonances. As determined earlier (see chapter 2), the use of the alternate-site

. 13 13 . .
labeling scheme removes C- C Interactions for the accurate measurement of

relaxation rates. The removal of these interactions does not limit sensitivity of

130 resonances. All NMR data were acquired without using the constant-time

format. While the constant-time format can remove spectral overlap in the

presence of 130-130 coupling, this method is subject to sensitivity loss. RNA

resonances suffer from poor chemical shift dispersion for different nucleotide
atoms. As RNA oligomers increase in size, the chance for spectral overlap
increases. In fact, not all the C2' and C4' resonances were analyzed. While the

alternate-site labeling scheme does not resolve this issue, using specific residue
13C NMR samples can aid analysis by removing the total number of resonances
observed (see chapter 4).

R1, R1 p, and 1H-13C hNOE data were obtained to analyze fast internal

motions of the GCAA RNA hairpin using the model-free formalism of Lipari-
Szabo (42-44). The results obtained reveal a relatively rigid structure on the ps -
ns timescale. The overall correlation time we was determined to be 3.00 ns which

is in good agreement with 2.97 ns obtained previously (55). Except for atoms

near the terminus and in the single-stranded 3' tail, general order parameters 82

146

are greater than 0.9, indicative of rigid nuclei. Only a few nuclei, which were

located near the terminus and in the tetraloop, required an effective correlation

time, 19,. In the tetraloop, only G5 and C5 C2' atoms were analyzed requiring 15

values of 892.4 :1: 291.2 and 266.0 1: 83.8 ps, respectively. Fast internal motions

using the model-free formalism of Lipari and Szabo are defined to be

approximately 10% or less than the overall isotropic motion (42). The re value

for G5 02' is almost 30% of the overall isotropic motion. While this can be

interpreted as slow motion on the ns timescale, this invokes the assumption of

anisotropic motion. However, G5 did not fit to the anisotropic condition of the

model-free formalism. Furthermore, Fiala and co-workers (55) determined

internal motional parameters using isotropic conditions of the model-free

formalism for the uniformly labeled 13C GCAA RNA hairpin. This suggests that
fast internal motions cannot be determined unambiguously for G5 with the data

collected. Fast internal motions for C5 C2' could arise from sugar pucker

transitions. Nowakowski et al. (62) determined proton chemical shift
perturbations due to sugar pucker on the ps timescale from molecular dynamic

simulations of the UUCG RNA hairpin. Also, slight decreases in the general

order parameter 82 for C5 02' were determined. These results suggest

conformational flexibility for this atom, which is in good agreement with previous

evidence of dynamics for C5 (14.20.55).

147

All tetraloop resonances except C5 02' required an Rex term using the
model-free formalism. The Rex contribution from the model-free formalism is

similar in magnitude to Rex contributions measured from Hahn echo experiments.

This result is satisfying as two technically different experiments report similar

results. Relaxation dispersion curves were obtained at 25 °C using rcCPMG, on-

, and off-resonance 130 spin relaxation experiments at 600 MHz (130 125 MHz).

Simultaneously fitting CPMG and R1 p data to determine exchange parameters

has seldom been done (38,39). In most cases, one type of experimentation is

suitable to measure exchange parameters. However, if kex values are within
intermediate ps timescales, both CPMG and R1 p measurements can be

effectively used to determine exchange parameters. The initial 13C R1,, results

obtained at 600 MHz hinted at this possibility. Thus additional rcCPMG and off-

13 . . . .
resonance C relaxatlon disperSIon curves were obtained, as well as rcCPMG

and on-resonance 13C dispersion curves at 900 MHz (130 225 MHz). Motional

parameters were determined by fitting relaxation dispersion curves to equations
3.3 and 3.6 from both fields simultaneously. Equations 3.3 and 3.6 are
approximations from generalized exchange equations that are applicable at all

timescales. To validate the use of equations 3.3 and 3.6, an a scaling factor was

determined which relates the Rex dependence to the static magnetic field. An 01

148

scaling factor of 1.7 and above was calculated for all resonances except for A7

02', for which no data could be obtained at the higher magnetic field. Because of

this result, caution should be taken for the analysis of A7 02'. The A7 02'

resonance is severely broadened as seen from the 1H-13C HSQC (Fig. 3.2).

Data analysis of this resonance was difficult due to data ambiguity as well as
acquisition of data only at 600 MHz. These problems could arise from fitting
dispersion curves using the fast-exchange equations when the general exchange

equations are appropriate. To unambiguously determine the exchange regime of

A7 C2', acquisition of relaxation dispersion curves at a second magnetic field is

needed. Because this resonance was not observed at 900 MHz due to fast

relaxation, acquisition of dispersion curves at lower magnetic fields could

alleviate this problem and help clarify the exchange regime for A7 C2'.

Relaxation dispersion curves were used to determine the exchange
parameters (Dex. kex, and R20. For the individual fit of A7 C2’ at 25 °C, a

. . . . 0 . .
minimum constraint of zero was Imposed for R1 p0'6 0. ThlS constraint was not

used when fitting kex to a single residue or global value. However, non-physical

R20600 values were obtained for all determined fits for A7 C2'. This can be

attributed to inconsistencies with rcCPMG data acquisition. From Fig. 3.8,

rcCPMG rates are observed to be nearly equal or greater than R1 p at low

effective fields at 600 MHz and 900 MHz. This is not the case for A7 C2' (Fig.

149

3.8e) where rcCPMG rates are significantly less than R1 p rates. These effects

could be attributed to fitting our rcCPMG data to the fast exchange equation

when the general equation is more appropriate. Yet, it was demonstrated that at

25 °C, kex values for C2' resonances were similar to C4' of the same residue for

G5 and A3. Also, exchange parameters for A7 C4' were unambiguously

determined using the fast exchange equations. The source of the

inconsistencies in rcCPMG data acquired for A7 C2' at 600 MHz is unclear at this

time.

No Rex dependence was observed for C5 02' at 600 or 900 MHz. This
result is unexpected due to the measurement of Rex for all other resonances
within the tetraloop. In fact, observation of substantial Rex contributions for C5

C4' as well as relatively small Rex contributions for G5 C4' demonstrates the

sensitivity of our measurements. Also, results from two out of ten NMR solution

structures solved by Jucker and co—workers (14) determined the aromatic ring of

C5 was exposed from the tetraloop region (Fig. 3.1b), along with sugar pucker
equilibrium for C5, as well as molecular simulations that observed sugar re-

puckering transitions from C3'-endo to C2'-endo upon base flipping for C5 (20).

From examination of equations 3.3 and 3.6, if no chemical shift difference

between A and B is observed, the Rex term goes to zero. It can be tentatively

150

concluded that the chemical shifts of the C3'-endo and CZ'-endo are the same for

C5 C2' resonance.

Evidence for correlated motions for ribose resonances in multiple residues

was determined from by fitting curves to a kex value. The sugar pucker

transitions in the GNRA tetraloop can be attributed to base dynamics.
Hoogstraten et al. (25) observed exchange contributions for the terminal adenine
CB atom within the GAAA tetraloop of the lead-dependent consistent with results

from Jucker et al. (14) that determined a heterogenous hydrogen bond network in

GNRA tetraloops. However, no Rex contributions were observed from on-

resonance R1 p curves (Fig. 3.9). A reason for this difference could be due to the

sequence difference. In both sequences, the apical residue stacks upon the third
base of the loop. However, the cytosine residue has been shown to flip out of
the tetraloop structure for two out of ten NMR structures obtained for GCAA (14).
No motions were observed for the third adenine in both sequences. This

conclusion would be better supported with the observation of dispersion curves
for C5 CB resonance. Unfortunately, no dispersion curves for this resonance
were acquired due to spectral ambiguity from 1H only chemical shift assignments

for GCAA. The use of standard HCCH-COSY pulse sequences would correlate

H5 resonances to H6 resonances, possibly clearing up spectral ambiguity.

rcCPMG data could not be accurately determined due to the presence of 2JCC

scalar coupling. To investigate us to ms timescales using CPMG based

151

experiments, isolation of the aromatic carbons will be crucial. For example, Pardi

and co-workers (63) have developed a method to highly enrich purine CB atoms

using 13C formate in bacterial cell growths.

As well as exchange timescales, chemical shift differences can be

determined from relaxation dispersion curves. Large (Dex values were
determined for A7 and C5 atoms as opposed to G5 and A5 atoms. Because (Dex
is proportional to the chemical shift difference, the data suggest that C5 C4' and

A7 C2' and C4' have large chemical shift differences. This can be seen from the

broadening of these resonances from the HSQC (Fig 3.2). Jucker et al. (14)
determined sugar pucker equilibrium estimates for the tetraloops studied. To
better illustrate the magnitude of chemical shift change within the tetraloop ribose
resonances, the tetraloop region of the 1.4 A GUAA RNA hairpin (15) with CPK

spheres sized proportional to minimum chemical shift differences is presented in

Fig 3.15. Here one can see the magnitude of chemical shift changes for C5 and

A7 atoms.

152

  
  

6 4. v
u c . 6C2'

. /
/

A7 C4'

  
     
 
     

_/
—/
GS C2'

Figure 3.15: Representation of the tetraloop region of GUAA RNA hairpin
determined to 1.4 A (PDB code 1msy) by Correll and Swinger (15). CPK

spheres for the C2' and C4' atoms are drawn proportional to Awmin values for the
corresponding resonances.

“C4'

0

A8 C2'

Relaxation dispersion curves at multiple temperatures were acquired to
analyze the activation energy of sugar pucker conformations within the tetraloop

of the GCAA RNA hairpin. Changes in temperature are expected to change the

determined kex. For the tetraloop of the GCAA RNA hairpin, temperature
dependent Rex contributions were observed to be proportional to kex because
(Dex and R20 were not significantly altered with temperature (Fig. 3.13). At 15 °C,

residue level fits for G5 and A7 could not be obtained. For A7, kex values for
02' and C4' resonances are not similar. Similar to data obtained at 25 °C, the

rcCPMG measured rates for A7 02' are lesser than R1 p For G5 and A5, kex

153

values determined at 20 °C were poorly defined which made data analysis

ambiguous, yet values for C5 and A7 were better defined.

It was previously demonstrated that a single kex could be obtained for both

resonances in a given ribose ring which were attributed to both resonances
reporting on the same conformational event of sugar pucker interconversion. If
this is indeed true, calculation of similar activation energies for both resonances

should be obtained. Using Arrhenius plots, activation energies for all tetraloop

C2' and C4' atoms were calculated except C5 02'. 02' and C4' atoms of A5 have

similar activation energies which suggest these atoms are reporting on sugar

pucker transitions. Likewise, C4' resonances of C5 and A7 have similar

activation energies which could suggest correlated motions. Activation energies

measured are higher that those obtained for 15M amide resonances in

ribonuclease A studied by the Loria group, where measured activation energies
for exchange yielded 15 kJ/mol and 31 kJ/mol (57). However, LiWang et al. (64)
determined the lower threshold for ribose pucker activation energy is 17 kJ/mol
using ribose molecules. It is possible that ribose pucker conformations within the
tetraloop region are more sterically hindered. Such constraints could raise the
activation energies measured.

To sum up, rigorous analysis of ribose dynamics in the tetraloop region of
the GCAA RNA hairpin were presented. Fast internal motions on the ps to ns
timescales were analyzed by the model-free analysis of Lipari-Szabo (42-44).

Motions on the us to ms timescale indicative of conformational exchange were

154

analyzed using rcCPMG, on-, and off-resonance R1 p NMR spin relaxation

experiments at multiple fields. This analysis was made possible using the novel
alternate-site isotope labeling scheme. Motional exchange parameters for 02'
and C4' atoms were determined from simultaneous fits of relaxation dispersion

curves at multiple fields. From these results, individual fits for C2' and C4' atoms

to a single kex value were determined. Because kex values for C2' and C4'

atoms of a residue were determined to be similar within error, data were refit to a

single kex value for a residue. Likewise, multiple residues displayed similar kex
values, thus a global kex values was determined for G5 and A5 as well as C5 and

A7. It can be concluded that exchange parameters are reporting on sugar pucker

transitions. To ascertain thermodynamics of ribose motions within the tetraloop,
activation energies for sugar pucker transitions were determined, further
supporting the idea of correlated motions. The work presented here details the
usefulness of the alternate-site labeling scheme to study ribose dynamics within
RNA molecules. This work has laid the foundation for studies in biologically
relevant molecules, such as the hairpin ribozyme, to evaluate structure/function

relationships.

ACKNOWLEDGMENTS
I would like to thank Max T. Rogers NMR administrators Kermit Johnson
and Dr. Daniel Holmes for assistance in programming the adiabatic pulse

sequence used for the 600 MHz spectrometer. I would like to thank CTA

155

Biomolecular NMR Facility administrator Dr. Aizhuo Liu for assistance in setting

up all pulse sequences used for the 900 MHz spectrometer. Lastly, I would like

to thank Jodi Boer for acquisition of rcCPMG and on-resonance R1 p dispersion

curves for aromatic resonances.

156

REFERENCES

10.

Leulliot, N. and Varani, G. (2001) Current topics in RNA-protein
recognition: control of specificity and biological function through induced fit
and conformational capture. Biochemistry, 40, 7947-7956.

Treiber, OK. and Williamson, JR. (2001) Beyond kinetic traps in RNA
folding. Curr Opin Struct Biol, 11, 309-314.

Fedor, M.J. and Williamson, JR. (2005) The catalytic diversity of RNAs.
Nat Rev Mol Cell Biol, 6, 399-412.

Kuimelis, R.G. and McLaughlin, L.W. (1998) Mechanisms of ribozyme-
mediated RNA cleavage. Chem Rev, 98, 1027-1044.

Varani, G. (1995) Exceptionally stable nucleic acid hairpins. Annu Rev
Biophys Biomol Struct, 24, 379-404.

Antao, V.P., Lai, S.Y. and Tinoco, |., Jr. (1991) A thermodynamic study of
unusually stable RNA and DNA hairpins. Nucleic Acids Res, 19, 5901-
5905.

SantaLucia, J., Jr., Kierzek, R. and Turner, DH. (1992) Context
dependence of hydrogen bond free energy revealed by substitutions in an
RNA hairpin. Science, 256, 217-219.

Antao, VP. and Tinoco, l., Jr. (1992) Thermodynamic parameters for loop
formation in RNA and DNA hairpin tetraloops. Nucleic Acids Res, 20, 819-
824.

Uhlenbeck, 0.0. (1990) Tetraloops and RNA folding. Nature, 346, 613-
614.

Murphy, F.L. and Cech, TR. (1994) GAAA tetraloop and conserved bulge
stabilize tertiary structure of a group I intron domain. J Mol Biol, 236, 49-
63.

157

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Witherell, G.W. and Uhlenbeck, 0.0. (1989) Specific RNA binding by Q
beta coat protein. Biochemistry, 28, 71-76.

Zwieb, C. (1992) Recognition of a tetranucleotide loop of signal
recognition particle RNA by protein SRP19. J Biol Chem, 267, 15650-
15656.

Heus, HA. and Pardi, A. (1991) Structural features that give rise to the
unusual stability of RNA hairpins containing GNRA loops. Science, 253,
191-194.

Jucker, F.M., Heus, H.A., Yip, P.F., Moors, E.H. and Pardi, A. (1996) A
network of heterogeneous hydrogen bonds in GNRA tetraloops. J Mol
Biol, 264, 968-980.

Correll, CC. and Swinger, K. (2003) Common and distinctive features of
GNRA tetraloops based on a GUAA tetraloop structure at 1.4 A resolution.
RNA, 9, 355-363.

Szewczak, AA. and Moore, RI. (1995) The sarcin/ricin loop, a modular
RNA. J Mol Biol, 247, 81-98.

Orita, M., Nishikawa, F., Shimayama, T., Taira, K., Endo, Y. and
Nishikawa, S. (1993) High-resolution NMR study of a synthetic
oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate
for the cytotoxic protein, ricin. Nucleic Acids Res, 21, 5670-5678.

Mueller, L., Legault, P. and Pardi, A. (1995) Improved RNA structure
determination by detection of NOE contacts to exchange-broadened
amino protons. . JAm Chem Soc, 117, 11043—11048.

Pley, H.W., Flaherty, KM. and McKay, DB. (1994) Model for an RNA
tertiary interaction from the structure of an intermolecular complex
between a GAAA tetraloop and an RNA helix. Nature, 372, 111-113.

Sorin, E.J., Engelhardt, M.A., Herschlag, D. and Pande, VS. (2002) RNA
simulations: probing hairpin unfolding and the dynamics of a GNRA
tetraloop. J Mol Biol, 317, 493-506.

158

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

Palmer, A.G., 3rd. (1997) Probing molecular motion by NMR. Curr Opin
Struct Biol, 7, 732-737.

Palmer, A.G., 3rd. (2004) NMR characterization of the dynamics of
biomacromolecules. Chem Rev, 104, 3623-3640.

Akke, M. (2002) NMR methods for characterizing microsecond to
millisecond dynamics in recognition and catalysis. Curr Opin Struct Biol,
12, 642-647.

Dayie, K.T., Wagner, G. and Lefevre, J.F. (1996) Theory and practice of
nuclear spin relaxation in proteins. Annu Rev Phys Chem, 47, 243-282.

Hoogstraten, C.G., Wank, JR. and Pardi, A. (2000) Active site dynamics
in the lead-dependent ribozyme. Biochemistry, 39, 9951-9958.

Palmer, A.G., 3rd, Kroenke, CD. and Loria, JP. (2001) Nuclear magnetic
resonance methods for quantifying microsecond-to-millisecond motions in
biological macromolecules. Methods Enzymol, 339, 204-238.

Eisenmesser, E.Z., Bosco, D.A., Akke, M. and Kern, D. (2002) Enzyme
dynamics during catalysis. Science, 295, 1520-1523.

Korzhnev, D.M., Orekhov, V.Y., Dahlquist, F.W. and Kay, LE. (2003) Off-

resonance R1 ,, relaxation outside of the fast exchange limit: an
experimental study of a cavity mutant of T4 lysozyme. J Biomol NMR, 26,
39-48.

Kim, S. and Baum, J. (2004) An on/off resonance rotating frame relaxation
experiment to monitor millisecond to microsecond timescale dynamics. J
Biomol NMR, 30, 195-204.

Shajani, z. and Varani, G. (2005) 13c NMR relaxation studies of RNA
base and ribose nuclei reveal a complex pattern of motions in the RNA
binding site for human U1A protein. J Mol Biol, 349, 699-715.

Carr, H.Y. and Purcell, EM. (1954) Effects of diffusion on free precession
in nuclear magnetic resonance experiments. Phys Rev, 94, 630-638.

159

32.

33.

34.

35.

36.

37.

38.

39.

40.

Meiboom, S. and Gill, D. (1958) Modified spin-echo method for measuring
nuclear relaxation times. Rev Sci Instrum, 29, 688-691.

Luz, Z. and Meiboom, S. (1963) Nuclear magnetic resonance study of the
protolysis of trimethylammonium ion in aqueous solution - order of the
reacton with respect to solvent. J Chem Phys, 39, 366-370.

Deverell, 0., Morgan, RE. and Strange, J.H. (1970) Studies of chemical
exchange by nuclear magnetic relaxation in the rotating frame. Mol Phys,
18, 553-559.

Loria, J.P., Rance, M. and Palmer, AG. (1999) A relaxation-compensated
Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange
by NMR spectroscopy. J Am Chem Soc, 121, 2331-2332.

Yamazaki, T., Muhandiram, R. and Kay, L. (1994) NMR experiments for
the measurement of carbon relaxation properties in highly enriched,

uniformly 130, 15N-labeled proteins: application to Ca carbons. J Am
Chem Soc, 116, 8266-8278.

Mulder, F.A.A., de Graaf, R.A., Kaptein, R. and Boelens, R. (1998) An off-
resonance rotating frame relaxation experiment for the investigation of
macromolecular dynamics using adiabatic rotations. J Magn Reson, 131,
351-357.

lVlulder, F.A., van Tilborg, P.J., Kaptein, R. and Boelens, R. (1999)
Microsecond time scale dynamics in the RXR DNA-binding domain from a

combination of spin-echo and off-resonance rotating frame relaxation
measurements. J Biomol NMR, 13, 275-288.

Meekhof, A.E. and Freund, SM. (1999) Separating the contributions to

5N transverse relaxation in a fibronectin type III domain. J Biomol NMR,
14, 13-22.

Massi, F., Johnson, B, Wang, C., Rance, M. and Palmer, A.G., 3rd.

(2004) NMR R1 p rotating-frame relaxation with weak radio frequency
fields. J Am Chem Soc, 126, 2247-2256.

160

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

Rupert, P.B., Massey, A.P., Sigurdsson, ST. and Ferre-D'Amare, AR.
(2002) Transition state stabilization by a catalytic RNA. Science, 298,
1421-1424.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 1. theory
and range of validity. J Am Chem Soc, 104, 4546-4559.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 2. analysis
of experimental results. J Am Chem Soc, 104, 4559-4570.

Mandel, A.M., Akke, M. and Palmer, A.G., 3rd. (1995) Backbone dynamics
of Escherichia coli ribonuclease Hl: correlations with structure and function
in an active enzyme. J Mol Biol, 246, 144-163.

Batey, R.T., Battiste, J.L. and Williamson, JR. (1995) Preparation of
isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol,
261, 300-322.

Johnson, J.E., Jr., Julien, K.R. and Hoogstraten, CG. (2006) Alternate-
site isotopic labeling of ribonucleotides for NMR studies of ribose
conformational dynamics in RNA. J Biomol NMR, 35, 261-274.

Milligan, J.F. and Uhlenbeck, 0.0. (1989) Synthesis of small RNAs using
T7 RNA polymerase. Methods Enzymol, 180, 51-62.

Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, 0.0. (1987)
Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic
DNA templates. Nucleic Acids Res, 15, 8783-8798.

Cunningham, L., Kittikamron, K. and Lu, Y. (1996) Preparative-scale
purification of RNA using an efficient method which combines gel
electrophoresis and column chromatography. Nucleic Acids Res, 24,
3647-3648.

Tollinger, M., Skrynnikov, N.R., Mulder, F.A., Forman-Kay, JD. and Kay,
LE. (2001) Slow dynamics in folded and unfolded states of an SH3
domain. JAm Chem Soc, 123, 11341-11352.

161

51.

52.

53.

54.

55.

56.

57.

58.

59.

Hwang, T.L., van Zijl, PC. and Garwood, M. (1998) Fast broadband
inversion by adiabatic pulses. J Magn Reson, 133, 200-203.

Millet, O., Loria, J.P., Kroenke, C.D., Pons, M. and Palmer, A.G., 3rd.
(2000) The static magnetic field dependence of chemical exchange
Iinebroadening defines the NMR chemical shift time scale. J Am Chem
Soc, 122, 2867-2877.

Kay, L.E., Torchia, DA. and Bax, A. (1989) Backbone dynamics of

proteins as studied by 15N inverse detected heteronuclear NMR

spectroscopy: application to staphylococcal nuclease. Biochemistry, 28,
8972-8979.

Borer, P.N., LaPlante, S.R., Kumar, A., Zanatta, N., Martin, A., Hakkinen,

A. and Levy, GO (1994) 13C-NMR relaxation in three DNA

oligonucleotide duplexes: model-free analysis of internal and overall
motion. Biochemistry, 33, 2441-2450.

Trantirek, L., Caha, E., Kaderavek, P. and Fiala, R. (2007) NMR 130-
relaxation study of base and sugar dynamics in GCAA RNA hairpin
tetraloop. J Biomol Struct Dyn, 25, 243-252.

Bryce, D.L., Grishaev, A. and Bax, A. (2005) Measurement of ribose
carbon chemical shift tensors for A-form RNA by liquid crystal NMR
spectroscopy. J Am Chem Soc, 127, 7387-7396.

Cole, R. and Loria, JP. (2002) Evidence for flexibility in the function of
ribonuclease A. Biochemistry, 41, 6072-6081.

Mulder, F.A., Mittermaier, A., Hon, 8., Dahlquist, F.W. and Kay, LE.
(2001) Studying excited states of proteins by NMR spectroscopy. Nat
Struct Biol, 8, 932-935.

Blad, H., Reiter, N.J., Abildgaard, F., Markley, J.L. and Butcher, SE.
(2005) Dynamics and metal ion binding in the U6 RNA intramolecular
stem-loop as analyzed by NMR. J Mol Biol, 353, 540-555.

162

60.

61.

62.

63.

64.

Massi, F., Grey, M.J. and Palmer, A.G., 3rd. (2005) Microsecond
timescale backbone conformational dynamics in ubiquitin studied with
NMR R1 rho relaxation experiments. Protein Sci, 14, 735-742.

Trott, O. and Palmer, A.G., 3rd. (2002) R1 p relaxation outside of the fast-
exchange limit. J Magn Reson, 154, 157-160.

Nowakowski, J., Miller, J.L., Kollman, PA. and Tinoco, l., Jr. (1996) Time
evolution of NMR proton chemical shifts of an RNA hairpin during a
molecular dynamics simulation. J Am Chem Soc, 118, 12812-12820.

Latham, M.P., Brown, D.J., McCallum, SA. and Pardi, A. (2005) NMR
methods for studying the structure and dynamics of RNA. Chembiochem,
6, 1492-1505.

LiWang, A.C., McCready, D.E., Drobny, G.P., Reid, BR. and Kennedy,
MA. (2003) Observation of a distinct transition in the mode of
interconversion of ring pucker conformers in non-crystalline D-ribose-2'-d

from 2H NMR spin-alignment. J Biomol NMR, 26, 249-257.

163

Chapter 4

Analysis of Ribose Dynamics at the Cleavage Site of the Lead-dependent
Ribozyme Using 13C NMR Spin Relaxation Experiments

164

INTRODUCTION

The lead-dependent ribozyme, or leadzyme, is a catalytic RNA molecule that

was derived from in vitro selection assays of yeast tRNAPhe (1), which was

shown to undergo cleavage upon the addition of Pb2+ (2,3). The mechanism of

the leadzyme was determined to be similar to other small, naturally occurring
ribozymes in using an internal 2' OH nucleophile, it cleaves the phosphate
backbone at a specific phosphodiester bond linkage to form 2',3'-cyclic
phosphate and 5'-OH termini (4). However, the leadzyme is unique in that the

2',3'-cyclic phosphate is further hydrolyzed to 3'-phosphate (4). As evidenced by

its name, the leadzyme is specific for catalysis in the presence of Pb2+. Even
though Pb2+ is required for catalysis, the addition of divalent and trivalent metal

. . . . 2+
Ions Including rare earth metals can enhance the catalytic rate over Pb alone

(5,6). The structure of the leadzyme (Fig 4.1, 4.2) is centered within an

asymmetrical internal loop containing the cleavage site flanked by A-form

helices. Within the internal loop, C5, G9, and G24 are conserved residues (7,8).

165

\G

5'9 C.GAC.C.G A 6094089
I ICC.) 0 I III A
6LIJGA G GGUUGA

Figure 4.1: Secondary structure of the lead-dependent ribozyme used in this
work. Cytidine residues are highlighted in bold; and the arrow denotes the site of
phosphodiester bond cleavage. Ovals (filled and open) denote non Watson-
Crick base pairs.

Kinetics studies have probed the catalytic mechanism of the leadzyme
(5,7-11). Uhlenbeck and co-workers (8) have shown that the log of the cleavage

rate is linear with pH in the range from 5.5 to 7.0, indicative of a basic group

being involved in the rate-limiting step of catalysis, presumably Pb2+-bound

hydroxyl. Unfortunately, full kinetic profiles cannot be obtained for the leadzyme
due to the formation of lead polyhydroxide species at high pH values.

Several structural methods including the NMR solution structure (12), X-
ray crystal structures (13,14), and computer modeling (15) of the leadzyme have
yielded conflicting results for the internal loop. Glycosidic torsion angles as well
as sugar pucker conformations for the three guanosines within the internal loop
are not consistent in the three analyses. Based on these inconsistencies, it was

hypothesized that conformational changes occur for the active site to activate the

166

leadzyme. The use of 8-bromo-guanosine (8BrG) analogs (9) as well as locked
nucleic acid (LNA) ribonucleotides (rNTPs) (10) have probed glycosidic and
sugar pucker conformations, respectively. Using 8BrG analogs forces the

glycosidic torsion angle into syn conformation through steric hinderence. The

results demonstrated that G7 and G9 strongly prefer anti conformation while rate

enhancement was observed when G24 was modified with 8BrG (9). Locked

nucleic acids are rNTPs that restrict the sugar pucker to C3'-endo. The
leadzyme with a locked nucleic residue at position 9 was shown to have a rate
enhancement when the sugar pucker was restricted to C3'-endo using the LNA
variant (10).

What the different structural analyses do agree on is a C3'-endo sugar

pucker for C5. Yet, in this conformation, the 2'-OH is not positioned for in-Iine

nucleophilic attack as required for this mechanism. It was hypothesized that
conformational fluctuations could participate in the catalytic mechanism of the
leadzyme. In fact, comparison of residues in the asymmetric unit of the crystal

structures for the leadzyme indicated that metal binding can force a sugar pucker

conversion for C5 (13,14).

As shown in previous chapters, heteronuclear NMR experiments are

useful in examining conformational fluctuations. Previous NMR work by Pardi
and co-workers (16,17) measured a pKa of 6.5 for A25 N1 as opposed to < 3.1
for other helical adenines. Using NMR line shape analysis, an exchange lifetime
of 28 :l: 4 us was determined for the protonated state of A25 N3 (16). Also,

167

Hoogstraten et al. (18) measured a strong Rex dependence for A25 CZ and CB

atoms for the leadzyme from power dependent R1 ,, measurements. For A25 C2

and C8 atoms, exchange lifetimes of 40 :t 1.8 ps and 47 :t 18 us were

determined, respectively. It was hypothesized that protonation of A25 N1 is

influenced by the base of C5 flipping out of the helix. The agreement of these
experiments suggests a model whereby base pair opening is required for
catalysis. Unfortunately, no dynamics could be collected for the base of C5 due

to spectral overlap.

Based on these findings, it can be suggested that the sugar pucker

conformation of C5 would be affected by the proposed base dynamics. As

demonstrated in previous chapters, utilizing the alternate-site labeling scheme
can probe such dynamics. In this chapter, sugar pucker transitions were

examined for a cytidine-labeled leadzyme incorporating the alternate-site labeling

scheme using power dependent R1 p experiments. The results demonstrate

active site fluctuations on-the us to ms timescale which are postulated to position

the reactive nucleophile for catalytic activity.

MATERIALS and METHODS

Preparation of 13C Cytidine rNTPs. Cytosine specific 130 rNTPs for the lead-

dependent ribozyme were prepared as described earlier (19,20). 13C rCMP was

168

separated from other rNTPs using an Akta Basic FPLC (GE Healthcare) with a
POROS Pl/20 column (20 pm, 4.6 mm x 100 mm) using a linear gradient from 50
mM NH4COOH, pH 3.0 to 500 mM NH4COOH, pH 2.5 over 3.5 min and a flow

rate of 5 mUmin collecting 10 mL fractions followed by UV absorbance. After
lyophilization of pooled fractions, 13C rCMP was phosphorylated to 13C rCTP
(19). The phosphorylation reaction was monitored using a Vydac 302lC4.6 lon-

Chromatography column (10 pm, 4.6 mm x 250 mm) with a linear gradient from

25 mM 1:1 NaHzPO4zNazHPO4, pH 2.8 to 125 mM 1:1 NaHzPO42Na2HPO4, pH

2.9 over 34 min and a flow rate of 1 mUmin. 130 rCTP was purified from
proteins using centrifugation, concentrated using a Rotovapor RE 120 (Buchi)
under vacuum at 40 °C to a final volume of 1 mL, and ethanol precipitated.

Transcription of the 13C Lead-dependent Ribozyme. The alternate-site
labeling scheme was incorporated into the cytidines of the thirty nucleotide lead-
dependent ribozyme by in vitro transcription using recombinant T7 RNA
polymerase and a synthetic DNA template (21,22) with 2',4'-13C2 rCTP and non-
labeled rATP, rGTP, and rUTP. Transcribed RNA was ethanol precipitated and
purified using denaturing polyacrylamide gel electrophoresis on a Bio-Rad model
491 preparative cell (23) with a flow rate of 1 mUmin with 4 mL fractions. RNA

fractions corresponding to the lead-dependent ribozyme as determined by gel

electrophoresis were pooled, concentrated as above to 1 mL, and desalted using

Centricon-3 units (Millipore). RNA was dried, exchanged into 99.9% D20 with

169

repeated lyophilization, and resuspended to a final concentration of 360 (M in

260 pL 99.96% 020, 10 mM sodium phosphate pH 5.5, 100 mM NaCl, and 200

(M EDTA in an advanced microtube matched with 020 (Shigemi, lnc.). Prior to

use, the NMR sample was incubated at 65 °C for 5 min and cooled on ice for 5
min.
NMR Data Setup, Acquisition, and Processing. All NMR data were acquired

on a Varian UnitleOVA 600 MHz (13C 150 MHz) spectrometer at 25 °C. A 1H-
13C heteronuclear single quantum coherence (HSQC) spectrum was acquired to
verify chemical shift assignments previously obtained (11). The 1H-13C HSQC

spectrum was acquired with 1024 x 512 complex points in the t2 and t1

dimensions, respectively, with corresponding spectral widths of 6000 and 7540

Hz, a 1.5 s recycle delay, and 64 steady state scans. The proton radio frequency

(RF) carrier was centered on the residual HDO signal and the 130 carrier

frequency was set at 85 ppm to allow differential folding of the ribose and base

13
C resonances.

For 130 relaxation experiments, all two-dimensional spectra acquired on
the 600 MHz spectrometer were obtained with 1024 x 128 complex points in the
t2 and t1 dimensions, respectively, with corresponding spectral widths of 6000

and 1800 Hz, a 2 s recycle delay, and 15 min of steady state scans. The proton

RF carrier was centered on the residual HDO signal, and the 13C carrier

170

frequency was set at 78.3 ppm. R1 data were acquired using minor variations of

published pulse sequences (24). R1 delay times ranged from 100 ms to 1050 ms

collected in a random format. Dispersion curves were acquired through the use

of relaxation-compensated CPMG (25,26), on-resonance R1 ,, (24), off-resonance
R1p (27,28), and Hahn echo (29) pulse sequences. The relaxation-compensated
CPMG experiments were acquired as a function of inter-pulse spacing 210p,
where 15p values ranged from 180 us to 4500 ps, corresponding to effective
spin-lock powers 781/21: [vcp=1/(4rcp)] of ca. 1400 Hz to 55 Hz. On-resonance
R1 p experiments were acquired as a function of RF power (01, where w1/21r
ranged from 1.5 to 6 kHz. Off-resonance R1 ,, experiments were acquired as a
function of Q at a (01/21: of 3 kHz, where @211 ranged from 3.5 to 9 kHz. Hahn

echo experiments were acquired with a TCP value of 40 ms.

All NMR data were processed using FELIX 2002 (Felix NMR, lnc.). Prior

to Fourier transformation in the t2 dimension, a 20% DC offset was applied, data

were zero filled, and a 3 Hz exponential line broadening function was applied.

Prior to Fourier transformation in the t1 dimension, t1 was extended by 20% using

linear prediction and a cosine-squared apodization function was applied.
Data Analysis. Resolved crosspeak intensities were integrated using FELIX

2002 and exported to Igor Pro 5 (Wavemetrics). In all experiments, one data

171

point was acquired three times to determine error. R1 relaxation rates were

extracted by fitting the integrated peaks to a single exponential decay. For

. . obs . .
d1spersron curves, R2 and R1 p rates were calculated usrng the equation

R2/R1I0Obs = 1/T * In (l(T)/lo), where T is the relaxation delay time, I is the

measured intensity, and I0 is the measured intensity with no relaxation delay.

The ID was acquired twice to ensure reproducibility. R1 ,, rates were calculated
from R1 p°bs using the equation R1 p°bs = R1 cos2 9 + R1 p sin2 9, where 9 = tan"1
(an/Q) is the tilt angle of the spin lock axis from the z axis, (01 is the spin lock
power in Hz and Q is the offset in Hz. Errors were propagated from a single (055
or vcp repeated three times (see chapter 3). Minimum values of errors were
. 2 0 0

Imposed to be 2% or greater. kex, (Dex = pApB(Aw ), R2 , and R1 ,0 values were
extracted by simultaneously fitting dispersion curves to CPMG and R1 ,, equations

(30) using Igor 5.0.4.8’s Global Fit. The CPMG and R1 p equations used are
given:

2 * * * *
R2(1/I'CP )=R2(1/TCP—>°°) + PAPB(Aw) lkex (1-((tanh(kex/(4 TOP)» 4 1rCP/kexl)

[4.1]
and

2 , 2 2
R1 p (weft) = R1 p (weir —>°°) + pApB(Aw) kex / (weft + kex ) [4.2]

172

where R2(1/th—>°°) and R1 p(a)efi—+°°) are the transverse relaxation rates in the
absence of exchange, respectively, pA and p5 are the exchangeable populated
states where the sum of pp, and p5 are equal to one, Aw is the chemical shift
difference in radians between the populated states pp, and p5, kex is the
exchange rate constant, 215p is the delay time between 180° pulses, and the
effective field, (095, in radians per second can be expressed as:

werr= ((012 + 02V” [43]

F-statistic critical values (a) were calculated to justify fitting dispersion

curves to exchange dependent equations as opposed to a horizontal line. F-

values for C2' and C4' resonances were obtained by comparing X2 values

obtained by fitting data sets to a horizontal line versus the dispersion equations.
F-statistic critical values were obtained using the website:
http://www.stattrek.com/tables/f.aspx. The confidence threshold was set to an a

value of >0.95.

RESULTS

Hoogstraten et al. (18) previously analyzed dynamics of nucleotide bases

within the active site of the lead-dependent ribozyme using power-dependent R1 p

dispersion curves. From this work, it was proposed that the base of C5

undergoes conformational fluctuations that extrude the base from the asymmetric

173

internal loop. Unfortunately, spectral overlap prevented the analysis for C5 using

NMR spin relaxation techniques. To directly probe motions of C5, ribose

dynamics were analyzed for cytidine residues in the lead-dependent ribozyme

using the alternate-site labeling scheme.
1 13 , , 13 . .
The H- C HSQC for the 2,4- C2 cytidine-only lead-dependent

ribozyme was acquired to verify chemical shifts previously obtained (11) (Fig.

4.3). All eight C2' and four C4' resonances, including the C2' and C4' for the C5
residue, were resolved for analysis. Hahn echo R2 vales were measured to
qualitatively determine resonances that have Rex contributions (31) (Fig. 4.4).
An increase in the R2 value consistent with exchange on the us to rns timescale
was observed for both 02' and C4' resonances of C5 as well as for C2 C4', but

not 02'. Also, a decrease in the R2 value was observed for C14 02' and 035 C2'

and C4'. This decrease is indicative of motions on the ps to ns timescale.
Overall, these results suggest motions on multiple timescales for sugar carbons

in the leadzyme. Relaxation dispersion curves were obtained to analyze ribose

pucker transitions for C5 in detail.

174

Figure 4.2: 1H-13C HSQC spectra of the cytidine-only specifically-labeled lead-
dependent ribozyme corresponding to 02' (top) and C4' (bottom) resonances.

Both C5 resonances are labeled. The * denotes the resonance of C35 C2'.

175

 

 

 

 

 

 

 

 

©
@' C, .
-76
4P5 I 4Io 78
1WNW)
6 0 _
L83
E
l
45 4’0 ;84
1H(r>pm)

176

‘30 (ppm)

50

40

 

R2 (3'1)

    

      
        

d

C6 C10 C11 C14 028 C30

.1 1.. 1;;

0 if? _
C2 C5

Residue

Figure 4.3: 13C R2 measurements from Hahn echo experiments for cytidine C2'
(grey) and (b) C4' (black) resonances at 25 °C.

Analysis of ps-ms Motions. Relaxation dispersion curves were acquired for C2’
and C4' atoms of cytidine residues in the lead-dependent ribozyme.
Inconsistencies were observed for rcCPMG data points for C6 02' (Fig. 4.5a).

Due to these inconsistencies, rcCPMG data points were deemed unreliable and

motional parameters were determined from R1p data points only. Motional
parameters were extracted from statistically significant fits to equation 4.2 for C5

and C35 C2' atoms. In Figure 4.4a, relaxation dispersion curves obtained for 05

177

C2' R1 p data are presented. Also shown are the rcCPMG and R1 p data for C5

C4'. Motional parameters could not be obtained for C4' due to the large errors.

In Table 4.1, motional parameters determined from relaxation dispersion curves

are presented. For C5 C2', the kex was determined to be (1.1 :t 0.4) x 104 5-1,
which corresponds to an exchange lifetime, Tex. of 93 :I: 36 us. This value is

larger than the tax values previously reported for A25 atoms around 40 to 50 us

(16,18).

178

350

 

 

 

40
Ii”- 30 flat I - I
:21 "5" I
.. I
10
I I I I I I I I I I I I I I I I
o 5000 10000 15000
781/21r(Hz)
b 50
40 {i I i
'3’- 30 I i I I
53' r
20
10
I I I I I I I I I I I I I I I I
o 5000 10000 15000
781/21c(Hz)

Fig. 4.4: CPMG (open circles) and R1 p (closed circles) data for C5 (a) C2' and
(b) C4' of the lead-dependent ribozyme. Solid line represents fit to equation 4.2

for R1 ,, data.

179

Table 4.1: Motional parameters derived via fitting Ce and C35 C2' R1 p data to
equation 4.2 at 25 °C.

 

C5 C2' C35 C2'
(Dex 1.8e5 1: 0.2e5 1.5e5 t 0.2e5
kex ' 1.1e4 :I: 0.434 2.5e4 :I: 0.364
R190 28.2 1 0.9 14.9 1 0.5
71am (ppm) 0.91 1 0.05 0.81 1 0.07
tex (us) 93 :I: 36 41 :l: 5

 

DISCUSSION

In this work, sugar pucker transitions in the lead-dependent ribozyme were

analyzed using 13C NMR spin relaxation experiments. Base dynamics of the
leadzyme were previously studied using on-resonance R1 p dispersion data (18).

Rex contributions were observed for several residues found within the tetraloop

and in the asymmetric internal loop covering a range of exchange lifetimes.

Interestingly, A25 CZ and C8 nuclei showed evidence of Rex contributions that

were attributed to proposed C5 base fluctuations (18). It was postulated that this

motion could be coupled to a sugar pucker interconversion from CS'-endo to C2'-
endo (12,14) that would position the 2'-nucleophile for catalytic attack on the
scissile phosphate.

For analysis of ribose dynamics, the leadzyme was transcribed using 2',4'-

13C2 rCTPs and commercial non-labeled rATPs, rGTPs, and rUTPs. Because

180

only cytidine residues are 13C enriched with the alternate-site labeling scheme

and there are only eight cytidines in the leadzyme, spectral overlap is decreased.

Near complete resolution for the C2' resonances was achieved. Only C14 and

C25 were slightly overlapped; however, they were resolved well enough to allow

for data analysis. Only four resonances were resolved for the C4' resonances.

Fortunately, both C5 13C resonances were completely resolved allowing analysis

of the critical nucleotide at the cleavage site.

Hahn echo experiments were acquired to verify us-ms motions for the

ribose ring. Hahn spin-echo experiments measure R2 values at low effective
fields. An increase in R2 for both 02' and C4' atoms of C5 were observed (Fig.

4.4). An increase in the R2 was also observed for C2 C4' which is adjacent to the

terminal residues. Because terminal residues are normally frayed, it can be

presumed that dynamics for C2 is due to terminal fraying. A decrease in the

measured R2 was observed for C14 02' and both C35 resonances, indicative of

ps to ns motions. Both residues are located at the end of the helix. To further
probe these motions, analysis using the model-free formalism (32-34) would be

bestsufled.
Conformational change has been hypothesized to position the 2'-OH of C5

to attack the scissile phosphate. Functional studies demonstrated that

conformational flexibility for C5 is required for catalysis. Cedegren and co-

181

workers (7) observed decreases in the observed catalytic rate when mutations

stabilized the base pairing of site 6 to site 25. For example, when C5 was

involved in a Watson-Crick base pair at site 25 with a guanosine, catalysis is
reduced by 50% (7). However, the catalytic rate was enhanced when
modifications were utilized that disrupted stable base-pair formation for site 6. In

fact, an increased catalytic rate over wild type was observed for an abasic

residue of A25 (7).

Interpretation of data for C35 is complex due to the existence of motions

on multiple timescales. Motional parameters were extracted from the relaxation
dispersion curve of C5 C2' fitted to equation 4.2. Motions in the ribose ring have
been attributed to ribose puckering transitions (see chapter 3). An exchange rate

of (1.1 d: 0.4) x 104 s.1 was calculated for C5 C2'. This corresponds to an

exchange lifetime of almost 95 us, which is longer than the exchange lifetimes for

A25 CZ (40 ps) and C8 (47 ps) aromatic carbons (18). It should be noted that

rcCPMG measured R2 data were not included in the analysis. To rigorously

analyze exchange for cytidine residues, acquisition of relaxation dispersion

curves at a second field could aid in the interpretation of dynamics for C5,
particularly at 900 MHz. Though motional parameters were obtained from using

R1 p data acquired at only one magnetic field, this initial result is promising

182

because it demonstrates that the ribose ring of C5 is dynamic consistent with the

assumption that fluctuations at this site are needed to activate the leadzyme.

A statistically significant relaxation dispersion curve was not observed for

C5 C4' (01 = 0.48) which can be attributed to the large error bars on these data
points. However, an increased R2 was observed from Hahn echo experiments.

It is possible that there is an exchange contribution present, yet the Rex

contribution is too small to accurately determine. Sugar pucker interconversions
have been shown to be coupled to both 02' and C4' atoms of a given residue

(see chapter 3). Acquisition of relaxation data at higher magnetic fields can

increase Rex contributions, which could clarify ambiguities.
The rex value obtained for the ribose C5 02' is longer than aromatic C2

and C8 for A25 previously obtained (18). One reason that these values are not

similar could be additional constraints affecting sugar pucker interconversion. In

the GCAA RNA hairpin, correlated motions for C5 and A7 residues and G5 and

A5 residues (see chapter 3) were observed. This could also be occurring in the

lead-dependent ribozyme. Dr. Minako Sumita of the Hoogstraten lab is

. . . . . . . . . 13
investigating ribose dynamics for guanosme res1dues in a guanosme C

specifically labeled leadzyme NMR sample. Based on previous work by Julien et

al. (10), the restriction of G9 to C3'-endo conformation using a guanosine LNA

greatly increased the observed catalytic rate. The measurement of ribose sugar

183

pucker interconversion for the guanosine residues could elucidate a pattern of

concerted motions that helps position the Z'-OH of C5 for proper orientation.

These results coupled with C5 dispersion data taken at multiple fields can yield

insight into the active site of the leadzyme.

In this chapter, preliminary dynamics for sugar carbons of the lead-

dependent ribozyme enriched with 130 cytidine residues using the alternate-site

labeling scheme were examined via NMR spin relaxation techniques. As

evidenced from increased R2 values from Hahn spin-echo measurements, an
exchange lifetime of 93 us was measured for C5 02', which is longer than values

reported for base carbons of A25. While intra-residue correlated motions were
not observed, these preliminary results suggest conformational flexibility for the
ribose ring of C5 which could aid in the catalytic mechanism of the lead-

dependent ribozyme. For detailed analysis of the active site in the lead-
dependent ribozyme, acquisition of relaxation dispersion curves at higher
magnetic fields will be critical to aid in the understanding the catalytic

mechanism.

184

REFERENCES

1. Pan, T. and Uhlenbeck, CC. (1992) In vitro selection of RNAs that
undergo autolytic cleavage with Pb2+. Biochemistry, 31, 3887-3895.

2. Brown, R.S., Dewan, J.C. and Klug, A. (1985) Crystallographic and
biochemical investigation of the lead(II)-catalyzed hydrolysis of yeast
phenylalanine tRNA. Biochemistry, 24, 4785—4801.

3. Krzyzosiak, W.J., Marciniec, T., Wiewiorowski, M., Romby, P., Ebel, JP.
and Giege, R. (1988) Characterization of the lead(ll)-induced cleavages in

tRNAs in solution and effect of the Y-base removal in yeast tRNAPhe.
Biochemistry, 27, 5771-5777.

4. Pan, T. and Uhlenbeck, 0.0. (1992) A small metalloribozyme with a two-
step mechanism. Nature, 358, 560-563.

5. Ohmichi, T. and Sugimoto, N. (1997) Role of Nd3+ and Pb2+ on the RNA
cleavage reaction by a small ribozyme. Biochemistry, 36, 3514-3521.

6. Sugimoto, N. and Ohmichi, T. (1996) Site-specific cleavage reaction
catalyzed by leadzyme is enhanced by combined effect of lead and rare
earth ions. FEBS Lett, 393, 97-100.

7. Chartrand, P., Usman, N. and Cedergren, R. (1997) Effect of structural
modifications on the activity of the leadzyme. Biochemistry, 36, 3145-
3150.

8. Pan, T., Dichtl, B. and Uhlenbeck, DC. (1994) Properties of an in vitro
selected Pb2+ cleavage motif. Biochemistry, 33, 9561-9565.

9. Yajima, R., Proctor, D.J., Kierzek, R., Kierzek, E. and Bevilacqua, PC.
(2007) A conformationally restricted guanosine analog reveals the
catalytic relevance of three structures of an RNA enzyme. Chem Biol, 14,
23-30.

185

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Julien, K.R., Sumita, M., Chen, P.H., Laird-Offringa, LA. and Hoogstraten,
CG. (2008) Conforrnationally restricted nucleotides as a probe of
structure-function relationships in RNA. RNA, 14, 1632-1643.

Legault, P., Hoogstraten, C.G., Metlitzky, E. and Pardi, A. (1998) Order,
dynamics and metal-binding in the lead-dependent ribozyme. J Mol Biol,
284, 325-335.

Hoogstraten, C.G., Legault, P. and Pardi, A. (1998) NMR solution
structure of the lead-dependent ribozyme: evidence for dynamics in RNA
catalysis. J Mol Biol, 284, 337-350.

Wedekind, J.E. and McKay, DB. (1999) Crystal structure of a lead-
dependent ribozyme revealing metal binding sites relevant to catalysis.
Nat Struct Biol, 6, 261 -268.

Wedekind, J.E. and McKay, DB. (2003) Crystal strUcture of the leadzyme
at 1.8 A resolution: metal ion binding and the implications for catalytic
mechanism and allo site ion regulation. Biochemistry, 42, 9554-9563.

Lemieux, S., Chartrand, P., Cedergren, R. and Major, F. (1998) Modeling
active RNA structures using the intersection of conformational space:
application to the lead-activated ribozyme. RNA 4, 739-749.

Legault, P. and Pardi, A. (1997) Unusual dynamics and pKa shift at the

active site of a lead-dependent ribozyme. J Am Chem Soc, 119, 6621-
6628.

Legault, P. and Pardi, A. (1994) In situ probing of adenine protonation in
RNA by 13c NMR. JAm Chem Soc, 116, 8390-8391.

Hoogstraten, C.G., Wank, JR. and Pardi, A. (2000) Active site dynamics
in the lead-dependent ribozyme. Biochemistry, 39, 9951-9958.

Batey, R.T., Battiste, J.L. and Williamson, JR. (1995) Preparation of
isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol,
261, 300-322.

186

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

Johnson, J.E., Jr., Julien, K.R. and Hoogstraten, CG. (2006) Alternate-
site isotopic labeling of ribonucleotides for NMR studies of ribose
conformational dynamics in RNA. J Biomol NMR, 35, 261-274.

Milligan, J.F. and Uhlenbeck, 0.0. (1989) Synthesis of small RNAs using
T7 RNA polymerase. Methods Enzymol, 180, 51-62.

Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, 0.0. (1987)
Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic
DNA templates. Nucleic Acids Res, 15, 8783-8798.

Cunningham, L., Kittikamron, K. and Lu, Y. (1996) Preparative-scale
purification of RNA using an efficient method which combines gel
electrophoresis and column chromatography. Nucleic Acids Res, 24,
3647-3648.

Yamazaki, T., Muhandiram, R. and Kay, L. (1994) NMR experiments for
the measurement of carbon relaxation properties in highly enriched,

uniformly 130, 15N-Iabeled proteins: application to Ca carbons. J Am
Chem Soc, 116, 8266-8278.

Loria, J.P., Rance, M. and Palmer, AG. (1999) A relaxation-compensated
Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange
by NMR spectroscopy. J Am Chem Soc, 121, Z331-2332.

Tollinger, M, Skrynnikov, N.R., Mulder, F.A., Forman-Kay, JD. and Kay,
LE. (2001) Slow dynamics in folded and unfolded states of an SH3
domain. JAm Chem Soc, 123, 11341-11352.

Mulder, F.A.A., de Graaf, R.A., Kaptein, R. and Boelens, R. (1998) An off-
resonance rotating frame relaxation experiment for the investigation of
macromolecular dynamics using adiabatic rotations. J Magn Reson, 131,
351-357.

Hwang, T.L., van Zijl, PC. and Ganrvood, M. (1998) Fast broadband
inversion by adiabatic pulses. J Magn Reson, 133, 200-203.

Millet, O., Loria, J.P., Kroenke, C.D., Pons, M. and Palmer, A.G., 3rd.
(2000) The static magnetic field dependence of chemical exchange

187

30.

31.

32.

33.

34.

Iinebroadening defines the NMR chemical shift time scale. J Am Chem
Soc, 122, 2867-2877.

Palmer, A.G., 3rd, Kroenke, CD. and Loria, JP. (2001) Nuclear magnetic
resonance methods for quantifying microsecond-to-millisecond motions in
biological macromolecules. Methods Enzymol, 339, 204-238.

Massi, F., Grey, M.J. and Palmer, A.G., 3rd. (2005) Microsecond
timescale backbone conformational dynamics in ubiquitin studied with
NMR R1 rho relaxation experiments. Protein Sci, 14, 735-742.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 1. theory
and range of validity. J Am Chem Soc, 104, 4546-4559.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 2. analysis
of experimental results. J Am Chem Soc, 104, 4559-4570.

Mandel, A.M., Akke, M. and Palmer, A.G., 3rd. (1995) Backbone dynamics
of Escherichia coli ribonuclease HI: correlations with structure and function
in an active enzyme. J Mol Biol, 246, 144-163.

188

Chapter 5

Assignment of 1H, 13C, and 15M Resonances of Domain A of the Hairpin
Ribozyme

189

INTRODUCTION

The hairpin ribozyme is a catalytic RNA that is derived from the minus
strand of the tobacco ringspot virus satellite RNA (1). In vivo, the function of the
ribozyme is to generate monomeric genomes from the rolling circle replication of
the viroid genome for packaging (2). The hairpin ribozyme is similar to other
small endonucleolytic ribozymes in that the cleavage reaction produces 2',3'-
cyclic phosphate and 5'-OH termini ends (3). Unlike other small ribozymes, such
as the hammerhead and Hepatitis delta virus, the equilibrium favors ligation of
the products rather than cleavage (4). An interesting characteristic of the hairpin
ribozyme is that metal ions are required for folding, but do not directly participate

in the chemical reaction. Catalysis for the hairpin ribozyme has been shown to

. 2+ .
occur in the presence of Mg as well as non-ligand exchangeable

Co(lll)[NH3]53+ (5,6). Because of this, it has been theorized that nucleotide

aromatic groups play an important role in the catalytic mechanism.

In vivo, the hairpin ribozyme uses a four-way helical junction to stabilize
the tertiary structure (Fig. 5.1) (4). However, a reduced structure known as the
minimal form that comprises the helix-loop-helix domains A and B that are linked
together by a hinge is capable of catalysis (7), as are constructs in which the
domains have been isolated and function in trans. (8). The scissile phosphate is
located within the internal loop of domain A. For catalysis to occur, the two
domains must interact with each other. This process is commonly referred to as
docking. The domains for the hairpin ribozyme have been characterized from

NMR solution structures of isolated domains A (9) and B (10), and from X-ray

190

crystal structures of the docked hairpin ribozyme complex (11-14), as well as

functional studies (4-7,15-24).

5' 3'

1

H2

3' H1
|||||||A||| ||||
5' v

0!

 

 

 

 

3.
H3

 

 

H4

 

 

3' 5'

Figure 5.1: Diagram of the hairpin ribozyme utilizing a four-way junction. The
internal loops are labeled, and the arrow denotes the site of cleavage.

In the NMR solution structure of the isolated domain A, an A-form RNA
structure with anti X glycosidic angles for all residues was found with a CS'-endo
sugar pucker for all stem residues (9). In the NMR structure, the residue 5' of the
cleavage site was cytidine as opposed to adenosine in our sequence (Fig. 5.2).

Within the internal loop, C3’-endo sugar pucker conformations were determined

191

for A9, A15, and C-1, C2'-endo sugar pucker for G5 and G+1, and an equilibrium
between the two forms for A7, U+2, and C+3 (see figure 5.2 for numbering). This
cytidine base pairs with A15, which is protonated due to a shift in the pKa to 6.2,
similar to results seen in the lead-dependent ribozyme (25,26). Also, G+1 was

shown to form a non Watson-Crick base pair with A9.

Conformational changes have been implicated in the catalytic mechanism

for the hairpin ribozyme. From X-ray crystal structures, Ferre-D'Amare and co-

workers (11) observed the extrusion of G+1 from the domain to base pair with
C25 in the internal loop of domain B upon docking, which is hypothesized to be

strengthened by base stacking with A35. Comparison of the docked active site

precursor, transition state mimic, and product structures of the hairpin ribozyme

demonstrates a rigid active site (12) where changes were observed for the

scissile phosphate and sugar moiety of A4, including a switch of pucker

conformation from C3'-endo to CZ'-endo. Investigation of ribose and base
dynamics could help elucidate the role that conformational flexibility of active site
nucleotides contributes to the overall catalytic mechanism of the hairpin
ribozyme.

Mutational studies have been important in highlighting residues that are
critical for the catalytic mechanism of the hairpin ribozyme, particularly within the

active site (27-30). For instance, Burke and co-workers (30) investigated binding

192

and cleavage assays using single base substitutions of A4 and U+2. While it
was determined that U+2 is not required for catalysis, the data suggested that
U+2 is involved in tertiary contacts that can properly align the active site. From

NMR analysis, the U+2 sugar pucker conformation was determined to be in

equilibrium and the residue was modeled to be bulged from the internal loop with
evidence of conformational flexibility on the ms timescale (9). Burke and co-

workers (21) used time resolved fluorescence resonance energy transfer

experiments to probe how U+2 mutations affect docking. Although cleavage

rates decreased significantly, nucleotide substitutions did not drastically affect

formation of the docked complex, including a U+2C modification which could form
a Watson-Crick base pair with G5. From X-ray crystal structures, U+2 was

determined to have cross-strand interaction with G5 (11,13,14). What still

remains unknown is how mutations within the internal loop prohibit catalysis, but
allow for global conformational changes such as docking. It is possible that the
mutations that alter the internal loop architecture can quench key dynamics that
are required for catalysis and not affect docking, a hypothesis which can be

clarified by comparing dynamics between wild type and mutant sequences. One

possible mutant sequence to test is U+2CIC+3U domain A (Fig. 5.2). Burke and

co-workers (31) demonstrated that U+2C/C+3U sequences inactivate G5

ribozyme strands in ligation assays, which could partially be rescued with the

193

introduction of a G5U mutation. Unlike the U+2C mutant, the double mutant was

shown to inhibit formation of the docked complex, which again could partially be

restored with G5U. Taken together, these results suggest that the role of U+2 is
to properly align G5 in the active site such that catalysis can occur efficiently.

With the U+2CIC+3U mutant sequence, internal loop dynamics may be

decreased such that key interactions cannot be made.

Several groups have measured 13C nucleotide base dynamics of RNA

molecules (32-36). In previous chapters, methods and results were described
that detailed the investigation of conformational exchange for ribose atoms in
RNA molecules using NMR spin relaxation experiments. These methods can aid
in understanding the catalytic mechanism of the hairpin ribozyme, particularly by
investigating conformational exchange within the active site. Even though the
NMR solution structure has been determined and chemical shift assignments for
that sequence have been reported, the sequence used in this thesis is different
(Fig. 5.2). Thus, chemical shift assignments for the wild type and mutant
sequences are needed for detailed analyses using NMR spin relaxation

experiments.

194

.11

C 99115
GCGC3'

b

iqulfrrGA19$95
AAGAGAGAAGCGC1

Figure 5.2: Secondary structure of the (a) wild type and (b) U+2C/C+3U mutant
domain A. The numbering scheme is similar to the hinge form of the hairpin
ribozyme in which the structure is constructed from separate strands. Arrow
denotes the site of catalysis. For the mutant sequence, the box denotes the
residues that were transposed which could result in Watson-Crick base pairs.
Standard methods employing multidimensional spectroscopy of RNA
molecules focus on two assignment strategies (37-40). The strategy of through-
bond correlation uses scalar coupling between covalently bound nuclei to identify
spin pairs, while through-space correlations of spin pairs are determined based
on distances. The use of standard uniform Isotope labeling can greatly aid in the

determination of chemical shifts. Specific heteronuclear experiments have been

designed to take advantage of heteronuclear spin pairs in RNA molecules. For

195

example, H1' and H2' ribose spin pairs can be identified using an HCCH-COSY

pulse sequence. The H1' region of the spectrum from this pulse sequence has

less resonance overlap as compared to a 1H-1H NOESY spectrum of the same
region. Even though correlated spectroscopy can identify spin pairs for a given
nucleotide, NOE based experiments are vital to determine sequential nucleotide

assignments. By combining the results obtained using both strategies, the

complete assignment of a RNA molecule can be determined.

In this work, partial 1H, 13C, and 15N chemical shift assignments have

been determined for the wild type and U+2CIC+3U mutant domain A of the

hairpin ribozyme. These assignments were obtained from exchangeable and
non-exchangeable spectroscopy experiments. Comparing exchangeable

spectroscopy, new imino proton peaks and amino proton peaks were detected

for the mutant U+2CIC+3U indicating a tightening of the internal loop. While tvvo-
dimensional experiments were acquired, the acquisition of three-dimensional
data as well as RNA-specific triple resonance experiments will allow for the

complete determination of chemical shifts for detailed analysis of active site

dynamics of domain A of the hairpin ribozyme.

MATERIALS and METHODS

Transcription of the 13C, 15M Domain A of the Hairpin Ribozyme. Uniformly

13C, 15N labeled ribonucleotides (Spectra Isotopes) were incorporated into the

196

tvventy-six nucleotide domain A of the wild type and mutant U+2ClC+3U hairpin

ribozyme by in vitro transcription using recombinant T7 RNA polymerase and a
synthetic DNA template (41,42). For the wild type sequence, transcribed RNA
was purified from an ethanol precipitate of the previous reaction using denaturing
polyacrylamide gel electrophoresis on a Bio-Rad model 491 preparative cell with
a flow rate of 1 mUmin and 4 mL fractions. Fractions corresponding to domain A
of the hairpin ribozyme as determined by gel electrophoresis were pooled,
concentrated using a Rotovapor RE 120 (Buchi) under vacuum at 40 °C to a final
volume of 1 mL, desalted using a G-25 Sephadex column collecting 1 mL
fractions, pooled again and dried. For exchangeable proton spectroscopy, wild

type domain A was resuspended to a final concentration of 1 mM in 270 “L

90%/10% H2O/D2O, 10 mM sodium phosphate pH 6.4, 150 mM NaCl, and 100

(M EDTA in an advanced NMR microtube matched with 020 (Shigemi). For
non-exchangeable proton spectroscopy, wild type domain A was resuspended in
99.96% 020 after repeated lyophilization and resuspension with 99.9% D2O to a
final concentration of 1 mM as mentioned above.

For the mutant U+2CIC+3U, Patrick Ochieng, a graduate student in the

lab, transcribed domain A and purified RNA using gel filtration chromatography

on an Akta Basic FPLC (GE Healthcare) using a HiLoad 26/60 Superdex 75

column (34 pm, 26 mm x 60 mm) (43) monitored at A260 with an isocratic flow

rate of 3 mUmin of 10 mM sodium phosphate pH 6.4, 100 mM NaCI collecting 5

197

mL fractions. Fractions corresponding to mutant domain A of the hairpin

ribozyme were pooled and exchanged with H2O with Amicon Ultra-4 centrifugal

filter units, 3000 MWCO (Millipore). Mutant domain A was resuspended to a final

concentration of 0.8 mM in 250 pL 90%/10% H2O/D2O, 10 mM sodium

phosphate pH 6.4, 150 mM NaCl, and 100 (M EDTA in an advanced NMR
microtube matched with 020 (Shigemi). Prior to use, both NMR samples were

heated to 65 °C for 5 min before cooling on ice for 5 min.

NMR Data Acquisition and Processing. All NMR data were acquired on a

Varian UnitleOVA 600 MHz or Bruker Avance 900 MHz spectrometer. The 900

MHz spectrometer was equipped with a cryogenically cooled probe (Bruker TCI).
Exchangeable proton spectra were acquired at 5 and 15 °C. In all cases,

the proton RF carrier was placed on the HDO residual peak. One-dimensional

imino-optimized spectroscopy was performed using a 1-1 echo pulse sequence

with 16388 complex points in the t1 dimension, a spectral width of 15003.8 Hz, a

3 s recycle delay, and 80 steady state scans. 1H-15N heteronuclear single
quantum coherence (HSQC) spectra were acquired with 1024 x 256 complex
points in the t2 and t1 dimensions, respectively, with corresponding spectral
widths of 13197 and 6078.76 Hz, a 1.5 s recycle delay and 128 steady-state

scans. The 15M carrier frequency was set at 118 ppm as determined from

indirect referencing to internal reference standard 10 mM of sodium 2,2 dimethyl-

Z-silapentane-5-sulfonate (DSS). 1H-1H 15N-edited homonuclear nuclear

198

Overhauser spectroscopy (NOESY-HSQC) spectra were acquired with 1024 x

128 complex points in the t2 and t1 dimensions, respectively, with corresponding

spectral widths of 11995.8 and 11995.8 Hz, a 1 s recycle delay, 256 steady-state
scans, and mixing times of 150 ms and 300 ms.
All non-exchangeable proton spectra were acquired at 25 °C for the wild

type domain A. The proton RF carrier was placed on the HDO residual peak.

Unless othenrvise noted, the 13C carrier was positioned at 111 ppm based on

indirect referencing to internal reference standard DSS.

1H-13C HSQC spectrum was acquired with 1024 x 256 complex points in

the t2 and t1 dimensions, respectively, with corresponding spectral widths of

6000.15 and 7540 Hz, and a 2 s recycle delay. 13C carrier frequency was set at
85 ppm, as determined from indirect referencing to internal reference standard
DSS (10 mM), to allow folding of the aromatic 130 resonances. 1H-13C HSQC
spectrum at 900 MHz was acquired with 512 x 256 complex points in the t2 and

t1 dimensions, respectively, with corresponding spectral widths of 9920.64 and

15847.86 Hz, a 1.5 s recycle delay, and 256 steady-state scans. 1H-1H HCCH-

correlated spectroscopy (COSY) sub-spectra were acquired with 1024 x 256

complex points in the t2 and t1 dimensions, respectively, with corresponding

spectral widths of 2999.18 and 2399.18 Hz, a 2 s recycle delay, and 256 steady-

state scans. 1H-1H HCCH-total correlated spectroscopy (TOCSY) sub-spectra

199

were acquired with 512 x 256 complex points in the t2 and t1 dimensions,

respectively, with corresponding spectral widths of 2999.18 and 2999.18 Hz, a 2

s recycle delay, and 384 steady-state scans. For carbon correlation, a 10 ms

DIPSl-3 mixing period was used. 1H-1H homonuclear 13C-edited nuclear

Overhauser spectroscopy (NOESY-HSQC) sub-spectra were acquired with 1024

x 256 complex points in the t2 and t1 dimensions, respectively, with

corresponding spectral widths of 5997.9 and 5997.9 Hz, a Z s recycle delay, 256

steady-state scans and mixing times of 150 ms and 300 ms. At 900 MHz, 1H-1H

homonuclear NOESY spectrum was acquired with 1024 x 200 complex points in

the t2 and t1 dimensions, respectively, with corresponding spectral widths of

9920.64 and 9920.64 Hz, a 1.5 s recycle delay, 256 steady-state scans and a

mixing period of 150 ms.

All two-dimensional exchangeable spectra in H2O were processed using

Varian VNMR 6.1. Prior to Fourier transformation, 3 Hz exponential line

broadening function was applied for the t2 dimension and a Gaussian window
function was applied for the t1 dimension. All other experiments were processed

using FELIX 2002 (Accelrys). Prior to Fourier transformation in the t2 dimension,

a 20% DC offset was applied, data was zero filled, and a 3 Hz exponential line

broadening function was utilized. Prior to Fourier transformation in the t1

dimension, a cosine-squared apodization function was applied.

200

RESULTS
One-Dimensional Spectroscopy. RNA molecules have different types of

exchangeable protons including those on imino (-NH) groups of guanosines and

uridines, and amino (-NH2) protons for cytidines, guanosines, and adenosines.

These protons are not observed in 1H NMR unless they are protected from fast

exchange with solvent, primarily through base pairing (37). The observation of
these exchangeable protons can provide information about secondary structure

of the RNA molecule. Figure 5.3 shows imino proton resonances detected at 15

°C for (top) mutant U+2CIC+3U and (bottom) wild type domain A of the hairpin

ribozyme. Guanosine imino protons usually resonate between 10 and 13 ppm
while uridine imino protons typically resonate between 12 and 15 ppm. From the
wild type imino proton spectrum, six guanosine imino protons and one uridine
imino proton were detected, which is in good agreement with the predicted
secondary structure. Guanosine imino protons detected upfield at 10 ppm have

been observed from sheared G:A base pairs (37). The guanosine imino proton

of G+7 was observed in both sequences at 10.5 ppm. From the wild type

sequence, the lone uridine imino proton for U+5 was detected at 14.16 ppm

whereas two uridine imino protons were detected in the mutant sequence, one at

14.2 (U+5) and another at 13.96 ppm (U+3). From 12 to 13 ppm, five distinct

guanosine imino protons were detected in the wild type sequence. Several

additional imino protons were detected in the mutant sequence. Interestingly,

201

four guanosine imino protons have similar chemical shifts in both the wild type

and mutant sequence in this chemical shift range. These peaks can be attributed

to base-paired guanosine imino protons of G3, G4, G11, and G13. For the

mutant sequence, an additional guanosine imino proton was detected at 12.36

ppm, which can be attributed to G5 located in the internal loop. This imino proton

was not observed in the wild type spectrum. Also, the guanosine imino proton
detected at 12.62 ppm in the wild type spectrum was shifted to 12.76 in the
mutant spectrum. This guanosine imino proton was tentatively attributed to G5.
In the mutant spectra, an imino proton peak was detected at 12.49 ppm. The

identity of this peak is unknown (see discussion). No imino proton peaks were

detected for G+1 in either spectrum. G+1 imino proton would resonate upfield

similar to G+7 due to non Watson-Crick base pairing with A9. These results

strongly suggest a properly folded RNA molecule with good agreement with
predicted RNA secondary structure. Based on overlap of chemical shift
assignments in both spectra and the addition of new peaks in the mutant
spectrum, these results display consistent secondary structure predictions with

variations localized near the internal loop.

202

 

 

 

 

 

 

*
1
*
1
'r"”‘214'0'7'3'1310'm”12'.0"""11.‘0'"""100
1H(ppm)

Figure 5.3: One-dimensional comparison of the imino proton region for the (top)

mutant U+2C/C+3U and (bottom) wild type isolated domain A at 15 °C. For the
mutant spectrum, * represents new peaks not observed in the wild type

spectrum. =l= represents a downfield shift for G5 H1 in the mutant spectrum.

Two-dimensional Exchangeable Spectroscopy. For the imino proton region,

the 1H-15N HSQC spectra acquired at 15 °C confirms the 1H assignments from
one-dimensional spectroscopy of the wild type and mutant U+2CIC+3U

sequences (Fig. 5.4). In addition, 15N chemical shifts were correlated to 1H

chemical shifts.

203

 

148:
1503
152-3
154-3
1563

1583

(wad) N9,

1603
162-3

1645

 

 

 

163

‘I'YUI'V' V‘V'I'II'I‘IIllfiiTIT

l7"...I'l"""""""l"""TVI'I'7""""""""'

14.0 13.8 13.6 13.4 13.2 13.0 12.8 12.6 12.4 12.2 12.0 11.8 11.6
1H(pr>m)

 

148:
1503

152-3

 

1663

 

 

I'VI'Y'T'VI'IIIII'l'I'IIU'U'll'Iil'It'l'U'"""""'l I""IUUIITIIUIIIITIU"I' 'U'I'I'TYI'I'V‘U

13.8 13.6 13.4 13.2 13.0 12.8 12.6 12.4 12.2 12.0 11.8
1H1ppm)

Figure 5.4: 1H-15N HSQC spectra of the imino proton region for (a) wild type and

(b) mutant U+2CIC+3U domain A of the hairpin ribozyme at 15 °C. Labeled
crosspeaks were determined from one- and two-dimensional spectra.

204

 

Analyzing exchangeable amino protons is less straightforward than imino

protons due to resonance overlap with aromatic protons. The use of two-

dimensional spectroscopy can reduce resonance overlap and identify amino 1H
chemical shifts. Two dimensional 1H-15N HSQC spectra for the amino proton

region at 15 °C are shown in Figure 5.5 for the wild type and mutant U+2CIC+3U

sequences. Resonances are observed in two distinct chemical shift ranges.

These ranges correspond to the hydrogen-bond proton (downfield) and non-

hydrogen bond proton (upfield) of the amino NH2 for cytidines base paired with

guanosines. Both protons correlate to the same 15N nuclei. For the wild type

sequence, five amino proton pairs are detected while seven amino proton pairs

are detected for the mutant sequence, which is in good agreement with predicted

Watson-Crick base pairs for domain A. Unlike imino proton resonances, 1H and

15N amino proton chemical shifts are dissimilar for wild type and mutant

sequences. In the mutant spectrum, a feature derived from an additional cytidine
amino group was observed. Because amino proton chemical shifts are dissimilar
between the wild type and mutant spectra, tentative residue assignments could
not be made.

Amino protons from purine residues are less commonly observed due to
intermediate exchange which results in severely broadened resonances.
Temperature and pH changes could allow for detection in some cases. Lowering

the temperature to 5 °C did not result in the detection of purine amino protons

205

(data not shown). It is possible pH changes could result in the detection of

purine amino protons; however, pH changes were not examined in this work.

206

 

96.0
96.2:
96.44
96.0

a
Hydrogen-bonded Non-hydrogen-bonded
C amino protons C amino protons

 

96.8

97.0
_, 972‘
97.44
97.6'
97.0
98.0
98.2
98.4;
98.6'
988‘
99.0

(wad) N9

 

   

 

 

 

96.5

97.01
97.5{
a 98.03
2 :
g :
é. 98.5j
99.0{

99.53

b Hydrogen-bonded Non-hydrogen-bonded
C amino protons C amino protons

7,3.
A {laugh
. . -
' r 5. 1’.
“ ‘ n L I
., . '3 '
. ..
,. ..
. _ _ l. 1 111‘

 

 

 

100.01

 

9.

......... , ........ ........ , ................................
o 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6
1H(r>pm)

Figure 5.5: 1H-15N HSQC spectra of the amino proton region for (a) wild type

and (b) mutant U+2CIC+3U domain A of the hairpin ribozyme at 15 °C. For the
mutant spectrum, * denotes crosspeaks from a non-predicted cytidine residue.

207

To identify Watson-Crick G:C base pairs, 1H.1H 15N-edited NOESY sub-

spectra were acquired (Fig. 5.6 and 5.7). From these spectra, amino protons of

cytidine NH2 groups were identified which confirmed the identification of spin

pairs determined from 1H-15N HSQC experiments. G:C base pairs were

identified from guanosine imino proton to cytosine amino proton crosspeaks.
Two NOE correlations were observed for each imino proton resonance. These

NOEs, again, confirm amino proton assignments for cytidine residues. With the

tentative identification of G5 and G5 chemical shifts, imino to amino proton

correlations allowed the identification of 04-4 and 0+2, respectively. Also, the

unknown guanosine imino proton at 12.49 ppm was observed to correlate with
unknown cytidine amino protons. This was not observed in the wild type
spectrum. The identity of this G:C base pair is unclear.

NOESY experiments can also identify A:U base pairs. Uridine imino
protons in A-form RNA have been shown to exhibit strong NOEs to H2 of base
paired adenines. A single NOE was observed for each uridine imino proton to

the base paired adenosine H2 in the wild type and mutant spectra. These

crosspeaks can be attributed to H2 of A5 and A7 for U+5 and U+3, respectively.

The inter-residue distance for imino protons in base pairs is z 4 A. For
NOESY experiments, this can give rise to weak NOEs at longer mixing times.

From these NOEs, a NOESY walk would aid in the sequential assignment of

208

Watson-Crick base paired regions, particularly G5, G3, G4, G11, and G13. With

these assignments, the remaining cytidine resonances could be identified. No
imino to imino proton NOEs were observed for spectra acquired with 150 and
300 ms mixing periods at 15 °C. Even at 5 °C, imino to imino proton NOEs were
not observed at 300 ms mixing times (data not shown). Because imino to imino
proton crosspeaks were not observed, complete imino proton sequential
assignments were not obtained for the wild type or mutant domain A. However,

partial assignments have been made and are listed in Tables 5.1 and 5.2.

Taken together, all 1H and 15N chemical shifts for guanosine and uridine

imino protons as well as cytidine amino protons have been determined, which
are consistent with a properly folded RNA molecule and agree well with predicted
secondary structures for the wild type and mutant sequences of domain A.

Preliminary residue assignments were determined for helix cytidine amino

protons, uridine and guanosine imino protons including G5.

209

 

   

   

 

 

 

 

:1: .
’3? 7.6-E ,
3 7.8-i
80; @@ 9 °@
8.2-3
8.4:; .51???
8.6-3
8.8“?" I l 1 l I l "T' i YTV'HF i ”irrfljm'rrr" 1 I l
86 84 82 80 78 76 74 72 70 68 66
1Huaprn)
‘ ‘ 0
7{ " .,
1 b °
8‘1 I a o .
3 3 °
9%
1
E 114;
12‘ .9.-
139 /
14—3 _

 

 

 

II'

III I—TI IIII IIIIIIIII

1’4f8’1’146'14f4"14. ‘14Io 1318 13.6

"i314"1312"13ibm1'218"‘1'236 '12I4"'1"2'i2"'1'"2'.'0"”
1H (ppm)
Figure 5.6: 1H-1H NOESY-HSQC sub-spectra of the wild type hairpin domain A

at 15 °C. Shown are the (a) amino to amino proton and (b) amino to imino proton
regions at 150 ms mixing time.

210

 

 

   

 

 

Y YTYI‘YIIIV'TIIYYTTIfYIIITll'rlITIIIIIIY,I IIIII

87‘8”"86 8.4 8.2 8.0 7‘8 "716“?!” "'7'.'é"“’7‘.6 “678”"66

(wdd) HL

121
13—:

14-

 

.
.

‘ m

.

1H (ppm)

 

b
o . o
0 -° o

”a

 

 

‘11'2" ' ' WEB V 7818“ ' 1616' 1‘ ‘ $81K ' ' ”1'81'2" ' ' 'i'é'fo' ‘ ' 953‘ ' 35.6 ’ ' Y $13.1 ‘ ' W53 WEI)"
1H (ppm)

Figure 5.7: 1H-1H NOESY sub-spectra of the mutant U+2ClC+3U hairpin domain
A at 15 °C. Shown are the (a) amino to amino proton and (b) amino to imino
proton regions at 150 ms mixing time.

211

Table 5.1: Wild type domain A 1H and 15N chemical shifts.

 

 

1 .
lmino proton H chemical 15N chemical
assignment shift (ppm) shift (pprrp
G+7 10.50 146.14
GX1 12.11 147.61
(3+6 12.62 147.61
GX2 12.70 147.46
GX3 12.98 147.68
GX4 13.00 147.47
U+5 14.19 162.93
1H h ' I 15
Amino proton C emlca N chemical

assignment shift (ppm)b shift gpgm)

 

CX1 8.03, 6.63 97.52
C+4 8.35, 6.97 97.58
CX2 8.42, 6.69 98.04
CX3 8.43, 7.10 97.87
CX4 8.59, 6.76 97.77

 

a. . b . .
Resonances are ordered based on lmlno proton or amino proton chemical
shifts. X denotes unknown assignment of resonance.

212

Table 5.2: Mutant U+2C/C+3U domain A 1H and 15N chemical shifts.

1 .
H chemical 15N chemical

 

 

lmino proton a
assignment shift (ppm) shift (ppm)
G+7 10.51 146.18
GX1 12.08 146.69
G3 12.36 146.65
GX2 12.49 147.62
GX3 12.70 147.48
G5 12.76 148.64
GX4 13.00 147.69
GX5 13.04 147.55
U+3 13.96 162.65
U+5 14.16 162.74
1H h ' I 15
Amino proton c emlca N chemical

assignment shift (ppm) shift (ppm)

 

CX1 8.03, 6.62 97.59
C+2 8.23, 6.89 98.35
CX2 8.36, 6.81 97.65
CX3 8.40, 6.91 99.41
CX4 8.40, 6.68 98.64
CX5 8.57, 6.79 97.91
C+4 8.70, 6.94 98.02

 

a. . b . 1 .
Resonances are ordered based on lmino proton or amino proton H chemical
shift. X denotes unknown assignment of resonance.

213

Two-Dimensional Spectroscopy of Non-exchangeable Resonances. To

identify 1H-13C proton spin systems and obtain 1H and 13C assignments, non-

exchangeable experiments have been utilized for the wild type sequence.

Mutant U+2C/C+3U 1H and 13C assignments would be compared to wild type

chemical shifts. It is possible that 1H and 13C chemical shifts would be roughly

similar between both sequences, as was the case for imino proton resonance
chemical shifts. With this approach, mutant chemical shifts could be easily
identified with noticeable chemical shift changes limited to resonances in the

internal loop.
1 13 . . . . 1 13 .
H- C spin pairs were identified from a H- C HSQC experiment at 25

°C acquired at 600 MHz (Fig. 5.8) for the wild type sequence. For the ribose
atoms, significant overlap is observed for H2'/C2', H3'/C3', H4'/C4', H5'/C5', and
H5"/C5" crosspeaks while the H1'/C1' crosspeaks are well dispersed. From the
latter region, twenty-three resonances have been identified out of a total of
twenty-six. For the aromatic atoms of cytidine and uridine, all twenty H5/C5 and
H6/C6 crosspeaks have been identified. Thirteen out of sixteen crosspeaks were
detected for purine H8/CB atoms. Although the regions of purine H8/C6 and

pyrimidine H6/C6 resonances overlap, they could be differentiated based on

multiplet structures. Because 13C labeled C6 are coupled to 13C labled C5, the

H6/C6 crosspeak is observed as a doublet in the 13C dimension. Moderate

214

overlap was observed for adenine H2/C2 resonances.

Similar results were

obtained for a 1H-1BC HSQC acquired at 900 MHz (data not shown).

 

 

 

  
 
 

 

 

H5'/C5' and T t
H5"/C5' .1. -* :— 65
m L— 70
H3'/C3' and , . -:
H2'/02' 45$ __ 75
" “é ~o t
f 80
- a )
(ta 3 v a : A
H8/CB and- ’5 ”4’04 L 85 g
H6/C6 : -" H1701, e
. .-
....;.‘ ._90 mo
O O t v-
_- 95
a , at. Cyt H5/C5 :
— 100
Ade H2/C2 :
o - 0’ . :
‘7. 9 Ura H5/C5 7105
l'*"""rff77'fivfivr1vfivfiffvv.I ......... l ........ 1", ...... I;110
9.0 8.0 7.0 6.0 5.0 4.0 3.0

1H (ppm)

Figure 5.8: 1H-13C HSQC spectrum of the non-exchangeable resonances of the
wild type domain A of the hairpin ribozyme at 25 °C acquired at 600 MHz.

Spectral regions are labeled.

Considerable overlap for the H2'/02', H3'/C3', H4'/C4', H5'/05', and

H5"/C5" spin pairs prevents accurate obtainment of chemical shift assignments

from these data. However, the use of sophisticated RNA correlation experiments

can alleviate ambiguity by the detection of crosspeaks from multiple spin

215

systems, particularly correlations to well dispersed spin systems. HCCH-COSY

experiments were used to correlate covalently bound 1H-13C spin pairs. Briefly,

the HCCH-COSY pulse sequence transfers proton magnetization to adjacent
13 13 . 1 1

protons through one-bond C- C coupling. The H- H HCCH-COSY sub—

spectrum that shows correlation of H1' and H2' proton chemical shifts is

presented in Figure 5.9a. Nineteen H1'-H2' resolved crosspeaks out of twenty-

six were observed. Two H1' protons with 5.57 ppm chemical shifts that were
resolved in the HSQC based on 13C chemical shifts were correlated to a single
H2' proton. This was also observed at 5.59 ppm. Because a two-dimensional

sub-spectrum was acquired, the acquisition of three-dimensional spectrum could

clarify which H1' correlates to H2'. Correlations among other ribose protons

could not be differentiated due to their similar chemical shifts. The 1H-1H HCCH-

COSY sub-spectrum that shows correlation of H5 and H6 chemical shifts is
shown in Figure 5.9b. Ten crosspeaks are observed consistent with the total

number of pyrimidine residues.

216

 

Figure 5.9: 1H-‘H HCCH-COSY sub-spectra of the wild type domain A at 25 °c
at 600 MHz. Shown are the (a) ribose H1’-H2' correlations and (b) pyrimidine
H5-H6 correlations.

217

 

 

 

 

”4.0

 

 

6.0

5.2

 

 

 

 

0|
2:.
)

1H (ppm

I

5.8

- 6.0

 

 

218

To complete accurate chemical shift assignments for ribose protons,
HCCH-TOCSY experiments were employed. HCCH-TOSCY experiments are
similar to HCCH-COSY experiments. However, the HCCH-TOCSY pulse

sequence employs a mixing period where magnetization can be transferred

through several 13C-13C couplings, thus connecting all 1H-13C pairs within a
single spin system. Combining HCCH-COSY H1'-H2’ correlations with HCCH-
TOCSY H3', H4', and H5' correlations to H1', the entire ribose 1H-13C spin
system can be assigned. From the 1H-1H HCCH-TOCSY sub-spectrum

presented in Figure 5.10, additional crosspeaks are observed when compared to

the 1H-1H HCCH-COSY sub-spectrum of the same region. These additional

crosspeaks can be attributed to correlations to H3', H4', and H5' to H1'.

219

 

 

 

 

 

 

 

 

~ 3.5
F
K b
-4.0
.: o :3 y.
.‘I ”3‘"; {-3 ..
' ‘ .- ’64:
2"! '5" 5 32:5. Lfiffl) 1'-
id , r “this“? . L 4.5 A
an 9‘ . E
5}]; L F
o 1,12, fi 9 ‘6»; C.
6v ' Q.
° V
;50 I
w _ ' ‘—
9‘.
r-
0 63?) F
632153;: .
it: ‘2}, f”. - 5.5
,‘ WEE.»- f
o {r- QVQ? 9 9
. ‘6.0
0 (Q5 :-
L
. l . . . . . v r v v l 1 t r v . . . , s I . . f f r
6.0 5.5 5.0 4.5 4.0 3.5
1
H (ppm)

Figure 5.10: 1H-1H HCCH-TOCSY sub-spectra of the wild type domain A at 25
°C at 600 MHz.

The ribose and aromatic 1H and 13C chemical shift were obtained using

correlated spectroscopy identifying intra-residue correlations. This approach,
however, does not correlate ribose and aromatic chemical shifts to adjacent
residues. NOE based experiments can be used to correlate intra-residue sugar
and base protons, as well as inter-residue correlations. Both correlations can be
used to obtain sequential assignment of residues. Sequential assignments can
be made through H8/H6 to H2' correlations which give rise to strong NOEs

although significant overlap limits the usefulness of acquired data. Sequential

220

assignments can also be obtained from H8/H6 to H1' correlations. Because H1'
chemical shifts are more dispersed than H2', assignment can be less ambiguous.
In A-form RNA, the distance between H8/H6 and H1' are close enough to detect

NOEs (z 5 A).

1H-1 H 13C-edited NOESY-HSQC sub-spectra were acquired for the ribose

and aromatic regions. From the ribose optimized 1H-1H NOESY-HSQC sub-

spectra shown in figure 5.11a, correlations among ribose atoms were determined
from strong NOEs, consistent with HCCH-COSY and HCCH-TOCSY sub-
spectra. Correlations can also be observed among intra-residue H2' to H8/H6
from weak NOEs. This is probably due to the optimization of the pulse sequence
which attenuates aromatic proton signals. lntra- and inter-residue correlations for
H1' to H8/H6 could not be unambiguously determined due to overlap even with a

300 ms mixing period (data not shown). To complement the ribose NOESY,
aromatic 1H-1H 13C-edited NOESY-HSQC sub-spectra were acquired (Fig.
5.11b). Consistent with the ribose NOESY, inter-residue correlations were
observed for H2' to H8/H6. A few weak NOEs were observed for H1' to H8/H6

correlations. Other NOEs may be present yet overlapped with more intense H5

to H6 crosspeaks.

221

Figure 5.11: 1H-1H 13C-edited NOESY-HSQC sub-spectra of the wild type
hairpin loop A at 25 °C at 600 MHz. Shown are the (a) ribose and (b) aromatic
spectra using a 300 ms mixing time.

222

 

79 1,1: '1:

i

,r.

I»
‘4 o
4

 

1th 'l f H'" 'u" . If 'I '-‘ ‘ '. .-..- 'cn
N. 1" ' 'l ' i V ’ .f ,
“NEH 5: '5}. """u'.' l‘.| .F l? '. -- all. ' r i
. o I.
O
' l

 

 

IIIIIUIIIIIIIIIIIIIIIIIIIIIIIIIT
&
O

I

l I

I
F”
o

 

 

 

 

 

 

C
P
77.0
i.
l q :
”8.0
I ''''''''' IT '''''''' l ‘7er Y 1111' 'l" f V r YYYYY
80 70 6.0 5.0 4.0 3.0
1H(ppm)
:4.0
b i
. . 75.0
o - '
o a C
C- 0 O _
lfi r-
.9 “ '
o .
O. 77.0
78.0
b
C
“9.0
r-..fi...-.l ......... I ........ e,......-.., .........

 

9.0 8.0 7.0 6.0

223

1H (ppm)

1H (ppm)

For NOE based experiments, signal intensities can be enhanced at higher

magnetic field strengths. The aromatic to sugar region of a two-dimensional 1H-

1H NOESY spectrum acquired at 900 MHz with a 150 ms mixing period is

presented in Figure 5.12. Additional crosspeaks were observed compared to
600 MHz. These crosspeaks can be attributed to H1' to H8/H6 correlations intra-
and inter-residue correlations. Unfortunately, significant overlap with H5 to H6
prevents unambiguous assignments. While using a longer mixing period could
enhance magnetization transfer for increased SIN, three-dimensional acquisition

would be more useful due to H1' and H5 separation.

All results were acquired as two-dimensional 1H-1H sub-spectra. With

the acquisition of three-dimensional spectra, overlapped resonances can be

resolved with 13C chemical shifts. Combining the information of ribose spin
systems with intra-residue H1' to H8/H6 correlations, the entire nucleotide 1H-

13C spin systems can be identified. The inter-residue H1' to H8/H6 correlation

can identify residue assignments. At this point, three-dimensional experiments
have been acquired for HCCH-COSY, HCCH-TOCSY, and NOESY experiments,

but have not been processed for detailed analysis.

224

 

“II/z ‘8‘
| u, I

u 1 h
f," s
|
A -’ '
. 1‘ / V
.4, .'_'VA
:‘ Iv '/ , -
- \ 2’ " l I-
\ .
l ' . .. ’ ' . ‘. ’-
5 - .24
’ ‘ «1 r n 2. r
‘ ,V '1 ’ ‘
l ‘ “ - '
I .. a" /. '7'. ’ ’ ’- f r
. \ ‘.(ra -. I, . n b
h.‘ - r; . * fl
' \ - 1r 7 in" ’ l ‘ ‘ ‘ . v 4 Q
\. ~ . 1 ' * ~.
‘ . . , l 5r 2
I _./.. .. ~C ‘ _\-- l. \ ['| ”3.1:
l ~ » ., ' .23
1...? ~,.’ ‘4 " ( l'] "l . l-
1 i l ' I ~‘ ' ~ ” l ’ 1 l \ \‘
‘. ‘2, . / ‘\ ~ I . E . r
¢ ‘Vl. .r ‘ .‘ .‘,v i‘ ‘A‘« "
2‘» ~. [A ‘. ~ 1. ,/ . P
J I
x \ - ‘
“v . x . ‘ _‘."
‘2‘ . \, 1‘. fl '-
i . ' ‘ .
. ' l ..‘,\*.
'/ -‘..- ‘ ':'~
.4 .\. I; .V ', ‘
l‘ ‘ I ,t
1 _.'
5 ‘ ' \ a (3" r-
4 _/.
‘f \
1 ’- ,‘. z
.' ' ,-
‘.« V A
- " ' ‘1 l '. l' f 7:: \
, \ 2" 1, x \ ,‘ , x’. k? l i Pr ,7 P
. i ‘ .r. I .2 .
g , 9' I ' ' ( (5‘. ‘/
. l -‘-p./- .‘V
v l ‘ ./ - l
,L ' _‘r'~‘
- «'3 -
l

 

 

 

8T6 ' ' 7.0
1 1 1H (ppm)
Figure 5.12: H- H NOESY spectrum of the wild type hairpin loop A at 25 °C
with a 150 ms mixing time at 900 MHz.

225

Table 5.3: Ribose 1H and 13C chemical shifts for wild type domain A.

, 1
H1 H C1'13C H2'1H
(ppm)a (ppm) (ppm)

 

1 5.30 93.19 4.42
2 5.39 92.12 4.57
3 5.39 94.17 4.38
4 5.42 93.87 4.34
5 5.47 94.66 4.48
6 5.47 93.97 4.40
7 5.49 94.36 4.53
8 5.53 91.82 4.68
9 5.57 89.77 a

10 5.57 91.53 4'30

11 5.59 91.92 4.393

12 5.59 93.19
13 5.63 91.04 4.67
14 5.68 94.07 4.39
15 5.73 93.19 4.53
16 5.73 93.87 3.97
17 5.75 91.73 4.81
18 5.82 91.14 4.63
19 5.87 92.61 4.63
20 5.88 93.29 4.53
21 5.92 91.82 4.57
22 5.93 92.51 4.77
23 6.07 92.80 4.71

aResonances are ordered based on H1' chemical shift

 

226

Table 5.4: Aromatic 1H and 13C chemical shifts for wild type domain A.

1
H5 Ha C513C H61H C613C
(ppm) (ppm) (PPm) (ppm)

 

1 5.05 97.29 7.36 140.56
2 5.11 97.29 7.61 140.95
3 5.23 97.78 7.47 141.63
4 5.35 97.88 7.81 141.82
5 5.42 103.35 7.92 142.61
6 5.44 97.88 7.57 141.04
7 5.60 104.52 7.83 143.19
8 5.62 104.91 7.79 143.97
9 5.76 98.27 7.85 141.92

10 5.87 98.37 7.90 142.41

H81H $380 H2 1H 02 13c

(ppm)b (ppm) (ppm)° (ppm)
7.11 136.45 1 7.33 153.25
7.20 136.55 2 7.56 153.93
7.36 137.53 3 7.69 155.20
7.53 136.26 4 7.92 155.01
7.59 139.38
7.71 139.77
7.73 137.72
7.74 137.14
7.75 138.90

10 7.93 140.95

11 8.04 139.97

12 8.15 139.48

13 8.21 142.80

Resonances are ordered based on aH5, bH8, CH2 1H chemical shift

 

(DCDVCDO'I-D-ODN—K

 

DISCUSSION
. 1 13 15 . . . .
Partlal H, C, and N chemical shift aSSIgnments were determined for

the isolated domain A of the hairpin ribozyme. These assignments were made
possible by assignment strategies using standard through-space and through-

bond correlated spectroscopy experiments for uniformly enriched nucleic acids.

227

Due to ambiguity from spectral overlap in two-dimensional sub-spectra, only

partial chemical shift assignments were documented for wild type and mutant
U+2C/C+3U domain A.

From one-dimensional exchangeable spectroscopy, imino proton and
amino proton peaks were observed. The observation of imino and amino proton
peaks is indicative of folded RNA oligomer. The quantity of guanosine and
uridine imino proton peaks from wild type and mutant sequences, which can be
attributed to involvement in Watson-Crick base pairs, is consistent with predicted

secondary structures. One- and two-dimensional exchangeable data exhibited

high similarity between both sequences of domain A used. Consistent with

predicted secondary structure, imino proton peaks for G3-NH and U+3-NH were

observed in the mutant U+2C/C+3U sequence. Also, Ge-NH was determined to

be shifted downfield in the mutant sequence at 12.76 compared to 12.62 in the
wild type sequence. This is plausible because G6 is adjacent to the internal loop
in the wild type sequence and sandwiched by A:U base pairs in the mutant

sequence. This shift can be attributed to additional base stacking interactions.

However, no base pairing was observed for 6+1. Because G+1 would form a
non Watson-Crick base pair, the G+1-NH chemical shift can be expected to

resonate near 10 ppm, similar to G+7. No additional peaks besides G+7 were

observed between 10 to 11 ppm in the mutant sequence.

228

The proposed role of U+2 is to position G3 properly in the active site.

From wild type one— and two-dimensional spectra, no imino proton interactions

were observed for G8. However, an intense imino proton peak was observed in
the U+2C/C+3U mutant spectra for G3 as well as a second uridine imino proton

not present in the wild type spectra, U+3. Interestingly, the U+2C/C+3U mutant

not only abolishes chemistry, it also disrupts proper formation of the docked

complex. Docking and catalysis can be restored with a G3U substitution,

suggesting flexibility at site +2 in the internal loop of the wild type sequence. In
fact, an abasic residue at site +2 only leads to =10 fold decrease in catalysis

while nucleotide substitutions (G, C, and A) lead to more significant decreases in

the catalytic rate (21). Because the U+2C/C+3U mutant has been shown to not
form a docked complex, this can be interpreted as quenched dynamics for G+1.

While the U+2C severely reduced the cleavage rate constant, efficient docking

still occurred. Differences observed between wild type and mutant samples from

exchangeable data in this work indicate changes were localized to the active site.

It is possible that the U+2C/C+3U mutant sequence restricts the flexibility of G+1,
particularly limiting its ability to form a cross domain base pair with C25.

Comparison of C8 dynamics of G+1 using 13C NMR spin relaxation techniques

229

between the wild type and U+2C/C+3U samples could discern quenched

dynamics for the U+2ClC+3U mutant.

Tentative sequential assignments have been made for wild type and

mutant U+2C/C+3U domain A. Unambiguous sequential assignments could not

be obtained due to absence of imino to imino proton NOEs from 1H-1H 15N-

edited NOESY-HSQC sub-spectra. Several methods were attempted to observe
imino to imino proton NOEs including increasing the mixing period to 300 ms,
reducing the temperature to 5 °C to limit exchange, moving the proton irradiation

frequency to 12.5 ppm to maximize enhancement for imino proton resonances,

and the acquisition of a 1H-1H NOESY spectrum at 900 MHz. All methods

produced results similar to Figures 5.6 and 5.7. It is not clear why imino to imino
proton NOEs were not observed for wild type and mutant domain A spectra. It is

possible that imino to imino proton NOEs could be more easily observed from

1H-1H NOESY spectra as opposed to 1H-1H 15N-edited NOESY-HSQC sub-

spectra. The removal of 15N-editing would decrease the pulse sequence length,

thus increasing the signal to noise ratio of observed crosspeaks.

Using correlated spectroscopy, proton intra-residue correlations for H1'
and H2' were determined. To clear up ambiguities, such as H3', H4', H5' (H5")
correlations to H1' and H2' assignments, the use of three-dimensional data

acquisition can remove spectral overlap within the ribose region. To determine

intra- and inter-residue correlations, 1H-1H 13C-edited NOESY-HSQC sub-

230

spectra were acquired. While intra-residue proton to proton as well as aromatic
H5 to H6 NOEs were easily observed, H1' to H8/H6 were not easily detected.
The detection of these NOEs would aid in the sequential assignment of domain
A. Proper identification of aromatic and sugar resonances is crucial for detailed
analysis of dynamics of domain A using NMR spin relaxation experiments.
Acquisition of a three-dimensional 1H-1H 13C-edited NOESY-HSQC sub-
spectrum at 300 ms using 600 MHz and/or 900 MHz could help remove spectral
overlap with aromatic H5 resonances.

A complementary strategy that can be employed is using residue specific
triple resonance experiments that correlate exchangeable and non-exchangeable
protons. Pardi and co-workers (44,45) have developed residue specific pulse
sequences for guanosine, cytdine, and uridine residues that correlate
exchangeable and non—exchangeable protons. The combined results from these
experiments as well as other correlated spectroscopy could greatly refine the
assignment process. Unfortunately, no information was obtained implementing

modified versions of these pulse sequences (46) (data not shown).

In this chapter, partial 1H, 13C, and 15N chemical shift assignments were

determined for wild type and mutant U+2C/C+3U sequences of the isolated

domain A of the hairpin ribozyme. From exchangeable spectroscopy, imino and

amino 1H-15N spin pairs were identified, which led to preliminary residue

assignments. New imino and amino proton peaks were detected for the mutant

U+2C/C43U domain A compared to the wild type domain A. These new peaks

231

were localized to the internal loop. These results support the proposed model
that reduced mutant activity may be linked to decreased flexibility of internal loop

residues.
1H-13C spin pairs were identified from spectroscopy of non-exchangeable

resonances. To obtain complete sugar intra-residue spin systems, three-
dimensional HCCH-TOCSY spectra would aid in unambiguous assignment.
While sequential assignments could not be obtained, three-dimensional 13C-
edited NOESY-HSQC spectra will be critical. With the obtainment of complete
chemical shifts, active site dynamics for the isolated domain A of the hairpin
ribozyme can be investigated. Comparing dynamics in wild type and mutant

sequences, the role of key residues in the catalytic mechanism can be better

characterized.

ACKNOWLEDGEMENTS

I would like to thank Patrick Ochieng for the transcription and purification of

mutant U+2C/C+3U mutant used in this work.

232

REFERENCES

10.

Hampel, A. and Tritz, R. (1989) RNA catalytic properties of the minimum
(-)sTRSV sequence. Biochemistry, 28, 4929-4933.

Prody, G.A., Bakos, J.T., Buzayan, J.M., Schneider, LR. and Bruening, G.
(1986) Autolytic processing of dimeric plant virus satellite RNA. Science,
231, 1577-1580.

Buzayan, J.M., Gerlach, W.L., Bruening, G., Keese, P. and Gould, AR.
(1986) Nucleotide sequence of satellite tobacco ringspot virus RNA and its
relationship to multimeric forms. Virology, 151, 186-199.

Fedor, M.J. (2000) Structure and function of the hairpin ribozyme. J Mol
Biol, 297, 269-291.

Nesbitt, S., Heggr LA. and Fedor, M.J. (1997) An unusual pH-
independent and metal-ion-independent mechanism for hairpin ribozyme
catalysis. Chem Biol, 4, 619-630.

Nesbitt, S.M., Erlacher, HA. and Fedor, M.J. (1999) The internal
equilibrium of the hairpin ribozyme: temperature, ion and pH effects. J Mol
Biol, 286, 1009-1024.

Hampel, K.J., Walter, MG. and Burke, J.M. (1998) The solvent-protected
core of the hairpin ribozyme-substrate complex. Biochemistry, 37, 14672-
14682.

Butcher, S.E., Heckman, J.E. and Burke, J.M. (1995) Reconstitution of
hairpin ribozyme activity following separation of functional domains. J Biol
Chem, 270, 29648-29651.

Cal, 2. and Tinoco, l., Jr. (1996) Solution structure of loop A from the
hairpin ribozyme from tobacco ringspot virus satellite. Biochemistry, 35,
6026-6036.

Butcher, S.E., Allain, F .H. and Feigon, J. (1999) Solution structure of the
loop B domain from the hairpin ribozyme. Nat Struct Biol, 6, 212-216.

233

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Rupert, PB. and Ferre-D'Amare, AR. (2001) Crystal structure of a hairpin
ribozyme-inhibitor complex with implications for catalysis. Nature, 410,
780-786.

Rupert, P.B., Massey, A.P., Sigurdsson, ST. and Ferre-D'Amare, AR.
(2002) Transition state stabilization by a catalytic RNA. Science, 298,
1421-1424.

Salter, J., Krucinska, J., Alam, S., Grum-Tokars, V. and Wedekind, J.E.
(2006) Water in the active site of an all-RNA hairpin ribozyme and effects
of Gua8 base variants on the geometry of phosphoryl transfer.
Biochemistry, 45, 686-700.

Torelli, A.T., Krucinska, J. and Wedekind, J.E. (2007) A comparison of
vanadate to a 2'5 linkage at the active site of a small ribozyme suggests
a role for water in transition-state stabilization. RNA, 13, 1052-1070.

Ryder, SP. and Strobel, SA. (1999) Nucleotide analog interference
mapping of the hairpin ribozyme: implications for secondary and tertiary
structure formation. J Mol Biol, 291, 295-311.

BerzaI-Herranz, A., Joseph, S., Chowrira, B.M., Butcher, SE. and Burke,
J.M. (1993) Essential nucleotide sequences and secondary structure
elements of the hairpin ribozyme. EMBO J, 12, 2567-2573.

Butcher, SE. and Burke, J.M. (1994) Structure-mapping of the hairpin
ribozyme. Magnesium-dependent folding and evidence for tertiary
interactions within the ribozyme-substrate complex. J Mol Biol, 244, 52-63.

Butcher, SE. and Burke, J.M. (1994) A photo-cross-linkable tertiary
structure motif found in functionally distinct RNA molecules is essential for
catalytic function of the hairpin ribozyme. Biochemistry, 33, 992-999.

Walter, F., Murchie, A.|., Duckett, DR. and Lilley, BM. (1998) Global
structure of four-way RNA junctions studied using fluorescence resonance
energy transfer. RNA, 4, 719-728.

Walter, F., Murchie, A.l., Thomson, J.B. and Lilley, OM. (1998) Structure
and activity of the hairpin ribozyme in its natural junction conformation:
effect of metal ions. Biochemistry, 37, 14195-14203.

234

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

Walter, N.G., Chan, P.A., Hampel, K.J., Millar, DP. and Burke, J.M.
(2001) A base change in the catalytic core of the hairpin ribozyme
perturbs function but not domain docking. Biochemistry, 40, 2580-2587.

Walter, N.G., Hampel, K.J., Brown, KM. and Burke, J.M. (1998) Tertiary
structure formation in the hairpin ribozyme monitored by fluorescence
resonance energy transfer. EMBO J, 17, 2378-2391.

Zhuang, X., Kim, H., Pereira, M.J., Babcock, H.P., Walter, MG. and Chu,
S. (2002) Correlating structural dynamics and function in single ribozyme
molecules. Science, 296, 1473-1476.

Hegg, LA. and Fedor, M.J. (1995) Kinetics and thermodynamics of
intermolecular catalysis by hairpin ribozymes. Biochemistry, 34, 15813-
15828.

Hoogstraten, C.G., Legault, P. and Pardi, A. (1998) NMR solution
structure of the lead-dependent ribozyme: evidence for dynamics in RNA
catalysis. J Mol Biol, 284, 337-350.

Legault, P. and Pardi, A. (1997) Unusual dynamics and pKa shift at the
active site of a lead-dependent ribozyme. J Am Chem Soc, 119, 6621-
6628.

Shippy, R., Siwkowski, A. and Hampel, A. (1998) Mutational analysis of
loops 1 and 5 of the hairpin ribozyme. Biochemistry, 37, 564-570.

Komatsu, Y., Kumagai, l. and Ohtsuka, E. (1999) Investigation of the
recognition of an important uridine in an internal loop of a hairpin ribozyme
prepared using post-synthetically modified oligonucleotides. Nucleic Acids
Res, 27, 4314-4323.

Perez-Ruiz, M., Barroso-DelJesus, A. and Berzal-Herranz, A. (1999)
Specificity of the hairpin ribozyme. Sequence requirements surrounding
the cleavage site. J Biol Chem, 274, 29376-29380.

Chowrira, B.M., Berzal-Herranz, A. and Burke, J.M. (1991) Novel
guanosine requirement for catalysis by the hairpin ribozyme. Nature, 354,
320-322.

235

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

Sargueil, B., Hampel, K.J., Lambert, D. and Burke, J.M. (2003) In vitro
selection of second site revertants analysis of the hairpin ribozyme active
site. J Biol Chem, 278, 52783-52791.

Hoogstraten, C.G., Wank, JR. and Pardi, A. (2000) Active site dynamics
in the lead-dependent ribozyme. Biochemistry, 39, 9951-9958.

Blad, H., Reiter, N.J., Abildgaard, F., Markley, J.L. and Butcher, SE.
(2005) Dynamics and metal ion binding in the U6 RNA intramolecular
stem-loop as analyzed by NMR. J Mol Biol, 353, 540-555.

Shajani, z. and Varani, G. (2005) 13c NMR relaxation studies of RNA
base and ribose nuclei reveal a complex pattern of motions in the RNA
binding site for human U1A protein. J Mol Biol, 349, 699-715.

Trantirek, L., Caha, E., Kaderavek, P. and Fiala, R. (2007) NMR 13C-

relaxation study of base and sugar dynamics in GCAA RNA hairpin
tetraloop. J Biomol Struct Dyn, 25, 243-252.

Shajani, Z., Drobny, G. and Varani, G. (2007) Binding of U1A protein

changes RNA dynamics as observed by 13C NMR relaxation studies.
Biochemistry, 46, 5875-5883.

Flinders, J. and Dieckmann, T. (2006) NMR spectroscopy of ribonucleic
acids. Prog Nucl Magn Reson Spectrosc, 48, 137-159.

Cromsigt, J., van Buuren, B., Schleucher, J. and Wijmenga, S. (2001)
Resonance assignment and structure determination for RNA. Methods
Enzymol, 338, 371-399.

Nikonowicz, ER and Pardi, A. (1993) An efficient procedure for

assignment of the proton, carbon and nitrogen resonances in 3C/15N
labeled nucleic acids. J Mol Biol, 232, 1141-1156.

Wijmenga, S. and van Buuren, B. (1998) The use of NMR methods for
conformational studies of nucleic acids. Prog Nucl Magn Reson
Spectrosc, 32, 287-387.

236

41.

42.

43.

44.

45.

46.

Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987)
Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic
DNA templates. Nucleic Acids Res, 15, 8783-8798.

Milligan, J.F. and Uhlenbeck, O.C. (1989) Synthesis of small RNAs using
T7 RNA polymerase. Methods Enzymol, 180, 51-62.

Kim, I, McKenna, S.A., Viani Puglisi, E. and Puglisi, JD. (2007) Rapid
purification of RNAs using fast performance liquid chromatography
(FPLC). RNA, 13, 289-294.

Simorre, J.P., Zimmermann, G.R., Pardi, A., Farmer, B.T., 2nd and
Mueller, L. (1995) Triple resonance HNCCCH experiments for correlating
exchangeable and nonexchangeable cytidine and uridine base protons in
RNA. J Biomol NMR, 6, 427-432.

Simorre, J.P., Zimmermann, G.R., Mueller, L. and Pardi, A. (1996)
Correlation of the guanosine exchangeable and nonexchangeable base

protons in 13C-/15N-|abeled RNA with an HNC-TOCSY-CH experiment. J
Biomol NMR, 7, 153-156.

Lukavsky, P.J. and Puglisi, JD. (2001) RNAPack: an integrated NMR
approach to RNA structure determination. Methods, 25, 316-332.

237

Chapter 6

Conclusions and Future Work

238

CONCLUSIONS

This thesis was motivated by an interest in investigating dynamics in RNA
molecules by use of NMR spin relaxation techniques. NMR spectroscopy is a
versatile tool which can detect motions across a broad range of molecular motion
timescales (1). While investigations of molecular motions have been extensively
probed in proteins, dynamics in RNA molecules have been less studied. Motions
in RNA molecules have mostly been limited to the nucleotide base C8 and C6
atoms (2-5), and little information has been obtained for the ribose atoms.

Studies of ribose dynamics have been hampered because of difficulties in

acquiring reliable data due to the coupling among 13C atoms of the ribose ring

when using uniform isotope labeling schemes (6). Other methods, such as
natural abundance acquisition (7,8) or chemical synthesis (9) have their
limitations as well. Because the ribose ring of RNA molecules has may play an
important role in the catalytic mechanism (10), a method is needed to accurately
extend dynamic studies throughout sugar molecule. With the combined analysis
of base and ribose carbons of RNA molecules, the role of dynamics can be
defined for RNA molecules during RNA folding or catalysis.

In chapter two, a method was outlined to make use of the pentose

phosphate pathway of E. coli cells to obtain ribonucleotides that have an

alternating 13C-12C pattern for the ribose ring, thereby isolating 13C atoms for

NMR dynamic studies. This pattern was obtained using E. coli strains (zwf)
deficient in the oxidative portion of the pentose phosphate pathway. Other

methods were identified to effectively isolate other carbons, including C3'. These

239

results along with previous techniques provide a method to specifically label all

13C atoms in ribonucleotides such that NMR dynamic studies are suitable.

The utility of the alternate-site labeling scheme for relaxation studies was
validated by comparing R1 and R1 p measurements for specifically (2',4'-13C2)
and uniformly labeled rAMP. Consistent with previous work (11), it was shown,
from R1 measurements, that 13C-13C dipolar interactions can be neglected at

short correlation times, but these interactions become more significant at longer

correlation times consistent with previous results. While 13C-13C dipolar

interactions are negligible for R2 measurements, 130130 scalar interactions limit
data analysis of transverse relaxation. This was demonstrated by the removal of

Hartmann-Hahn 13C-13C interaction from R1 p measurements. The specific

labeling scheme removes 13C-13C dipolar and scalar coupling, greatly

simplifying the determination of relaxation parameters from 1H-13C spin systems.

From these results, it is clear that the alternate-site labeling scheme can be used
in RNA molecules to study ribose dynamics on multiple timescales.
In chapter three, dynamics were studied in the GCAA RNA hairpin via the

alternate-site labeling scheme. Previous NMR studies and molecular simulations

reported sugar conversion for C5 from C3'-endo to C2'-endo in conjunction with

Ca base flipping out of the ground state structure, as well as a heterogeneous

hydrogen bond model for the tetraloop (12,13). With the specific labeling

240

scheme, ribose dynamics were investigated on multiple timescales, particularly
on the us to ms timescale.

Motions on the ps to ns timescale were investigated using the Model-free
approach pioneered by Lipari-Szabo (14,15) from 13c R1, R1 p and
heteronuclear NOE values at 600 MHz. The GCAA RNA hairpin was found to be
a rigid molecule on this timescale with internal order parameters, 82, of at least

0.9 for non-terminal atoms. Some atoms were determined to have internal,

effective correlation times indicative of fast motions on the ps to ns timescale.

Interestingly, all tetraloop atoms except C6 C2' exhibited an Rex contribution,

indicative of us to ms motions. To accurately ascertain the Rex contribution,

relaxation dispersion curves were analyzed.
Motional parameters were determined from fits to relaxation dispersion

curves obtained at multiple field strengths. With the combined approach of

CPMG, on-, and off resonance R1 p measurements, as well as multiple fields, the

complete dispersion profiles for nearly all atoms in the GCAA RNA tetraloop

hairpin were determined, particularly the tetraloop atoms. Exchange lifetimes

were determined for C2' and C4' atoms of all tetraloop atoms except 05 C2'. For
G5, A7, A8, the C2' and C4' exchange lifetimes were determined to be similar

within error. The C2' and C4' atoms of a given residue could be fit to a single Tex,

which implies that atoms of a residue are reporting on a single conformational

event. This motional event was proposed to be sugar pucker transitions, where

241

previous results determined the sugar puckers of the GCAA RNA tetraloop to be

in equilibrium (12). Also, exchange lifetimes clustered into two groups across the

tetraloop, which suggested two different motional timescales. The C5 and A7
atoms were fit to a single Tex while G5 and A5 were fit to another Tex. This
suggests that the motions of C5 and A7 are coupled and G5 and A5 are coupled.
It is plausible that the C5 and A7 motions are reporting on the base flipping

motion of C5, while the G5 and A5 are reporting on the dynamic hydrogen bond

network. These results demonstrate the usefulness in measuring conformational
changes for RNA molecules using NMR experiments, whereas other methods
would not be able to detect such motions.

Relaxation dispersion curves were obtained at multiple temperatures to
ascertain the temperature dependence of the exchange rate constant for C2' and
C4' in the tetraloop. The activation energy of sugar pucker transitions was
determined from Arrhenius plots. While the data are somewhat ambiguous due

to large error bars, the fitted activation energy values suggest that sugar pucker

transitions require =30 kJ/mol. Not surprisingly, the activation energies for G5
and A5 atoms were clustered within a narrow range, while the activation energies

of C5 and A7 C4' were reported to be similar within error. These results depict

the first analysis of ribose dynamics for C2' and C4' of RNA molecules. Taken

along with nucleotide base dynamics, a complete assessment of motions within

242

RNA molecules can be obtained yielding a wealth of information, such as

correlated motions.
In chapter four, the alternate-site labeling scheme was incorporated into

cytidine residues of the lead-dependent ribozyme. Preliminary results suggest
an Rex contribution for C5 C2', which is the nucleophile in the catalytic
mechanism. An exchange lifetime of 93 :l: 36 us was determined, suggesting

possible motional correlation to the protonation/deprotonation of A25 N1, which
forms a non Watson-Crick base pair with C5.
. . 1 13 15 . .
In chapter five, preliminary H, C, and N chemical shifts are reported

for wild type and U+2C/C+3U mutant isolated domain A of the hairpin ribozyme.

From comparison of exchangeable spectroscopy data between the wild type and
mutant domain A, additional peaks were observed in the mutant sequence.

These peaks were attributed to the residues in the internal loop. Because the

U+2C/C+3U mutant is not able to form a docked structure, and the U+2C single

mutant does form a stable docked structure, it is plausible that the U+2C/C+3U

mutant reduces or quenches dynamics.

FUTURE WORK
While this thesis has documented methods and analyses in RNA
molecules, many questions still remain. Several of these issues have been listed

below:

243

 

Further Characterization of the GCAA RNA Hairpin. The usefulness of the
specific labeling scheme was demonstrated to obtain motional parameters for C2'
and C4' of the ribose ring of the tetraloop region. It was concluded that C2' and
C4' report on the same motion of sugar pucker interconversion. To further
examine sugar pucker transitions, motional parameters can be obtained for C3'

atoms of the tetraloop residues to further probe ribose dynamics. This can be

done by obtaining ribonucleotides from wild type E. coli cells with 4-13C glucose

as the sole carbon source. These ribonucleotides can then be incorporated into
any RNA oligomer of choice, i.e. the GCAA RNA hairpin.

Concerted motions for the ribose ring were determined, even among
multiple residues. However, concerted base and ribose motions were not

detected. In the GCAA RNA hairpin, C6 was predicted to de-stack from the

tetraloop structure. However, dynamics for C5 C6 were not observed which was
due in part from the unambiguous 1H only assignments for the GCAA RNA

hairpin. Using 1H-1H HCCH-COSY sub-spectra, H5 and H6 correlations can be

used to clarify H6/C6 spin system for accurate acquisition of relaxation dispersion
data.

Investigation of Dynamics for the Lead-dependent Ribozyme. Preliminary

data have been presented that suggest exchange contributions for 02' of C5 in
the lead-dependent ribozyme. To further characterize this motion, the acquisition

of CPMG and R1 p measurements at higher fields, particularly 900 MHz, could aid

244

 

in the analysis of leadzyme dynamics. As shown previously, acquisition at higher

magnetic fields increases the Rex contribution, aiding in the analysis of small Rex
contributions. Also, to investigate base motions, a uniformly labeled 13C

cytidine-only leadzyme NMR sample could unambiguously detect aromatic C5
C6 motions.

Investigation of Dynamics for the Hairpin Ribozyme. Preliminary 1H, 13C,

and 15N chemical shifts were obtained for wild type and mutant U+2C/C+3U

hairpin ribozymes. To complete the chemical shift assignments, three-
dimensional HCCH-TOCSY and NOESY will be essential. Three dimensional
datasets have been acquired at both 600 and 900 MHz. Processing and analysis
of these datasets would greatly improve the effectiveness of chemical shift

assignment.

With the unambiguous assignment of 1H and 13C resonances for wild type
and U+2C/C+3U mutant sequences, active site dynamics can be investigated on

multiple timescales using R1, R1 p, and heteronuclear NOE measurements as

well as relaxation dispersion curves at multiple field strengths for base and ribose

atoms using uniform and specific labeling schemes, respectively, particularly A1,

G+1, U+2, and G5. These experiments may reveal never-before-seen dynamics

within the active site that NMR spectroscopy is uniquely suited to measure.

245

With the ground state dynamics defined, active site perturbations due to

docking can be probed using labeled domain A with unlabeled domain B in the
presence and absence of Co(lll)[NH3]53+. To prevent cleavage, a G5AIA35U

mutant will be used which has been shown to dock but does not cleave on the
timescale of NMR experiments (personal communication). These experiments
have the unique ability to probe timescale of docking perturbations.

To conclude, the work detailed in this thesis has probed ribose dynamics
in RNA molecules. The methods documented herein in conjunction with other

studies can aid in the understanding the catalytic mechanism of ribozymes.

246

REFERENCES

1. Palmer, A.G., 3rd. (2004) NMR characterization of the dynamics of
biomacromolecules. Chem Rev, 104, 3623-3640.

2. Hoogstraten, C.G., Wank, JR. and Pardi, A. (2000) Active site dynamics
in the lead-dependent ribozyme. Biochemistry, 39, 9951-9958.

3. Blad, H., Reiter, N.J., Abildgaard, F., Markley, J.L. and Butcher, SE.
(2005) Dynamics and metal ion binding in the U6 RNA intramolecular
stem-loop as analyzed by NMR. J Mol Biol, 353, 540-555.

4. Shajani, Z. and Varani, G. (2005) 13C NMR relaxation studies of RNA
base and ribose nuclei reveal a complex pattern of motions in the RNA
binding site for human U1A protein. J Mol Biol, 349, 699-715.

5. Shajani, Z. and Varani, G. (2008) 13C relaxation studies of the DNA target
sequence for hhai methyltransferase reveal unique motional properties.
Biochemistry, 47, 7617-7625.

6. Shajani,1Z. and Varani, G. (2007) NMR studies of dynamics in RNA and
DNA by130 relaxation. Biopolymers, 86, 348- 359.

7. Borer, P.N., LaPlante, S.R., Kumar, A., Zanatta, N., Martin, A., Hakkinen,

A. and Levy, (3.0. (1994) 13c-NMR relaxation in three DNA
oligonucleotide duplexes: model-free analysis of internal and overall
motion. Biochemistry, 33, 2441-2450.

8. 1Spielmann, H. P. (1998) Dynamics of a bis-intercalator DNA complex by

1H- detected natural abundance C NMR spectroscopy. Biochemistry, 37,
16863- 16876.

9. SantaLucia, J., Jr., Shen, L.X., Cai, 2., Lewis, H. and Tinoco, L, Jr. (1995)
Synthesis and NMR of RNA with selective isotopic enrichment in the
bases. Nucleic Acids Res, 23, 4913-4921 .

247

10.

11.

12.

13.

14.

15.

Rupert, P.B., Massey, A.P., Sigurdsson, ST. and Ferre-D'Amare, AR.
(2002) Transition state stabilization by a catalytic RNA. Science, 298,
1421-1424.

Boisbouvier, J., Wu, Z., Ono, A., Kainosho, M. and Bax, A. (2003)

Rotational diffusion tensor of nucleic acids from 13C NMR relaxation. J
Biomol NMR, 27, 133-142.

Jucker, F.M., Heus, H.A., Yip, P.F., Moors, E.H. and Pardi, A. (1996) A
network of heterogeneous hydrogen bonds in GNRA tetraloops. J Mol
Biol, 264, 968-980.

Sorin, E.J., Engelhardt, M.A., Herschlag, D. and Pande, VS. (2002) RNA
simulations: probing hairpin unfolding and the dynamics of a GNRA
tetraloop. J Mol Biol, 317, 493-506.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 1. theory
and range of validity. J Am Chem Soc, 104, 4546-4559.

Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation
of nuclear magnetic resonance relaxation in macromolecules. 2. analysis
of experimental results. J Am Chem Soc, 104, 4559-4570.

248