PART I
TIE THREE DIMENSlONAL STRUCTURE
OF u-CHYMOTRYPSIN AT pH 4.2

PART 91

. THE STRUCTURE or-
m-CHYMOTRYPSW AT pH 5.7

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PART 1: THE THREE DIMENSIONAL STRUCTURE OF
a-CHYMOTRYPSIN AT pH 4.2

PART 11: THE STRUCTURE OF a-CHYMOTRYPSIN AT pH 6.7
presented by

RICHARD LEE VANDLEN

has been accepted towards fulfillment
of the requirements for

PH.D. degree in CHEMISTRY

 

QTIQ SWAN‘

Major professor

 

Date S‘H . 17‘ I912-
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0-7639

 
 
 
      
 

  

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ABSTRACT
PART I: THE THREE DIMENSIONAL STRUCTURE OF
a-CHYMOTRYPSIN AT pH 4.2

PART II: THE STRUCTURE OF 3- CHYMOTRYPSIN AT pH 6.7

BY

Richard Lee Vandlen

The three dimensional structure of the proteolytic enzyme
a-chymotrypsin was determined to 2.8A resolution by X-ray
crystallographic techniques. Crystals of a-chymotrypsin with
space group P21 are grown from ammonium sulfate solutions at
pH 4.2 and have four molecules in the unit cell or two mole-
cules in the asymmetric unit. The cell dimensions are
a = 49.24, 2': 67.20, g = 65.94A, and B = lOl.79°. A change
of the pH from 4.2 to 6.7 of the ammonium sulfate solution in
which the crystals are stored produced substantial changes in
the x-ray diffraction pattern of the native enzyme. The
structure of a-chymotrypsin at pH 6.7 was studied by difference
electron density techniques.

The structures of the two independent molecules of
a—chymotrypsin in the asymmetric unit were solved by the use
of six heavy atom isomorphous derivatives. Intensity data
were collected by a computer controlled diffractometer. The

electron density of the enzyme was interpreted in terms of

 

 

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Richard Lee Vandlen

the known amino acid sequence of 241 residues which comprise
a-chymotrypsin. A model of the enzyme molecule was constructed.
The path of the polypeptide chain through the globular molecule
is essentially random with the formation of a-helices, pleated
sheets, B-bends, nonpolar regions and aromatic clusters. The
location of several sulfate molecules bound to the enzyme was
established. The atomic coordinates were refined by optimizing
the fit of the model to the observed electron density distri-
bution.

The two independent molecules in the asymmetric unit are
related by a local two-fold axis to form a 'dimeric' molecule.
Various reciprocal interactions between the two molecules are
made in the dimer interface region. There are significant
differences in structure between the two independent molecules
as shown by variations in the heavy atom derivatives and by a
difference electron density map between the two molecules.

Many of the observed deviations occur around the interface
region and in the vicinity of the active sites of the two
enzyme molecules.

Difference electron density maps between the electron
densities of the enzyme at pH 4.2 and pH 6.7 revealed a number
of molecular changes which are both intramolecular and inter-
molecular in nature. The largest changes involved the move-
ment of the side chain of histidine 40, the rotation of the
terminal carboxyl group of tyrosine 146 away from an inter-
action with the imidazole ring of histidine 57 of the second

molecule in the asymmetric unit, and complicated movements

 

 

 

near t1
changes
regions
adjustn

molecul

Richard Lee Vandlen

near the favored uranyl binding site. Somewhat smaller
changes in the structure are observed around the active site
regions of both molecules and other places. In addition,
adjustments of the solvent structure on the surface of the

molecule were revealed.

PART I: THE THREE DIMENSIONAL STRUCTURE OF
a-CHYMOTRYPSIN AT pH 4.2

PART II: THE STRUCTURE OF a-CHYMOTRYPSIN AT pH 6.7

BY

Richard Lee Vandlen

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1972

bfl237b

To my parents, Mr. and Mrs. V. D. Vandlen, for
their constant encouragement and support through-

out the many years of my education.

ii

l
I
|
I
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For
ship thr
his Sinc

The
Horimoto
Rating I
Structur

To
liebman,
II. Step

My hel

ACKNOWLEDGMENTS

For his constant guidance, support, enthusiasm and friend-
ship throughout all aspects of this study, the author extends
his sincere appreciation and thanks to Dr. Alexander Tulinsky.

The author wishes to thank Dr. N. V. Mani, Dr. Carl
Morimoto, Dr. L. H. Wright and Dr. William Brinigar for stim-
ulating discussions and for their many contributions to the
structure determination of o-chymotrypsin.

To his fellow students, Dr. Penelope Codding, Mr. Michael
Liebman, Miss Irene Moustakali, Mr. Aristides Mavridis and
Mr. Stephen Kraft, the author appreciates their assistance and
many helpful discussions.

Support of the Molecular Biology Section of the National
Science Foundation during the course of this study is grate-
fully acknowledged.

Thanks are also due to Mr. John Fox of the Computer
Laboratory for his generous assistance with many computer-

related problems.

iii

 

II.

III.

IV,

0 Sr]

TABLE OF CONTENTS

GENERAL INTRODUCTION. . . . . . . . . . . . . . . .

PART I:

I.

II.

III.

IV.

THE THREE DIMENSIONAL STRUCTURE or
d-CHYMOTRYPSIN AT pH 4.2

INTRODUCTION . . . . . . . . . . . . . . . .

1.
2.

3.

The Chymotrypsin Family of Proteins. .
Physical and Chemical Properties of

Chymotrypsin . . . . . . . . . . . . .
Structural Studies . . . . . . . . . .

EXPERIWNTAL . C C O I O O O O O O O O O O O

1.
2.
3.
4

Methods. . . . . . . . . . . . . .
Three Dimensional Data Collection. .
Heavy Atom Derivatives and Phase Angle
Calculations . . . . . . . . . . . . .
Electron Density Map Calculation . .

DESCRIPTION OF THE MOLECULE. . . . . . . . .

1. Folding of the Polypeptide Chain . . .
2 o B-Bends o o o o o o o o o o o o o o o o
3. The Active Site Region . . . . . . . .
4. Non-polar Cavity . . . . . . . . . . .
5. Aromatic Clusters. . . . . . . . . . .
MOLECULAR INTERACTIONS . . . . . . . . . . .
l. The Structure of the a-CHT Dimer . . .
2. Local Two-fold Interactions. . . . . .
3. Location of Bound Sulfate Molecules. .
4. Differences Between the Two Molecules.
5. Uranyl Binding Interface . . . . . . .
STRUCTURE REFINEMENT . . . . . . . . . . . .
l. Refinement of the Coordinates. . . . .
2. Real Space Refinement. . . . . . . .
3. Comparison With the MRC Structure of
"Average" a-CHT. . . . . . . . . . . .

iv

15
23

25
25
31
32
38
41
46

46
53

62
69

76

76
79

84

PART IL

VI.

VII.

VIII.

PEFEREN:

PART II: THE STRUCTURE OF u-CHYMOTRYPSIN AT pH
VI. INTRODUCTION. . . . . . . . . .

1. Effect of pH on Protein Structure
2. Effect of pH on a-CHT . . .

VII. EXPERIMENTAL. . . . . . . . . . . . .

. Crystal Preparation . . . . . . .

. Data Collection and Processing.

. Difference Electron Density and
Error Assessment. . . . . . . . .

1
2
3
VIII. DISCUSSION OF THE RESULTS . . . . . . .

Histidine 40. . . . . . . . . . .
Dimer Interactions. . . . . . . .
Active Site . . . . . . . . . .
Uranyl Interface. . . . . . . .
Other pH Changes. . . . . . . .
Final Comments. . . . . . . .

\IO‘U'IubUNI-d

REFERENCES 0 O O O O O O O O O O O O O O O O O 0

APPENDIX. Side Chain Environment and Clarity in

The Electron Density of a-CHT. . . .

General Features of the Difference Map.

87

87
90

95

95
98

100
104

104
107
112
114
118
118
120

124

129

Table

 

II.
III.

IV.

VI.
VII,

VIII.

IX.

Table

II.
III.
IV.
V.
VI.
VII.
VIII.

IX.

LIST OF TABLES

Heavy Atom Parameters at 2.8A Resolution . . .
Summary of Side Chain Environment. . . . . . .
Best Defined B-Bends in a-CHT. . . . . . . . .
Non-Polar Cavity, a-CHT. . . . . . . . . . . .
Aromatic Clusters. . . . . . . . . . . . . .

Transformation Refinement Summary. . . . . . .
Bound Sulfate Positions. . . . . . . . . . . .
Ionizable Groups in Globular Proteins. . . . .

Cell Dimensions for pH 6.7 a-CHT Crystals and
Native Crystals. . . . . . . . . . . . . . .

Summary of Changes of a-CHT Upon Change of pH.

vi

17

29

33

42

45

50

63

88

99
121

 

10.

12.

Figure

l.

10.
11.

12.

13.

14.

LIST OF FIGURES

Axial diffraction patterns for native a-CHT and
for the Pt heavy atom derivative.

Heavy atom refinement of a—CHT.

Figure of merit distribution curve.

Approximate shape of

(a) Stereo drawing of a Type I B-Bend formed by
residues SER 115 to VAL 118.
of a Type II B-Bend formed by residues ASP 194

to GLY 197. . . . . .

Schematic drawing of the important residues

around the active site.

'dimeric'

(b) Stereo drawing

molecule of a-CHT.

Schematic representation of non-polar cavity.

Stereoscopic drawing of the tryptophan

aromatic cluster. . .

Schematic packing diagrams of a—CHT in the
(a) projection on yz,

unit cell;
plane . . . . . . . .

(b) on xz

Intermolecular interactions across dyad A .

Local two-fold interactions between the two

molecules, (a) PHE 39,

Electron density of the active site region

of one molecule . . .

Electron density of the helical region near

MET 180 . . . . . . .

Detail of the peptide residues near the

uranyl binding site .

(b) peptide chain.

vii'

12
21
22

27

34

37

40

44

52

54

57

58

68

73

20.

21.

22.

Figure

15.

16.

17.

18.

19.

20.

21.

22.

(a) Typical pH—rate profile for Chymotrypsin
(b) Functional relation-

catalyzed reactions;

ship of pH on dimer formation.

Axial scan along c* direction for Tosyl,

and pH 6.7 crystals of a-CHT

Radial distribution curves for pH 6.7 and

native crystals. . . .

Gaussian curves showing effect of a small

shift on the difference density.

Schematic drawing of important residues

around HIS 40. . . . .

Electron density around HIS 40

Electron density in vicinity of TYR'

and HIS 57 . . . . . .

Electron density map around SER 195.

viii

146

native

92

97

101

106

108

111

111

115

inter
in thl
cesse:
technj
molecn
more i
sis ha
Obtain
Of an

dEtail

GENERAL INTRODUCTION

The past decade has seen the growth of a tremendous
interest in the chemistry and physics of biological systems,
in the control and adaptation of mechanisms of life pro-
cesses, and in the very essence of life. Of all of the new
techniques that have been developed to study biological macro-
molecules, X-ray crystallography has emerged as one of the
more important tools. As intensity data collection and analy-
sis have improved and matured, it has now become possible to
obtain, under the right conditions, the tertiary structure
of an enzyme or protein to sufficient resolution to allow
detailed conclusions concerning relationships between struc-
ture and functionality to be made with some certainty.

Coupled with extensive chemical and biochemical studies,
reasonable, intelligent proposals can be made concerning
mechanisms of reactivity, catalysis, and molecular interac—
tions and the physical forces which are intimately involved.
Refinement of the proposals can then be achieved from new
experiments designed on the knowledge of the spatial relation-
ships of the amino acids involved.

The harvests of many years of dedicated work using the
above principles are beginning to appear on a variety of dif-
ferent biological systems. From a knowledge of the amino
acid sequences of a large number of cyctochrome molecules

1

 

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from various species, coupled with the structure determina-
tion of the oxidized and reduced forms, some very interesting
proposals concerning the course and rate of the biological
evolution of proteins have been made by Dickerson and
coworkers (1,2). Likewise, years of patient study of the
hemoglobin-myoglobin family of proteins by M. F. Perutz have
led to some very important insights pertaining to the physio—
logical processes involved in the transport of oxygen through-
out the body (3). Not only has Perutz established the rela-
tionship among the coordination state of the heme iron, the
disposition and structural changes associated with the sub-
units and the affinity of the molecule for various ligands,
but recent work has also made significant advances in the
elucidation of the causes of many blood disorders, such as
sickle cell anemia, which are due to abnormal mutant hemo—

globins (4).

PART I

THE THREE DIMENSIONAL STRUCTURE OF

a-CHYMOTRYPSIN AT pH 4.2

I . INTRODUCTION

1. The Chymotrypsin Family of Proteins

The Chymotrypsin family of proteins is one of a series
of homologous enzyme families, found in mammalian digestive
juices, whose principal task is the degradation of protein
polypeptide chains. The active species of each family con-
tains a unique serine residue, an absolute requirement for
enzymatic activity. In addition to the Chymotrypsin family,
other 'serine' proteinases are trypsin, elastase, subtilisin,
and thrombin. Although each enzyme has widely differing
specificities, it has been suspected for some time that all
react by similar mechanisms. This proposal is strengthened
by noting that each enzyme also has a histidine residue as a
component in the active site region and that the sequences
of amino acid residues around the serine are very similar
to one another (5).

The regulation and control of these families are also
similar. Each family has an inactive precursor or 'zymogen',
and one or more active forms. The enzyme is synthesized and
stored in the body in the zymogen form, becoming activated
when needed by the enzymatic cleavage of specific peptide
bonds or fragments of the polypeptide chain. Such a control
mechanism is needed since the enzymes, especially trypsin
and Chymotrypsin, are autocatalytic in nature.

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Of all biological protein systems, that of the chymo-
trypsin family is probably the most extensively studied system.
This wide popularity is due to the relative ease of isolation
and purification of the enzyme from bovine pancreas. First
isolated by Northrup, Kunitz, and Herriott (6), the family
shows a fairly high degree of stability under reasonable con-
ditions and can be stored almost indefinitely as a freeze-
dried powder without loss of enzymatic activity. The
Chymotrypsin family in bovine pancreas consists of two
inactive precursors, chymotrypsinogen A and B, and four very
similar, but characteristically different, active species of
each of the zymogens: alpha, gamma, delta, and pi chymo-
trypsin A and B. The zymogens appear to be 'isozymes' with
an overall amino acid similarity of 86% (5). The active
species are formed by enzymatic cleavage of one or two peptide
bonds or fragments from chymotrypsinogen followed by specific,
but poorly understood, conformational changes (7). The pi
and delta forms result from the cleavage of a single peptide
bond and fragment respectively (SER 14 - ARG 15), while the
alpha and gamma forms are the end products of activation of
chymotrypsinogen, with an additional dipeptide fragment
removed (THR 147 - ASN 148). Chemically, a and Y Chymotrypsin
are identical: Operationally, the existence of the two forms
resides in the formation of different crystalline modifica—
tions. The a species forms monoclinic crystals at acid pH

(about pH 4) and the Y species forms tetragonal crystals at

__.._—_..__-—- “-——"——

neutral or alkaline pH. The physical differences between

these two species are not definitively known (7).

2. Physical and Chemical Properties of Chymotrypsin

Alpha Chymotrypsin A (hereafter denoted as a-CHT) is a
proteolytic enzyme of chemical composition C1113N3000349H1752812
consisting of 241 amino acids arranged in three peptide chains
A, B, C of 13, 131, and 97 residues, respectively. The
resultant molecular weight is approximately 25,300 amu. The
245 amino acid sequence of chymotrypsinogen was determined by
Hartley (8,9) and Meloun, et a1. (10) in 1964 and revised in
a minor way in 1969 (ll). a-CHT is formed from chymotrypsino—
gen by the removal of the dipeptides between leucine 13 and
isoleucine 16 and between tyrosine 146 and alanine 149. The
resultant three peptide chains are held together by five
disulfide bridges between cysteine residues.

A study of the thermodynamic relationships between the
conformation of o-CHT and its physiological function has been
made by Lumry and others (12,13). By monitoring enthalpy and
entropy changes as a function of temperature, pH, salt, and
other experimental variables, a number of different folded
states are found to exist. The magnitude of the changes in
conformation can not be explicitly determined by solution
studies because the thermodynamic variables observed are
extremely sensitive to changes in the water structure sur-
rounding the enzyme. Nonetheless, the existence of some

subtle conformation changes during catalysis has been prOposed

but the nature of these changes is currently not well under-
stood (12).

The reactivity of a-CHT resides in its ability to cleave
amide bonds adjacent to the carboxyl group of large, bulky
aromatic amino acids. The enzyme catalyzed reaction is many
orders of magnitude faster than comparable non-enzymatic
digestion of proteins. The prOposed mechanism for hydrolysis
involves a three-step reaction

P1

E+S-—9E84EP2—+E+P2 (1)
where E is the enzyme and S is the substrate, ES is an
enzyme-substrate complex, EP2 is a second enzyme complex
involving the active site serine and P1 and P2 are products
of the enzyme catalyzed reaction. Recent studies have shown
that the hydrolysis reaction is much more complex than that
represented in equation 1 with a number of ionization and
conformational equilibria also involved (14). The activity
of the enzyme displays a bell—shaped pH rate profile with a
maximum at pH~7.2, and is interpreted as evidence that two
ionizing groups control the activity (15).

Unlike other enzymes, the specificity requirements of
a-CHT are not very stringent and its affinity for binding
substrates and inhibitors is not as large as might be
expected, especially when compared to the binding affinities
of other enzymes toward their inhibitors. In this sense,

a-CHT can be considered to be a 'poor' enzyme. Although

amino acid residues with aromatic rings are most frequently

the natural substrates, there is no absolute requirement for
aromatic rings, as shown by the fact that saturated hydro-
carbon rings such as cyclohexane bind as effectively as
aromatic groups. The reasons for the relatively poor speci-
ficity are not completely understood; however, the products
of chymotryptic catalysis are also inhibitors of a—CHT. Thus,
to be reasonably efficient but yet not become completely
inhibited by its products, the specificities for inhibitors
and substrates should be relatively lower than in other
enzymatic systems, where substrates and inhibitors often

have completely different chemical identities.

3. Structural Studies

Crystallographic studies have been reported for nearly
all members of the chymotrypsin family. The parent struc-
ture, chymotrypsinogen, has been determined by Kraut and
coworkers to 2.5A resolution (16). The crystal structure of
the alpha form was determined by Blow and coworkers at
Cambridge (17,18) and the structure of the gamma form has
been reported by Davies and coworkers at the National Insti-
tutes of Health (19). The n and d-CHT forms are crystalline
forms isomorphous with the Y species.

Our crystallographic studies on a-CHT were begun in
1962. In the intervening years, Blow and coworkers have
reported a number of papers relating to the structure of a-CHT.
Since there were substantial differences in the mode of oper-

ation and in the philOSOphy of approach between the work of

Blow and ours, this encouraged us to continue with the ini-
tial work. In addition to collection of X-ray intensity

data by diffractometer methods as contrasted to photographic
means, there were also a number of other experimental and
procedural differences between the two efforts which have
enabled us to elucidate some structural prOperties heretofore
unreported that are important in the functionality of the
enzyme. For instance, a systematic study of classes of
inhibitors and substrate-like molecules has been made in

order to ascertain modes of binding and resultant conforma-
tional changes in the enzyme. A detailed study concerning

the physical and chemical properties of the enzyme molecules
was undertaken also. It is known that environmental forces
play a substantial role in governing the activity of the
enzyme--such as changes in pH, ionic strength, buffer com—
ponents, and temperature. Other physical probes and tech-
niques for the study of protein structure and conformation

can only Show that changes are occurring; x-ray crystallo-
graphic studies that have been undertaken can show definitively
what conformational changes occur when an environmental change
is made. These results lead to new insights concerning con-
formational prOperties of enzymes and other biological
macromolecules. In addition to the above, the occurrence

of two molecules of a-CHT in the crystallographic asymmetric
unit gives a unique opportunity to study conformational
variability of a protein molecule in a systematic and detailed

manner .

 

ff.

rf

II . EXPERIMENTAL

1. Methods

Three times crystallized, salt free, lyophilized c—CHT
was obtained from Worthington Biochemical Corporation and
used without further purification or characterization.
Crystals suitable for x-ray work were obtained in a manner
similar to that described by Northrup, et a1. (6). The a—CHT
was dissolved in distilled water adjusted to pH 3.5 with
HZSO4 to produce a protein concentration of approximately
22 milligrams of protein per millimeter of solution. A 75%
saturated ammonium sulfate solution was added to the protein
solution until a slight cloudiness appeared. A small amount
of water at pH 3.5 was added to just clear the turbidity.
The final protein concentration is approximately 6 mg per
ml with a pH of 4.2. Large, diamond shaped crystals up to
1.5 mm by 0.75 mm by 0.5 mm grow from this solution in three
to four weeks at room temperature. The solution from which
the crystals are grown is replaced by a 75% (NH4)ZSO4 solu-
tion at pH 4.2 and the tubes of crystals stored in a water
bath maintained at 13°C. Crystals stored in this manner
retain their excellent quality for many years.

Crystals of a-CHT belong to the monoclinic space group
P2l as determined from precession photographs. The cell
dimensions for the native enzyme determined from diffractometer

9

measuremen
g=4$
B = 101
Four enzym
two molecu
tains appr
characteri
Crystals,
Since the
Material,
in X-ray n1
All c
use of a P
trOlled b3
Storage ar
raw inteng
With an A
further p

crys
glass Cal)

King (20)

aPPI‘OX 1m a

the "all
6"er 0f n

a drop Of

‘!

fie capi]

s“.
fiblliZE

10

measurements are

a = 49.24 (7) A, b 67.20 (10) A, g = 65.94 (9)Aw

8 = 101.79 (8)°
Four enzyme molecules are contained in the unit cell, with
two molecules in each asymmetric unit. The unit cell con-
tains approximately 60% protein and 40% mother liquor. A
characteristic of a-CHT is the tendency to form twinned
crystals, with the twinning occurring along the c* direction.
Since the crystals contained varying amounts of twinned
material, only crystals with small twins were generally used
in x-ray measurements.

All of the X-ray work to be described was performed by
use of a Picker Four Circle Automatic Diffractometer con-
trolled by a Digital Equipment Corp. PDP-8 computer. Program
storage and retrieval utilized a 32K DEC Disk File, with the
raw intensity measurements being recorded on magnetic tape
with an Ampex TMZ Tape Transport unit. The tapes were then
further processed at the main computer center.

Crystals selected for x-ray examination were mounted in
glass capillaries in a manner similar to that described by
King (20). Generally, the crystals used in this work were
approximately 1.0 x 0.6 x 0.4 mm. The crystals are held to
the wall of the capillary by the surface tension of a small
drOp of mother liquor. To prevent the crystals from drying,
a drop of liquid was placed above and below the crystal before
the capillaries were sealed. The crystals were allowed to

stabilize positionally for at least twenty-four hours in the

 

capilla
allowed
of the .
of the I
Prc
the recc
principa
diffract
I10 indic
changes
ticular
Scans an
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work was

2. Three

Thre
CEdure (
h
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55c.»

11

capillary before use. The distinct morphology of the crystals
allowed the unique 2 axis to be mounted along the long axis

of the capillary which becomes coincident with the phi axis

of the diffractometer when the crystal is aligned.

Preliminary X-ray examination of every crystal included
the recording of the diffraction pattern along the three
principal axes of the crystal. A visual comparison of the
diffraction pattern to that of the native crystals sufficed
to indicate the quality of the crystal and whether significant
changes in the diffraction pattern had occurred for the par-
ticular derivative of interest. Typical native crystal axial
scans and the corresponding scans for a heavy atom derivative
are shown in Figure 1.

Heavy atom derivatives were usually prepared by adding
a solution of the heavy atom reagent to a tube of crystals
so as to obtain a molar ratio of heavy atom reagent to protein
of approximately 10 or 20 to 1. The crystals were soaked with
the heavy atom derivative for at least two weeks, and in the
case of the uranyl derivative, one month, before any X—ray

work was attempted.

2. Three Dimensional Data Collection

Three dimensional intensity data were collected on the
Picker diffractometer using a limited omega step scan pro-
cedure (21) with automatic realignment capabilities and
balanced filters for the measurement of intensities and

background (22). The normal mode of data collection consisted

 

 

FiQUre

12

~—-— RV-O-SH-l. 80"

LII

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

M J

F-TT-S-l, Native 350

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Axial diffraction patterns for native a-CHT and
for the Pt heavy atom derivative.

 

of recor
omega ac
largest
sity. T
HE step
filter.
rical pe.
would OCI
The data
tional 51
reflectic
Anot
is the at
Crystal 1
automatic
BeCaQSe 1
my a d]
the Capi:
orientat;
EonitOri]
togmall

lectiOn.

13

of recording the diffracted intensity at each of six steps in
omega across the center (m = 0°) of the reflection. The four
largest steps of the six were summed to form the raw inten-
sity. The background for each reflection was measured at
the step with the largest intensity with a balanced cobalt
filter. Occasionally, due to crystal movement or asymmet-
rical peak shapes, the maximum intensity of the reflection
would occur at an omega step on either end of the scan range.
The data collection program would then make one or two addi-
tional steps to find the peak maximum. Approximately 2000
reflections per day could be measured in this fashion.
Another powerful feature of the data collection program
is the ability to detect crystal movement and to 'realign' the
crystal by obtaining a new least squares orientation matrix
automatically before continuing with data collection.
Because the crystals are mounted in glass capillaries with
only a drop of moisture adhering the crystal to the wall of
the capillary, most crystals tend to undergo some small
orientational motions. These movements are detected by
monitoring the intensities of three reflections sensitive
to small angular displacements every hour or so of data col—
lection. When the intensity of any monitor drops below a
certain specified fraction of its initial intensity, the auto-
matic realignment program takes control. A certain number
of reflections, well distributed in reciprocal space, are
then centered in a procedure which locates the best angular

coordinates of each reflection with respect to the

 

diff:
descx
to ti
auton

resun

are h

ment

the q

quart

14

diffractometer angles w, x, and 26. A new matrix which
describes the orientation of the crystal lattice with respect
to the diffractometer axial system is then obtained by an
automatic least squares procedure (23) before data collection
resumes.

Generally, intensity measurements made in this manner
are highly reliable and Show good reproducibility. Assess-

ment of the quality of the data can be obtained by evaluating

the quantity R, as defined as
N N
R=£I IFiI - <F>l/ z <|F|>
i

where F1 and <F> are the observed structure amplitudes of
the reflections being compared and their average value,
respectively. This factor is nearly always less than

R W 0.025 for redundant data from the same crystal or even
different crystals of the same derivative (22,24).

There are approximately 10,500 reflections within one
quarter of the limiting sphere defined to 2.8A resolution.
Since the intensities of protein crystals tend to decay when
exposed to the X-ray beam for extended periods of time, the
2.8A set of reflections for each heavy atom derivative and
the native enzyme was generally collected from five crystals.
The intensity data were measured in shells of 26 and a suit-
able number of overlap reflections between each shell were
measured to check scaling procedures. Excluding unobserved

and low order reflections with d-spacings greater than 15A,

 

9044 ref

angle ca;

3. Heav:

Poss
basis of
IhOi) pha
some 12 c'
best five
After aPF
ties, ap;
were Obta
ence Patt

culdtiol'l
[A

Where IF
F
derivativ
C‘YStal f
BeCa
Space 9rd
individua
IMSEM
origin f0

functions

15

9044 reflections (or 86% of the total) were used for phase

angle calculations and electron density maps.

3. Heavy Atom Derivatives and Phase Angle Calculations
Possible heavy atom derivatives were screened on the
basis of two dimensional projection work by using the centric
(hOL) phases determined for the native enzyme (25). Although

some 12 different heavy atom derivatives were prepared, the
best five derivatives were chosen for three dimensional work.
After apprOpriate data reduction and scaling of the intensi-
ties, approximate heavy atom positions for each derivative
were obtained from the individual three dimensional differ-
ence Patterson maps (26). The coefficients used in the cal-
culation of the Patterson maps were of the form

2

IA (hkinz = IF (2)

p+HI - IFPI

where IF is the structure amplitude of the heavy atom

|
p+H
derivative and IFPI is the corresponding value of the native
crystal for the reflection (hkl).

Because the origin along the y axis is arbitrary in
space group P21, the heavy atom positions obtained from the
individual Patterson maps did not necessarily correspond to
the same origin. To relate all derivatives to the same
origin for phase calculations (27), two types of correlation
functions between the various heavy atom derivatives were

computed. In the first case, Patterson-like coefficients of

the type (28)

 

were u
deriva'
gives 1
as pos:
tives a
as the

0f the

(

WhiCh 9
two dif
of all

the Sam

16

2
IF I - IF I (3)
p+H1 p+H2

I and IF
p+H1 p+H2

derivatives 1 and 2 used in the correlation. This function

were used, where IF I refer to the heavy atom
gives the self-vectors of the heavy atoms in each derivative
as positive peaks and the mixed cross-vectors between deriva-
tives as negative peaks. This function was not as useful
as the second correlation map computed by using coefficients
of the type (29,30)

( IF

IF I ) ( le+H | - IF I ) <4)

I
p+H1 p 2 p

which gives only the vectors between the heavy atoms in the

two different derivatives. Subsequently, the y coordinates

of all heavy atom derivatives were adjusted to relate to

the same origin, arbitrarily assigned to be the major uranyl
position in the uranium derivative (Table I).

Phase angles for all observed reflections were computed
using a program originally written by Hilary Muirhead, et a1.
(31) and modified to our particular needs. Alternate cycles
of phase angle calculation and least squares refinement of
the heavy atom parameters (32) were performed, first using
low resolution data (6A and 3.5A) and later using all the
data to 2.8A resolution. The analysis of each heavy atom
derivative yields a probability distribution for the value
of the phase of the native enzyme. Two types of phases can
be obtained from the product of these individual probability

distributions, the most probable phase angle, “max and the

Ih‘nnllv Cali-.I . ‘I-QI-h.

 

 

17

 

0.00 000.0 000.0- 000.0 00 I o
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0.0 0.0 0.0 0.0 0.0 0.0 000.0 000.0- 000.0 00 I 0
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.cosc0ucoo

.H 00noh

 

best p2
gravitj

86
maps we

tional

and

19

best phase angle, abest' which is the phase of the center of
gravity of the probability distribution.
Between cycles, three dimensional difference Fourier

maps were computed for each derivative to locate any addi-

tional minor heavy atom sites utilizing coefficients

( IFpI ) exp [1ap] (S)

lFP+HI
and

exp (iapH) - IFPI exp (iop) ] - fH (6)

I IFp+H|
where up is the current estimate of the most probable native

phase, a is a similar quantity for the heavy atom and fH is

pH
the calculated heavy atom structure factor. Maps prepared

by using coefficients of the first type have peaks corres-
ponding to all atoms included in the calculation and other
minor occupancies. The second map is a true difference map
with peaks only for substitutions not included in the calcu-
lations.

The relative scale factors, occupancies, positional
parameters and thermal parameters were refined for each heavy
atom position. Upon inclusion of the high resolution data,
the thermal parameters of the major occupancy sites were
allowed to vary in an anisotropic manner. When convergence
was nearly completed in the least squares refinement process,
a sixth derivative with measurements of anomalous dispersion
was included in order to fix the absolute configuration of
the molecule (33). A summary of the heavy atom derivatives

used in the phase determination process and other pertinent

20

refinement parameters are given in Table I and Figures 2 and
3. The final figure of merit, the mean of the cosine of the
error in the phase angle, for all observed reflections to
2.8A resolution is 0.76.

The six heavy atom derivatives used to determine the
phases of the native enzyme by the multiple isomorphous
replacement method were obtained from the following chemical
reagents: KthI4 to form the derivatives denoted as Pt and
Pt(+), phenyl mercuric acetate to make the Hg derivative,
U02(N03)2 to make the U and U(+) derivatives and KAuI4 with
uranyl nitrate to form the mixed derivative U+Au.

Except for the uranyl derivatives, the various heavy
atom sites and occupancies in the derivatives were very sensi-
tive to the concentration of the reagent used in the soaking
process, and to a somewhat lesser degree, to the time the
crystals were allowed to soak in the presence of the heavy
metal solution. By use of the reagent KthI4, for example,
it was possible to prepare two different, isomorphous deriva-
tives Pt and Pt(+). For these two derivatives, Table I shows
that enhanced binding occurs at some of the sites, especially
1 and 1', in the Pt(+) derivative. In contrast to the two
or three weeks usually needed to prepare a derivative, the
U(+) derivative was formed after prolonged soaking (over one
year at high concentrations of reagent). To minimize the
effects of the concentration dependence, crystals from the
same tube were used in the collection of the intensity data

whenever possible.

21

 

 

 

60*-
.\(3.44
5.0 I-
3

mm III 4.0 I"
rmeml

&O-

20- 25$

W" 12.5 as as 5.0 42 36 3- 23 25A

1 I I I' E i 'I ' 1 l .I
.02 .04 .06 .08 .10 .l 2 .14 .lb .18 . 20
sin I
A

Figure 2. Heavy atom refinement of a-CHT. Curve shows ratio
of rms calculated heavy atom contribution, f, to
rms lack of closure error as a function of sin B/A.
The overall ratio for each derivative is given in
parentheses. The larger the ratio, the better the
phase determining power of the derivative. Only
for the last point of the U derivative are the
calculated errors larger than the heavy atom con—
tributions.

.P- Pt(+): 0- Pt; A - Hg; 0- U; D- U+Au;
O- U(+) .

22

0.9 —
03 -
<‘>
0.7 -

05*-

 

3
l

 

1 l 1 I

In 40‘ .06 00 J0 J2 J4 J6 J0

flng
A

125 as 03 50 u 10 3.1 u 211 A
l I l l l
20

Figure 3. Figure of merit distribution curve. Plot of the
average value of the figure of merit, m, as a
function of sin e/A.

 

Thr
the heav
two-fold
of a-CHT
differenc
metry op.
of sites
the two-:
tion. 11
have any
in 62, 5
show par1
Hg derivi
derivatix
metry is
occupanc;
on the p‘

from the

23

Through this work, it has been observed that some of
the heavy atom derivatives did not show the non-crystallographic
two-fold symmetry known to be present between the two molecules

of a-CHT in the asymmetric unit. Column 6 contains the

2
differences in positions in angstroms after a two-fold sym-
metry operation is applied to one of the members of each pair
of sites grouped by brackets in Table I. The derivation of
the two-fold axis and its equation are given in a later sec-

tion. In each derivative, there are some sites which do not

have any two-fold related positions, denoted by the dash

++
2 )
show particularly poor two-fold symmetry, while those in the

in 62. The heavy atom sites in the uranyl derivative (00

Hg derivative are particularly good. For all heavy atom
derivatives, the standard deviation from exact two-fold sym-
metry is approximately 0.5A. The standard errors of the
occupancies (Z) are approximately 2 electrons and.the errors
on the positional coordinates are 0.1 to 0.2A, as calculated

from the phase refinement.

4. Electron Density Map Calculation

The 2.8A resolution three dimensional electron density
map of native a-CHT was computed using the 8900 reflections
with figures of merit greater than 0.30 as determined during
phase calculations. The 'best' Fourier synthesis (34) was
calculated in sections perpendicular to the x axis in grid
divisions of a/76, b/106 and c/l30. The 'best' Fourier uses

coefficients of the type

where I
angle.

undistc
were d:
0.5 eA'
plots 0
and non
stacked
Purpose
the mod
the twc
using K

REpetit

24

m IF I exp (id

p ) (7)

best

where m is the figure of merit and a is the best phase

best
angle. The resultant electron density was printed in an
undistorted way on a scale of 2cm per Angstrom. Contours
were drawn by a Calcomp plotter with the lowest contour at
0.5 eA-3 and with subsequent intervals of 0.25 eA-B. The
plots were traced onto transparent cellulose acetate sheets
and mounted on plexiglass sheets. The sheets were then
stacked into a "Richards' Optical Box" for model building
purposes (35). A half-silvered mirror arrangement allowed
the model to be fitted to the electron density. Models of
the two a-CHT molecules in the asymmetric unit were built

using Kendrew atomic skeletal units produced by the Cambridge

Repetition Engineers, Ltd.

III. DESCRIPTION OF THE MOLECULE

1. Folding of the Polypeptide Chain

Molecules of a-CHT are packed four per unit cell, two
per asymmetric unit. The two molecules in the asymmetric
unit come into close physical contact and are related to each
other by a non-crystallographic two-fold axis running nearly
parallel to the crystallographic a* direction. Schwert (36)
and Schwert and Kaufman (37) observed that the sedimentation
coefficient of a-CHT was concentration and pH dependent,
suggestive of the formation of a monomer-dimer equilibrium.
Molecular weight measurements verified the existence of the
dimeric unit. Because of the intimate association in solu-
tion and in the crystal, the two molecules in the asymmetric
unit can be pr0per1y termed a dimer. Many close contacts
obscure the molecular boundaries in the dimer interfacial
region. However, the boundaries elsewhere are generally well
defined, with solvent regions of low electron density between
adjacent dimeric molecules. Some small peaks present in
these channels and near the protein molecule suggest the
presence of ordered solvent molecules.

The dimeric unit formed from two molecules of a-CHT
is roughly an elongated ellipsoid with slight cusps in the
surface of the ellipsoid near the interface region. The
ellipsoid is approximately 45A by 33A by 67A with the axes of

25

 

the ell
tions a
molecul
individ
Spheric.
and an
the molt
present:
cut out
rough c1
resides.
The
Clearly
Whole mc
anchored
are “not
are at t

26

the ellipsoid corresponding to the crystallographic direc-
tions 3*, b and E, Figure 4 shows the shape of the dimeric
molecule obtained by considering the Kendrew model. The
individual molecules in the asymmetric unit are more nearly
spherical except along the side where the dimer is formed
and an indentation is made in the sphere. While the rest of
the molecular surface is relatively smooth, the contact region
presents a rough appearing surface, as though a swath was
cut out of the otherwise spherical molecule. It is in this
rough crevice-like region that the active site of the enzyme
resides.

The path of the polypeptide chain of the enzyme can be
clearly discerned from the electron density for nearly the
whole molecule. Only the last four residues of the A chain,
anchored with a disulfide bridge between residues 1 and 122,
are unobserved in the electron density map. These residues
are at the top of the molecule and make no specific inter—
actions to stabilize them with the rest of the molecule or
with neighboring molecules. In many places, bumps which
occur on the backbone density correspond to the carbonyl
group of the amino acid residues. In those regions where
the polypeptide chains are in close proximity, there is some
overlap of the electron densities from the chains, due either
to hydrogen bonding between the chains or possibly to a lack
of resolution.

Nearly all of the side chains are resolved from the main

chain or form obvious protuberances from the main chain

27

 

 

Figure 4. Approximate shape of 'dimeric' molecule of a-CHT.
A local two-fold axis between the two molecules
is indicated by the O symbol. Interactions
between the two molecules represented by arrows.
The asterisk shows approximate location of the
active site.

 

elect
ially
define
found
electx
the nc
dues a
the sj
molecu
less a
side c
into t
I
the re
Chymot
to whe
the Sn
a“ ind
densit
densit
engerV
althot
Others
is SUH

OCcurI

h
‘1

\SO'

28

electron density. The large bulky aromatic groups, espec-
ially tryptophanes and phenylalanines, are very clearly
defined. The histidine and tyrosine side chains, frequently
found near the surface of the molecule, have characteristic
electron densities but are somewhat less well defined than
the nonpolar aromatic groups. In many cases, branched resi-
dues appear as regions of forked electron density. Although
the side chain definition is best within the interior of the
molecule, the exterior side chain positions are only somewhat
less well defined. Surprisingly, nearly all of the lysine
side chains are observed, even though most of them extend
into the solvent.

The appendix gives the amino acid sequence of a-CHT,
the residues being numbered according to the sequence of
chymotrypsinogen. Each side chain is classified according
to whether it is in the interior of the molecule (I), on
the surface (S) or exposed to the solvent (E). In addition,
an indication of the clarity of each side chain in the electron
density map is given. This table is based on the electron
density of one of the molecules in the asymmetric unit; the
observations are generally applicable to the other molecule,
although the resolution of some side chains is better and for
others is worse in the other molecule of the dimer. The appendix
is summarized in Table II. The distribution of the commonly
occurring amino acids with side chains in a-CHT is given.
Also, the distribution of each residue in the three environ-

ments specified above and the clarity of each residue is

29

Table II. Summary of Side Chain Environments and Clarity in
the Electron Density of a-CHT.

 

Environment* C1arity#
Residue Total I S B Good Fair Poor
ALA 22 7 10 5 10 10 2
ARG 3 0 1 2 2 0
ASN 13 0 8 5 5 1
ASP 9 2 3 4 3 2
CYS 10 2 8 0 10 O 0
GLN 10 l 4 5 3 2
GLU 5 O l 4 0 2
HIS 2 0 2 0 O 0
ILE 10 4 6 O S 0
LEU 19 13 3 3 11 6 2
LYS 14 0 3 ll 7 7 0
MET 1 0 l 1 0
PHE 1 4 3 0
PRO 3 6 O l O
SER 27 5 7 15 17 5 5
THR 22 4 11 7 12 7 3
TR? 8 4 4 0 2 0
TYR 4 1 2 1 2 0
VAL 22 13 8 2 12 3

 

*Environment: I - Interior, not readily accessible to solvent;
S - On surface of molecule, partially accessible
to solvent;
E - Extending into solvent.

1 Clarity: Good - Complete and definitive electron density
. for side chain.
Fair - Reasonable density for side chain,
configuration less well defined.
Poor - Little or no electron density for side
chain.

30

tabulated. It can be seen that only a few residues are
poorly described in the electron density map. Overall, about
85-90% of the amino acids have fair to good side chain densi-
ties.

While the backbone of the protein was generally clearly
defined, the model building process depended heavily upon the
knowledge of the chemical sequence for the identification of
the side chains. The five disulfide bridges are readily
apparent as cylinders of relatively high electron density
(approximately 2-3 electrons/A3) forming a bridge between
two adjacent polypeptide chains. .

Three regions of the polypeptide chain posed substantial
problems when attempting to fit the sequence to the electron
density: residues 10-13 had no apparent electron density,
implying no preferred conformation; residues 67-70 on the
edge of the molecule in a solvent region: and residues 34—37
were difficult to fit to the observed density in one mole-
cule. In the latter case, the conformation was determined
as implied from the electron density of the second molecule
for this region.

The path of the polypeptide chain through the globular
shaped molecule consists of stretches of fully extended pep-
tide chain punctuated with abrupt changes in the direction
of the peptide chain, with the chain doubling back upon
itself. The path of the chain appears to be random, produc-

ing the resultant globular shaped molecule. Approximately

31

one half of the molecule is formed by the folding of the B
chain upon itself with the other half formed by the C chain.
The two chains occasionally intermingle for short stretches
of the polypeptide chain. Short regions of the classical
types of protein chain conformation--he1ix and pleated sheet--
are observed in the molecule. There are only two short
regions of the peptide chain which form helices; (1) a well
defined a-helix of approximately 3 turns involving the
residues LEU 234 to ASN 245 at the carboxyl terminal of the C
chain on the surface of the molecule away from the dimer
interface and (2) residues SER 164 to GLY 173, also on the
surface of the molecule, forming a short, distorted helix of
about 1.5 turns in length. This helix appears to be a hybrid
of an a-helix and a 310 helix. Several regions of extended
peptide chain are found on the surface and through the inte-
rior of the molecule. Three of the longest extended chain
regions consist of residues 80 to 93, 100 to 115 and 154 to
164. In several places, the extended chains are arranged in
an antiparallel fashion with interchain hydrogen bonding to
form short pieces of pleated sheet. One such pleated sheet
showing a slightly curved surface is formed by the chain seg-

ments 85 to 95, 105 to 110 and 52 to 55.

2. B-Bends
The peptide chain can change direction abruptly by
forming 'B-bends' as described by Venkatachalam (38). The

B-bends, similar to 310 helical bends, are produced by a

32

tight hydrogen bonding system between the carbonyl oxygen

of the first residue and the amide nitrogen atom of the fourth
residue in a tetrapeptide sequence. In a-CHT, there are
twelve well defined B-bends which are listed in Table III,
with several more peptide segments in a configuration similar
to a B-bend but not quite so well defined. In agreement with
the calculations of Venkatachalam, those bends where the amide
plane between the second and third residues is flipped 180°
(type II) with the carbonyl oxygen pointing towards the side
chains have a glycine as the third residue. Similar peptide
conformations have been observed in nearly all proteins whose
structure have been reported, the most recent being carbonic
anhydrase (39) and ferricytochrome C (1). It has been sug-
gested that B-bends might function as 'directing' sections

to provide prOper orientation of distant segments of the
polypeptide chain during the initial protein folding process
(40). Figures 5a and 5b show stereoscopically the two types

of bends which occur in a-CHT.

3. The Active Site Region

The active site of the enzyme is found along the side of
the molecule which forms the interface between the two mole-
cules. It is situated at the bottom of a 'jaw like' depres-
sion formed by a junction of the two halves of the folded
molecule just off the surface of the molecule. The site is
partially screened on two sides from the solvent by several

amino acid residues arranged as a partial cover to the

33

Table III. Best Defined B—Bends in a-CHT.

 

Residues Sequence Type*
23-26 VAL-PRO-GLY-SER II
27-30 TRP-PRO-TRP-GLN I
35-38 ASP-LYS-THR-GLY I
48-51 ILE-GLU-ASN-TRP I
56-59 ALA-HIS-CYS-GLY I

115-118 SER-GLN-THR-VAL I

177-180 LYS-ASP-ALA-MET I

191-194 CYS-MET-GLY-ASP II

194—197 ASP-SER-GLY-GLY II

203-206 LYS-ASN-GLY-ALA II

217-220 SER-SER-THR-CYS I

221-224 SER-THR-SER-THR I

 

*See reference 38.

depression. On the tOp are the residues 190 to 193 and 215
to 219, while the bottom is closed by residues 94 to 97.

The amino acid residues which form the active site components
have been unambiguously identified by chemical and crystal—
lographic studies on a-CHT and other homologous enzymes such
as trypsin (41) and elastase (42).

Reaction of a-CHT with di-isopropylfluorophosphate was
found to selectively phosPhorylate a specific serine residue,
SER 195, completely blocking all activity of the enzyme (43).
Also, the sulphonyl fluoride compounds of Fahreney and Gold
(44) were found to form ester linkages with SER 195.

Studies of the catalysis of p-nitrophenyl—acetate (45)

indicated that the reaction proceeds through an acyl-serine

34

 

Figure 5a. Stereo drawing of a Type I B-Bend formed by
residues SER 115 to VAL 118, Main chain carbonyl
oxygen atoms indicated by lines through the atoms.
Hydrogen bonds formed between carbonyl oxygen and
amide hydrogen indicated by dotted lines. The
carbonyl group of GLN 116 at the top of the loOp
is directed out of the page, while the adjacent
side chains are directed into the page.

PRO
'00

   
   

OLY l9!

 

OLY I00

Figure 5b. Stereo drawing of a Type II B-Bend formed by
residues ASP 194 to GLY 197. SER 195 carbonyl
directed in same direction as side chains.

35

intermediate. Direct evidence for the involvement of a his-
tidine residue (HIS 57) was provided by Schoellman and Shaw
(46) with the specific alkylation of the imidazole ring of
the histidine with loss of enzyme activity by tosylamino-Z-
phenylethylchloromethylketone.

In addition to the primary requirement of serine and
histidine for activity, there are several secondary require-
ments also. It has been shown that the activity is dependent
upon the protonation of the N-terminal amino group of iso-
leucine 16 (14,47). The carboxylate ion of ASP 194, directly
adjacent to SER 195, forms an internal salt bridge with the
former. This salt bridge in some way controls the conforma-
tion of the active site and its maintenance is crucial to
the activity of the enzyme. One of the changes which has
been observed when chymotrypsinogen, the inactive precursor,
is converted to the active a-CHT by tryptic cleavage of the
bond adjacent to ILE 16, is the repositioning of the side
chain of ASP 194. In chymotrypsinogen, this side chain is
said to be hydrogen bonding to HIS 40 and, upon conversion
to the active form, the side chain swings up to form a buried
salt bridge with the newly created N-terminal amino group of
ILE 16 (16). The formation of this bridge may be a control-
ling step that converts the inactive form to the active form
of the enzyme.

The enzyme becomes inactive at pH values higher than
8 to 9. Various experiments have shown that ILE 16 becomes

deprotonated at these pH values leading, presumably, to the

36

disruption of the stabilizing ion pair and to changes of

the active site conformation (49). By analyzing stOp flow
kinetic data of the binding of proflavin to a-CHT as a func-
tion of pH, Fersht was able to estimate the energy of the
salt bridge between ASP 194 and ILE 16 to be approximately
2.9 kcal/mole (50).

The other residue in the active site region which has
been implicated as necessary for activity is ASP 102 (11).
ASP 102 is located somewhat removed from the surface of the
enzyme and relatively inaccessible to solvent molecules larger
than water. Depending upon its ionization state, and that of
HIS 57, the carboxylate ion can either form a salt bridge
to HIS 57 or hydrogen bond with it. Alternatively, the
carboxylate ion can also form hydrogen bonds to the hydroxyl
group of SER 214 or the main chain amide of HIS 57. Chemical
attempts to block the carboxyl groups of a-CHT with glycine
methyl ester result in 15 of the 17 carboxyls of a-CHT being
modified. One of the unmodified carboxyl groups has been
shown to be ASP 194, involved in the ion pair with ILE 16
(51). The other, unmodified, carboxyl group is presumably
ASP 102 since it is located in such a position that it is
relatively inaccessible to the reagent.

A schematic drawing of the approximate orientations of
the catalytically important groups around the active site and
a possible hydrogen bonding network linking the various resi-
dues together is shown in Figure 6. The conformation of the

HIS 57 and SER 195 side chains is such that, directionally,

Figure 6.

37

1L6 I0

   

ASP

   

I04

‘8' l0!

Schematic drawing of the important residues around
the active site. The side chain atoms are con-
nected with solid bonds, main chain atoms by Open
bonds. Bridging $04= molecule shown between SER
195 and TYR' 146. Possible hydrogen bonds indi-
cated by dashed lines. Ox gen atoms -@; nitrogen
atoms -0; carbon atoms - .

38

only a 'poor' hydrogen bond can be formed between histidine
and serine. As shown, the carboxylate group of ASP 102 can

form hydrogen bonds with the N nitrogen atom of HIS 57,

62
the main chain amide of HIS 57, or to the hydrogen atom of

the hydroxyl of SER 214. A sulfate molecule is also found

in a position to hydrogen bond to SER 195. While these resi-
dues are directly involved with the chemistry of activity,
there are undoubtedly many other residues in this region
which are important in the alignment and binding of substrates
to be hydrolyzed. Excellent review articles concerning the

activity and specificity of a-CHT have been written by Hess

(14,52).

4. Non-Polar Cavity

A very interesting structural feature became apparent
when the model of the enzyme was built. Some seven different
chain segments from the sequence come together to form the
surface of a large, somewhat irregular, sphere with the side
chains from 18 non-polar residues extending toward the center
of the sphere. The interior of this sphere is an empty
cavity approximately 8 to 9A in diameter. All of the resi-
dues which participate in the formation of this 'oily' cavity
have non-polar side chains consisting mainly of isoleucine,
leucine, and valine groups. Three tryptophan residues are
also involved, one on either end of the cavity, which seem
to act as lids to the cavity, and one near the center, some-

what removed from the cavity surface. One side of the cavity

39

is bounded by the inner side of the a-helix at the carboxyl
terminal of the C chain involving residues 231 to 242. Other
chain segments which are involved are residues 47 to 53, 103
to 105, 121 to 124, and 209 to 212.

The non-polar cavity can be schematically represented
as four shells or levels of residues as shown in Figure 7.
Levels 1 and 4 form lids at either end of the cavity with
levels 2 and 3 around the center of the cavity. The arrange—
ment of residue names in each level approximates the side
chain positions around the cavity. The cross-hatched region
is empty space. Possible entrances to the cavity by solvent
molecules are blocked by THR 241 and VAL 238.

The occurrence of such a large structural feature at some
distance from the active site raises some interesting ques—
tions concerning the physical forces which interact during
the folding of large biological molecules. Normally, the
business end or active site of a molecule is located at a
specific region on the enzyme molecule, involving the inter-
action of, usually, only a few critical amino acid residues.
It is believed that the function of the rest of the molecule
with its pleated sheets, helix, 310 bends and other protein
substructures is to hold the critical residues in the correct
conformation for activity. In addition, these protein sub-
structures may play an important role in directing the fold-
ing of the chain into its proper conformation of lowest free

energy.

m... IE.
‘W

ILEIOS
LEU |\05
LEVEL 2 LEU 234 TRP’BI
LEI; 242
AL 235 MAL 230
LEVELS

LEVEL4

 

Figure 7. Schematic representation of non—polar cavity.
Level 1 is near the surface of the protein and
level 4 is in the interior. The cross-hatched
regions correspond to the vacant space of the
cavity.

 

The
the conf
the stru
evolutio
residues
polar ca
bin and
invarian
cavity,
servativ
shows th
with son
fied as 1
Cf these
Way thi
while th
ShrfaCe J
PariSQns
becauSe J
served d
an impori

protein I

a. ArOmJ
Ano
an °bVi01

lit to 11

 

41

The importance of this non-polar cavity to stabilize
the conformation of a-CHT can be inferred by a comparison of
the structures of other proteolytic enzymes thought to be
evolutionarily related. A comparison of the amino acid
residues of a-CHT involved in the formation of the non-
polar cavity with those of various species of trypsin, throm-
bin and elastase (45) in Table IV shows a high degree of
invariance in the amino acid residues concerned with the
cavity. The Substitutions which do occur are usually con-
servative, e.g., an isoleucine for leucine, etc. LEU 234
shows the greatest variability among the various species
with some side chain substitutions which are usually classi-
fied as polar groups (lysine and tyrosine). The side chains
of these polar residues could be positioned, however, in such
a way that the polar regions are directed toward the solvent,
while the non-polar regions of the side chain lay along the
surface of the cavity. It becomes obvious from these com-
parisons that the hydrophobic stabilization which arises
because of this arrangement is important enough to be con-
served through evolutionary processes, and as such, must be
an important factor in the tertiary conformation of these

protein molecules.

5. Aromatic Clusters
Another protein substructure that manifests itself in
an obvious manner is the clustering of aromatic groups simi-

lar to that observed in other protein structures (53,1). One

42

 

 

 

 

 

Table INK Comparison of Residues Around Non-Polar Cavity in
a-CHT to Several Homologous Enzymes.
Level a-CHT BT DFT Th BCA BCB PE
TRP 237 TRP -- TRP TRP TRP TRP
l
THR 241 THR THR VAL THR THR VAL
TRP 51 TRP -- TRP TRP TRP TRP
ILE 103 ILE ILE ILE ILE ILE ILE
LEU 105 LEU LEU LEU LEU LEU LEU
2 LEU 234 TYR -- LYS LEU LEU TYR
VAL 235 VAL -- LEU VAL MET ILE
VAL 238 ILE ILE ILE VAL VAL ILE
LEU 242 ILE ILE ILE LEU LEU. ILE
ILE 47 ILE ILE ILE ILE ILE ILE
VAL 53 VAL -- -- VAL VAL MET
LEU 123 LEU LEU -- LEU LEU LEU
3
PRO 124 PRO PRO -- PRO PRO PRO
ILE 212 ILE ILE ILE ILE ILE VAL
VAL 231 VAL -- VAL VAL VAL VAL
TRP 29 TYR -- TRP TRP TRP SER
VAL 121 ILE ILE -- VAL VAL GLY
4
VAL 200 VAL VAL VAL VAL VAL HIS
LEU 209 LEU -- GLN LEU LEU VAL
BT - Bovine Trypsin BCA - Bovine CHT-A

DFT - Dogfish Trypsin

Th

BCB - Bovine CHT—B

- Thrombin PE - Pig Elastase

43

of the most pronounced groupings is that formed by the three
tryptophan residues 27, 29 and 207. These residues form a
nest-like arrangement with the proline residues 4, 8, and
28, acting as additional boundaries to the nest. Figure 8
shows this tryptophan arrangement.

A number of crystallographic experiments have been per—
formed to study the interaction of aromatic substrate-like
molecules and competitive inhibitors with a-CHT. It has been
observed that, in addition to binding of these small mole-
cules in the active site region, many of these aromatic mole-
cules also are localized at another specific site on the
enzyme (54). This site has been identified to be near this
tryptophan nest.

Other aromatic clusters are found with HIS 40 and TRP 141.

Their side chains are nearly parallel to one another with a
distance of approximately 5A between the two rings. Some-
what more distant, but nearly caplanar to the above residues
is PHE 41. Near one end of the non-polar cavity, TRP 51,
PHE 89 and TRP 215 are found clustered together near the
active site region. As observed with the non-polar cavity,
nearly all of these aromatic residues are invariant through-
out the related proteolytic enzymes compared, with any sub-
stitutions being, again, conservative in nature. A comparison
of these residues for several related enzymes is given in
Table V.

If one considers the dimeric molecule of a-CHT, then

two other aromatic clusters are formed. Near the active site,

44

 

 

 

Figure 8. Stereoscopic drawing of the tryptophan aromatic
cluster.

45

the aromatic side chains of HIS 57 from one molecule and

TYR 146 from the other come into close proximity with their
rings lying nearly in the same plane. Also in the interface
region, there are two phenylalanine 39 residues, one from

each molecule, which must interact with each other.

Table V. Comparison of Residues in Aromatic Clusters.*

 

 

Cluster a-CHT BT DFT Th BCA BCB PE
PRO 28 PRO -- PRO PRO PRO PRO
TRP 27 VAL -- SER TRP TRP TRP
1
TRP 29 TYR -- TRP TRP TRP SER
TRP 207 ' -- -- TRP TRP TRP TYR
HIS 4O HIS HIS LEU HIS HIS HIS
2 PHE 41 PHE PHE GLN PHE PHE PHE

TRP 141 TRP TRP TRP TRP TRP TRP

 

TRP 51 TRP TRP TRP TRP TRP TRP

3 PHE 89 ILE ILE ILE PHE PHE VAL
TRP 237 TRP TRP TRP TRP TRP TRP

 

TYR 171 ALA ALA TYR TYR TYR TYR
4 TRP 172 TYR TYR PHE TRP TRP TRP

TRP 215 TRP -- TRP TRP TRP PHE

 

*Enzyme abbreviations same as in Table IV.

IV. MOLECULAR INTERACTIONS

1. The Structure of the a-CHT Dimer

The crystallographic asymmetric unit of a-CHT contains
two enzyme molecules in intimate association with one another.
The two molecules have been found to be related to one another
by an approximate two-fold rotation axis which is nearly par-
allel to the a* direction. The location of this non-
crystallographic symmetry element with respect to the crystal—
lographic axes was obtained by a comparison of the electron
densities of the two molecules.

The transformation between the two molecules can be

expressed by

d
X

_. J
2 = C x + d (9)

l
where each point x1 in one molecule is related to the point
x2 in the second molecule by a rotation matrix C and a trans-

lation vector 3. Values of C and 8 are sought which minimize
)3 (p (x) - p (x ))z (10)
l l 2 2
where pl and 02 are the electron density values at the points
x1 and x2, respectively. The rotation matrix C contains coef-
ficients which are functions of the three independent Eulerian
angles 0, w, 6 and d is a column vector containing the trans-

lation components d1, d2, and d3 with respect to a set of

reference axes. Given an initial set of these variables,

46

47

refined values can be obtained from the least squares mini-
mization of equation 10.

Initial estimates for these variables were obtained by
taking portions of the electron density corresponding to a
peptide chain from one molecule, superimposing it on the
second molecule and recording the necessary transformation
parameters. Because the rotation axis is perpendicular to
the yz planes of the electron density, the two molecules can
be compared section by section in this direction, and approxi-
mate transformation parameters for each section obtained.

Refinement of the transformation parameters between the
two molecules of a-CHT in the asymmetric unit was performed
using a computer program written by Dr. Carl Morimoto. The
program was based on one used by Cohen, et a1. (56), which
was used in a low resolution comparison of the electron
densities of y-CHT and a—CHT. The 2.8A resolution electron
density map was stored in planes perpendicular to the a*
direction. A molecular boundary was defined so that during
the refinement, only those portions of the electron density
which corresponded to the two molecules of interest were used.
This molecular boundary excluded intermolecular solvent regions
and portions of the electron density associated with neighbor-
ing molecules. The final, refined values of the transforma-
tion parameters were

0 = 0.0°
W
0 = 0.0°

179.60° i 0.02°

48

d1 = -0.207 t 0.009A
d2 = 39.87 i 0.01A (11)
d3 = 49.39 t 0.01A

The position of a local two-fold axis can be derived from
these parameters. For a given x coordinate, the two-fold

axis passes through the points y and 2 at

2 0.1565 ($0.0009) x + 0.3683 (£0.0019) (12)

and y 0.2957 (10.0018)

where x, y and z are expressed in terms of fractional units

of the crystallographic unit cell. Because of the choice of
reference axes for the transformation parameters, it was not
possible to refine the angles 0 and 0 meaningfully. The
estimated standard deviations were obtained from the variance/
covariance matrix of the last cycle of refinement. The val—

ues for d1, d2, and d are in Angstrom units with respect

3
to the a*, b, and c directions of the crystal.

The assessment of the quality of agreement between the
two molecules is expressed in terms of the root mean square

difference between them.
I z (01(X) — 02(x))2 11/2 (13)

The rms difference using 102,468 electron density grid points
within the molecular boundary was 0.27 electrons per cubic
Angstromflas compared to an rms electron density of 0.407 eA-3.
The rms difference is due to structural differences between

the two molecules and to errors in the electron density map.

It was observed that there were a substantial number of

49

regions within the molecular boundary for which the differ-
ence in electron densities was significantly greater than the
rms difference. Another least squares calculation was per-
formed excluding regions where the differences in electron
density between the two molecules ( Ipl(x) - 02(x)| ) was
greater than 0.70 eA-B. About 13% of the electron density
points within one molecule were removed by this criterion.
The calculated position of the two-fold axis remained essen~
tially the same, but the rms discrepancy factor decreased to
0.24 eA-3 while the rms electron density remained nearly the
same (0.40 eA-3).

As a measure of the agreement between the electron densi-
ties of the two molecules, a correlation coefficient was com-
puted during the least squares refinement procedure to indicate
the progress of the refinement. The correlation coefficient
c12 was computed as

.2 (01-31) (02-52)
c = (14)
12 l 2 (pl-E51)2 2 (02-32)2 11/2

 

where p1 and p2 are the electron densities of molecule 1 and

2, and 02 are the average electron densities, with the

D1
summations being taken over all points within the molecular
boundaries. For the case where the electron densities are
exactly the same, c12 would have a value of 1.0; no correlation
between the electron densities would be represented by a value

of 0.0. By its definition, this coefficient is independent

of any relative scale factor between the electron densities

50

being compared (56). The correlation coefficient between the
two molecules using the initial transformation parameters

was 0.772. The coefficient increased to 0.82 after one cycle
of refinement; this indicated an obvious improvement in the
initial parameters. Further refinement did not improve this
value. The final values for the various agreement factors
are summarized in Table VI. In addition, values for the
various indicators are given for those calculations which were
made where electron density points less than 0.25 eA"3 were
excluded. The indicators in this case behaved in a manner
comparable to those obtained from calculations made using

all points within the molecular volume.

Table VI. Transformation Refinement Summary.

 

 

 

 

 

Calculation* Arms (eA-B) firms (eA-3) c12 NBEESESOf
1. 0.269 0.407 0.782 102,468
2. '0.244 0.400 0.814 88,734
3. 0.300 0.499 0.820 63,780
4. 0.268 0.495 0.854 53,681

 

*l. Calculations made including all points within the defined

molecular boundary.

2. Same as 1 except excluding regions containing differences
in electron density >0.7 eA‘3.

3. Same as 1 but excluding electron density points

<0.25 eA'3.

4. Same as 3 but excluding regions containing differences

greater than 0.7 eA'3.

51

The presence of one local two-fold axis along with the
crystallographic two-fold screw axis generates another local
two-fold axis, related to the first by a translation of one-
half the unit cell along the z direction. Because these
symmetry elements are not crystallographic elements, the two
molecules related by the axis are not required to be identi-
cal. _As will be seen later, the two independent molecules
show many chemical and physical differences in reactivity,
structure, sulfate binding and other effects. In addition,
the environments of the two-fold axes are different.

Figures 9a and 9b show schematically the packing of the
molecules projected on the yz and xz planes, respectively.
Molecules I and 1' form the asymmetric unit and are related
by the local two-fold axis. Molecules II and II‘l belong to
the second asymmetric unit and are related by the crystallo-
graphic two-fold screw axis to molecules I and 1‘, respectively.
The two dyad axes, A and B are indicated. It can be seen that
dyad A, defined by equation 12, runs through the middle of the
'dimeric' molecule of a-CHT. The individual molecules, when
related by a two-fold axis, are complementary in shape and
fit together rather intimately. Between the close contacts
at the top and bottom of the two monomeric molecules (Fig-
ure 9b), a large, partially Open region is formed. The
region is generally accessible to solvent and to smaller sub-
strate and inhibitor molecules. It is generally hydrophilic
in nature, with SER 195, ASP 35, GLN 73, HIS 57, TRY 146 and

other polar residues within the region. In addition, several

52

 

 

 

 

 

 

 

 

 

A
*
,»~ ‘ ~ (0 )
" I
C) lflz 2
A I ,
t , .
I
*l’ ’k I (b)
I ‘x I
V
, .
. l
x— - ACTIVE smas y_ - mm moms sns A.B - mos

Figure 9. Schematic packing diagrams of a-CHT in the unit
cell; (a) unit cell contents projected onto yz
plane; (b) projection onto xz plane.

53

water molecules and sulfate ions are bound in the cavity

between the two molecules.

2. Local Two-Fold Interactions

The molecular interactions which occur along this inter-
face region around dyad A can be classified into two specific
types: electrostatic and non-electrostatic, the latter includ-
ing the effects of hydrophobic interactions and hydrogen bond-
ing. The electrostatic interactions consist mainly of 'salt'
bridges formed by a negatively charged group on one molecule
and a positively charged group on the other. The position
and orientation of the groups allow the opposing charges to
be effectively neutralized, and results in a significant net
attractive force between the ionic groups. It is expected
that these salt bridge interactions would be dependent upon
the pH of the surrounding medium; as the various residues
involved become protonated or vice versa, the ionic inter-
actions would be disrupted.

The various interactions which occur between the two
molecules are diagrammed in Figure 10. The residue names are
in an approximately correct position with respect to the two-
fold axis position indicated by the horizontal line. The
residue names with the prime symbol (Molecule 1' in Figure 9a)
belong to the second molecule in the asymmetric unit and all
references to the second molecule hereafter will be denoted
by this symbolism. The interactions which are purely electro—

static are those involving the carboxylate group of ASP' 64

54

n
L.

.auwumHo “0m nonuocn oco Eoum ovomHmmfio czoom mum coauom

Iumucfi Hmooumflomu on» com mma .mmm pom ova Hocmom cwm3umo sofiuom
Iuoucw one .Ammsmc mooflmou omaflumv .H can Acmefiumssv H oaoomaoe
coozumo mum mcofiuomumuoH .s pomp mmouom mQOwuomumucw unasooaOEuousH .oa musmwm

co a9 - a! .54
2.. .EC - 5 2:

ma .mum- t~ .mmm

 

m! .35.... mm. .mmm
«2 Lu: _ _ «Puza a
ma cum -tu cum ~25: .vom .¢om an E: to
8. cum 9.. .22...

hr. .9: Iw¢_ KP». nn. amd Ion .m>I_

2.. <2 - «o 12

55

and the amino terminal of ALA 149, the e-amino group of

LYS' 36 and the carboxylate of ASP 153, and the carboxyl
terminal of TYR 146 and the imidazolium ion of HIS' 57,
although the spatial orientation of the groups involved in
the latter interaction is not the most favorable for complete
neutralization of the respective charges. Each of the corre-
sponding two-fold related electrostatic interactions is well
defined also, except the LYS 36 - ASP' 153 interaction.

In addition to these direct ionic interactions, sulfate
ions partiCipate in forming a hydrogen bonding bridge between
the phenolic hydroxyl of TYR' 146 and the hydroxyl group of
SER 195. The location of the sulfate ion between these two
groups, discussed in more detail in a subsequent section, is
also close to the HIS 57 side chain and might play a role in
neutralizing the charge which would be present on the imidazole
ring of HIS 57 at pH 4, the pH at which the crystals were
grown. Another 804= ion is found in the vicinity of LYS' 36,
ALA 149 and ASP' 64, participating in a complicated charge
interaction and hydrogen bonding scheme.

The non-electrostatic interactions are located close to
the dyad axis and involve the same residues from both mole-
cules. In the example of the PHE 39 self-interaction, the
two-fold axis passes nearly through the center of the ring of
one of the residues. If two exact monomers of a-CHT were
brought together to form a dimer, the phenylalanine rings of
the two PHE 39 residues would overlap one another. To avert

this impossibility, the rotations of one or both of the

56

phenyl rings occur so that the final position of each ring
is related to the other by a 180° rotation around the Ca-CB
bond, to locate the second ring at a sufficient distance from
the first to preclude appreciable steric hindrance. This
necessary rotation does not appear to affect the neighbor-

ing residues in the chain significantly, although HIS' 40 is
in a slightly different conformation than its counterpart in
the first molecule. Figure 11a shows the final arrangement

of the two phenyl rings.

By comparing the electron densities around the active
site, some substantial differences were observed. The larg-
est differences occurred around the interacting methionine 192
residues (Figure 12). If the sulfur atoms showed exact two-
fold symmetry, an extremely close S-S contact distance of
approximately 3A would be formed. To relieve this close
interaction, one methionine residue is translated approxi-
mately 1.5A with respect to the other along the a* direction.

A similar rearrangement in the two molecules is observed
around the residues GLY 216 - SER 217 - SER 218. The chain
segments from the two molecules corresponding to these resi-
dues would approach too close to one another if exact two-
fold symmetry were displayed. As a result, one chain becomes
displaced in a perpendicular direction from the two-fold axis.
This arrangement is shown in Figure 11b with the residues from
the second molecule significantly displaced from the two-fold

axis.

57

 

"II II.
( .

  

OLY ll.

 

)7ng

tuu’ an

Figure 11. Local two-fold interactions between the two
molecules. (a) Stereo drawing showing final
arrangement of PHE 39 and PHE' 39. (b) Section
of peptide chain near two-fold axis, 0 . The
lower peptide chain is obviously displaced from

the two-fold axis and assumes a slightly different
conformation.

Figure 12.

Electron density of the active site region of one
molecule. Contours at 0.25 eA‘3 beginning at
0.50 eA‘3. Difference electron density between
the two molecules shown in white; difference
contours at 0.15 eA‘3 beginning at 0.6 eA‘3.
Solid white contours represent excess density

in the molecule shown while broken contours
represent excess density in the other molecule.

58

 

59

It is interesting to note that some of the interactions
which are electrostatic in nature show, in general, precise
local two-fold symmetry, while all of those involving non-
ionic interactions do not show the same precise symmetry.

It can be presumed from an energetic vieWpoint that it is the
electrostatic interactions which are the main stabilizing
forces for the_formation of the dimer. Obviously, the forces
required for changes in the chain conformation are smaller
than those involved in the electrostatic interactions. The
conformational changes necessary to accommodate formation of
the dimer are relatively minor in scope but point out the
readiness with which protein conformations can change when
necessary. Similar rearrangements of several residues around
a local two-fold axis have been observed in the structure of
rhombohedral 2 Zinc Insulin (57). The crystallographic asym-
metric unit of insulin consists of two molecules related by

a local two-fold axis perpendicular to a three-fold axis.
Some significant deviations from the local two-fold symmetry
were noted, including the reorientation of a phenyl ring as
observed here. Various other deviations were observed which
occurred at substantial distances from the rotation axis.

The other two-fold axis (B) relates dimeric molecules
to other dimeric molecules in the crystal lattice. There are
no specific, well-defined interactions across this two—fold
axis as there are across dyad A. Only one interaction, pos-

sibly due to hydrogen bonding between the side chain amides

60

of GLN 204 and GLN' 240 and the corresponding two-fold inter-
action, could exist. The environment around this axis con-

sists almost entirely of solvent.

3. Location of Bound Sulfate Ions

After a plausible model of a-CHT had been constructed,
several regions of electron density for which no protein resi-
dues were assigned remained. It has been common practice in
protein crystallography to label unidentified peaks as water
molecules or, generally, solvent molecules with no attempt to
differentiate between water and salt ions. Since many pro-
tein crystals are grown from relatively high ammonium sulfate
solutions, it seems reasonable that some of the solvent peaks
could well be localized sulfate ions.

To definitively identify those peaks which were due to
sulfate ions, an attempt was made to exchange selenate ions
for sulfate ions. Ammonium selenate and ammonium sulfate
have similar molecular prOperties, including sizes, ionization
constants and solubilities in water. Since the selenate ion
has 18 more electrons than the sulfate ion, the difference in
X-ray scattering power of the selenate and sulfate ions should
be observable in the diffraction pattern of the protein if
displacement occurs.

Ammonium selenate, purchased from K and K Laboratories,
was purified by repeated crystallization from water before
use. The crystal soaking solution of 75% saturated (NH4)ZSO4

was slowly exchanged with an equivalent (NH4)ZSeO4 solution.

61

Initially, the crystals were observed to crack, but upon sit-
ting for several days, the cracks had disappeared and the
crystals appeared to 'heal'. No apparent changes in the X—ray
diffracting quality of the crystals as compared to that of

the native crystals were observed. Three dimensional 2.8A

resolution data were first collected on crystals with a low

4

reduction and scaling in the usual manner, a three dimensional

SeO4= / SO molar ratio (approximately 5 to 1). After data

difference map was computed. The map contained only two pre-
dominate peaks which superimposed upon electron density regions
thought to be due to solvent in the interface between the two
independent molecules. The two peaks were of the same height
(0.5 eA-3) and were related by the two-fold rotation axis.

The position coincided with the bridging sulfate ion between
the phenolic hydroxyl of TYR' 146 and SER 195, near HIS 57.

Since this was the only dominate position substituted by SeO4 ,

and since the crystals cracked when the 804 was being replaced

4
ion is an important factor in the formation of the crystal

by SeO ions, it can be concluded that this bridging sulfate
lattice and the stability of the dimeric molecule.

The soaking solution ratio was then further increased
until the solution was nearly completely (NH4)ZSeO4. This was
effectively achieved by removing the crystals and placing them
in 75% SeO4= solution. The crystals floated in this solution,
since the crystal density was now less than the solvent den-

sity. (The density of the crystal is about 1.24 g/ml and

that of the selenate about 1.47 g/ml.) Substantial changes

62

again occurred in the diffraction pattern in comparison to
that obtained with the 5 to l selenate/sulfate molar ratio
crystals.

Another set of three dimensional data was collected and
processed. The difference map revealed six additional pairs
of sites which corresponded to suspected sulfate positions in
the two molecules, at somewhat lower occupancies (No.22 eA-3)
than the initial substitution site. One additional set of
data was collected on crystals grown entirely from ammonium
selenate solutions. No additional sulfate positions were
located and the occupancies were approximately the same as
those obtained from the former experiment. This suggests
that all 804= ions present are exchangeable. A summary of the
sulfate positions and their interactions with the enzyme mole-
cule is given in Table VII (58). Some of this work was car-

ried out in conjunction with Dr. L. H. Wright of this

laboratory.

4. Differences Between the Two Molecules

The fact that the two molecules in the asymmetric unit
are not identical in structure has been indicated in a number
of ways. The first suggestion of the non—equivalence of the
two molecules was in their differing reactivity to the heavy
atom reagents. In the event that the chemical reactivities
were the same, each member of the heavy atom pairs related
by the two—fold axis (equation 12), should have the same posi-

tion and occupancy. For the major occupancy sites of the

Table VII.

 

Bound Sulfate Positions.

 

SO4 Peak Heights (eA-B) Protein Contacts
1 0.57 Bridge between SER 195
l' 0.50 and TYR' 146
g. 0'28 ASN 95, LYS 177
3 0.24
3, 0.27 Near THR 224
4 0.23 Adjacent to guanidinium
4' 0.17 group of ARG 154
5 0.23 Near NH + of ALA 149
5' 0.16 chain terminus
g, g:%: ASN 245 side chain
7 0.18
7, 0.13 Near ASN 236

 

Pt(+), Pt and Hg derivatives (Table I) reasonable agreement

is observed for the two-fold related positions. Consideration
of the minor occupancy sites shows a large discrepancy between
occupancies, if indeed there is a two—fold related substitu—
tion at all.

The heavy atom derivatives based on the U02++ ion pre-
sented a different picture. Here, the major substitution site
shows only a minor site 2A from the two-fold equivalent site.
It will be shown in the next section why the large discrepancy
in occupancies and positions exists. It is apparent from the

heavy atom occupancies that there are some substantial dif-

ferences between the two molecules.

64

The results from the selenate exchange work also indi-
cated a similar difference in structure. In general, the
occupancies between related sulfate ions were fairly consis-
tent, but the positions varied substantially more from exact
two-fold symmetry than did the heavy atom positions (58). In
addition, SO4= ion #2 in Table VII had no two—fold counterpart.
The electron density of the native crystals grown from sul-
fate solutions indicated that excess electron density
(~0.96 eA-3 and 0.71 eA_3) was positioned in these regions,
but only one position became substituted with selenate ion,
even in crystals grown in selenate solutions. From this, it
can be surmised that one position contains an ordered sulfate
molecule while the other position probably contains an ordered
water molecule. The residues involved are on the surface of
the molecule, and the electron density map suggests that the
local environments around each position are different. The
side chain of LYS 177 which interacts with the S04= in one
molecule shows a different configuration in the molecule which
does not bind sulfate ion.

The transformation parameters between the two molecules
obtained by the least squares refinement were used to compute
a difference map between the electron densities of the two
molecules in the asymmetric unit. Positive and negative con~
tours were drawn when the difference electron density exceeded
$0.6 eA’3, about 2.5 times the standard error of the differ-
ence density. Successive contours were drawn at intervals of

0.15 eA-3. Figure 12 shows the electron density in the region

65

around the active site with the difference electron density
superimposed in white. Solid contours represent excess den-
sity in the molecule shown, while broken contours represent
excess density in the second molecule. Solid and broken con-
tours adjacent to one another represent a shift or motion of
the particular residue in one molecule with respect to the
other. Isolated solid or broken contours may represent a sol—
vent molecule in one enzyme molecule which is not in the other.
Alternatively, single solid or broken contours superimposed
upon the electron density peaks could represent the possibility
that one enzyme is more ordered than the other. The net effect
of this would be increased electron density peak heights in one
molecule compared to those of the second.

The largest difference observed around the active site is
the shift of MET 192 which was described earlier. The posi-
tive and negative contours are clearly visible in Figure 12.

A positive and negative region on the alpha carbon of SER 195
indicates a slightly different position for this atom in the
two molecules. Also, differences are noted in the position

of the HIS 57 main chain and in the main chain of ASP 102.

Of equal importance is the fact that the side chains of the
catalytically important side chains SER 195, HIS 57, and

ASP 102 possess no significant differences in orientation
between the two molecules. A priori, this observation would
suggest that the two molecules should have the same reactivity,
if reactivity is only a function of the proper orientation of

the important residues. However, close examination of the

66

individual electron densities shows small, and perhaps sig-
nificant differences. In addition, the active site region
of the second molecule contains more solvent molecules than
the first molecule.

Further evidence for differences in the active site
region comes from studies of the binding of inhibitors and
substrate analogues to crystals of a-CHT. In some cases, the
substitutions occurred to an equal extent in both active
sites, with identical changes in the native structure. How-
ever, derivatives formed with N-pipsyl-L-Phenylalanyl
chloromethyl ketone and N-formyl-L-Phenylalanine, among
others, showed substantial differences in occupancies and
modes of binding (59,25).

Recent crystallographic and chemical work with
y-chymotrypsin has elucidated the pr0posed mode of binding
of oligopeptide inhibitors to a-CHT (60,61). It has been
observed that a polypeptide inhibitor forms an extended,
antiparallel configuration with the main chain of residues
SER 214, TRP 215, and GLY 216. Presumably, these residues
are necessary for substrate alignment before hydrolysis of the
peptide bond near SER 195. As discussed earlier (see Figure
10 and associated text), these residues also show significant
deviations from two—fold symmetry. If the position of these
residues is critical, then the two molecules might be expected
to show differing behavior toward substrates and inhibitors

due to the two different active site conformations.

67

Another region of the electron density which shows both
large deviations from two-fold symmetry and also rather exact
two-fold symmetry in the same region is contained in the con-
formation of residues 165 to 174. Residues 165 - 170 are
involved in a distorted helix near the surface of the mole-
cule. Figure 13 shows the differences between the two mole-
cules in this region. Many of the differences involve the
main chain amides and carbonyl groups of the peptide linkages
and correspond to different orientations of the planar peptide
bonds. Also shown in the figure is the very obvious reorienta-
tion of the lysine side chain of residue 169. The solid con—
tours represent its position in the first molecule and the
broken contours represent its position in the second molecule.
The change in conformation can be obtained by an approximate
45° rotation around the C6 — Ce bond of the e-amino group of
lysine as shown by the arrow. Similar side chain reorienta-
tions are observed throughout the map for many residues, espec—
ially the side chains of lysine, glutamine and asparagine on
the surface of the molecule.

The structure determination of insulin showed that many
of the tyrosine residues had water molecules hydrogen bonded
to the phenolic hydroxyl group (57). A similar observation
was made in the structure of a—CHT, the results of which are
amplified by the difference map. The peak marked W in Fig~
ure 13 corresponds to a water molecule hydrogen bonded to
TYR 171. The large solid white contours indicate that there

is no water molecule on the tyrosine of the second molecule.

Figure 13. Electron density of the helical region near
MET 180. Same contours as in Figure 12.

 

68

 

69

The reason for this observed difference is not obvious struc-
turally but this example shows in a clear and direct manner
the variability of the tertiary structure of a-CHT. In con-
trast, the absence of any differences in the residues TRP 172
to THR 174 shows the high degree of two-fold correSpondence

which can be obtained.

5. Uranyl Binding Interface

In addition to the interactions which occur along the
local two-fold axes, there is another region where substantial
intermolecular interactions occur. This region is referred
to as the uranyl binding site, denoted g in Figure 9a. The
region is formed by the interaction of one molecule and the
two-fold screw axis equivalent of the second molecule in the

asymmetric unit, or between molecule I and molecule 11'.

+
2

carboxylate ions of GLU 21 in molecule I and the carboxylate

The uranyl ion (00 +) is found located between the two
ion of ASP 153 in molecule II'. It appears that the uranium
atom obtains an octahedral oxygen coordination sphere, with
the negative charges of the carboxyl groups being neutralized
by the U02++ ion. The uranyl ion then forms an electrostatic
interaction with the protein as compared to the covalent
interaction with the other heavy atom derivatives. (The
platinum and mercury derivatives generally form covalent com-
plexes with the sulfur containing amino acids, methionine and

cysteine.) This unusual binding arrangement for the uranyl

derivative is probably responsible for the low thermal

70

parameters and high occupancies observed for the uranium
derivative during the phase calculation and refinement process.
In the native structure, the above carboxyl groups might
be forming a very interesting complex. Kirkwood and Schumaker
(62) have shown that the electrostatic force caused by the
fluctuations of protons between similar ionizable groups on
the surface of a protein may result in a nonspecific attraction
between two identical molecules. Timasheff has subsequently
shown that with a certain geometrical configuration of iden—
tical ionizable groups, a specific attraction may be estab-
lished as a result of the proton fluctuations (63). The
largest effect will occur in the pH region of the pK of the
ionizable group. He suggested that in a dielectric medium
similar to that found on the surface of proteins, a net
attractive energy of one to two kilocalories per mole may be
available due to such an interaction. For neutral carboxyl
groups, a model which might represent this type of inter-

action is

while if one of the carboxyl groups is deprotonated the model

would be

71

where the lone hydrogen could be found between either pair of
Opposite oxygen atoms, or somewhere in between, with equal
probabilities. Calculations have shown that the attractive
interaction would be strongest at the pH corresponding to the
pK of the particular group, or the pH at which the carboxyl
group is protonated 50% of the time.

The pK's of the two carboxylic acid groups involved in
the interaction, aspartate and glutamate, have been reported
to be 4.1 and 4.5, respectively (64). The pH at which the
native a-CHT crystals are grown is approximately 4.2, inter-
mediate between the pK's of these groups. It is likely that
this interaction promotes or enhances the association of
asymmetric units during the crystallization process.

Supporting this possibility is the observation that best
growth of crystals of CHT occurs around pH 4 and that crystals
of a-CHT fail to grow, or grow very poorly, at pH values above
or below the range of hydrogen ion concentrations which cor-
respond to the pK's of these residues.

This intermolecular interface region is in striking
contrast to the dimeric interfacial region between the two
molecules I and I' in the asymmetric unit. In the latter, an
interaction between two groups on the two molecules, i.e.

TYR' 146 with HIS 57, was accompanied by a corresponding
reciprocal interaction between TYR 146 and HIS' 57. Such is
not the case here. The U02++ heavy atom binding studies showed
there was only one site which became appreciably occupied. To

produce this interaction there is an accommodation or

72

conformation change on the part of one or both Of the mole-
cules involved (different orientations of the side chains Of
ASP 153 and GLU 21). In addition, the location of the mole-
cules in the unit cell precludes the reciprocal interaction.
Since the proton fluctuation site is a very special site and
is formed only when the residues possess a certain geometri-
cal relationship, the possibility of a reciprocal site is
diminished. This fact can be seen in Figure 14. The alpha
carbon atoms of the peptide chain for molecule I are con-
nected with solid lines, while the equivalent atoms belonging
to molecule II‘ are connected with dotted lines. The side
chains of GLU 21 and ASP' 153 are shown, with the uranyl ion
binding at position 2. It can readily be seen that the recip—
rocal interaction, ASP 153 - GLU' 21 could not occur due to
the large distance (about 8A) between their respective side
chains. It should also be noted that the orientation of
ASP 153 is different in the two molecules. The different
orientation brings the carboxyl group into a position such
that it can form a salt linkage with LYS' 36 Of molecule 1'.
This is an interaction across the dimeric interface with no
two—fold equivalent interaction!

The electron density around the uranyl binding site has
a large number Of peaks, unaccounted for by the protein chain.
Due to the polar nature of the interacting site, it is sus-
pected that the extra peaks are due to localized, partially
ordered solvent molecules. It has been shown by the selenate

exchange work that there is only one sulfate ion in this

Figure 14.

 

73

THRISI

Detail of the peptide residues near the uranyl
binding site. Solid circles represent the
a-carbon positions of the peptide chains. Open
circles are oxygen atoms. The solid lines con-
nect residues of molecule I while the dashed
lines are for molecule 11' (see Figure 9a). The
UOZ++ site is designated 0.

74

region. The lone $04= ion is adjacent to the guanidinium
group of arginine 154. Since the guanidinium group carries
a positive charge at pH 4, an ion pair is probably formed with
the sulfate ion with charge neutralization. The remaining
peaks in this region are water molecules or NH4+ ions from the
ammonium sulfate solutions.

In general, the electron densities corresponding to the
various side chains in this region have approximately the
peak heights Observed for side chains protruding into the
solvent elsewhere. The peak heights for exterior side chains
range from 0.5 to 0.8 eA"3 compared to 0.75 to 2.0 eA-3 for
interior side chains. Before the amino acid sequence was
traced in the electron density, the large peak heights of the
two residues, ASP' 153 and GLU 21, was puzzling. The peaks
now assigned to the two carboxyl groups always had the largest
peak heights in the electron density maps, with heights
approaching 3 eA-3 in some Of the low resolution maps. This
relatively high density can be possibly explained in two ways:
(1) The proton-charge fluctuation interaction stabilizes these
residues to such an extent that their disorder is very small,
and hence, their peak heights high; or, less likely, (2) Errors
are induced in the phases by the uranium heavy atom substitu-
tion. It is known that the sites occupied by heavy atoms used
in the phase calculations are occasionally distorted in the
electron density map of the native structure. This distortion

is usually manifested as a residual negative or positive den-

sity at the heavy atom position. In the present case, the

75

electron density in the native map at the U0 ++ binding site

2
is nearly zero; likewise, other heavy atom binding sites in
the native electron density do not appear to have any partic-
ularly distorted features. While the effects of the second
alternative can not be ruled out entirely, the extra rigidity
induced by the proton fluctuation interaction might be suf-
ficient to explain the large peak heights of these two resi—
dues.

In contrast to the large peak heights Observed for
GLU 21 and ASP' 153, the local two-fold counterparts, GLU'

21 and Asp 153 have low peak heights (0.35 eA'3

3

for GLU' 21
and 0.6 eA- for ASP 153% There are no particular forces
stabilizing the side chain position of GLU' 21 except hydro-

gen bonding to the nearby ARG' 154 side chain.

V . STRUCTURE REFINEMENT

l. Refinement Of the Coordinates

The fitting of the amino acid sequence with models to
the observed electron density is subject to the interpreta-
tion of the builder and other difficulties. The model of
o-CHT was built over a two-month period with the aid of
Dr. William Brinigar Of Temple University and Dr. Carl
Morimoto of this laboratory. After the three dimensional
model had been completely assembled, adjustments were made
to eliminate close contacts and questionable arrangements
of side chains. The relative x, y, z coordinates of all
non-hydrogen atoms were measured. Two of the coordinates (y
and 2) were measured by sliding a 'grid' sheet, ruled at the
scale Of 2 cm to the Angstrom, into the Richard's box at the
approximate x value of the atom to be recorded. Using the
half-silvered mirror, the image of the atom was projected onto
the grid sheet and the y and z coordinates were recorded for
every atom. The relative x coordinates for each atom were
likewise measured after the model was rotated with reSpect
to the mirror by 90°. The reproducibility of the coordinate
measurements was approximately i'0.25A for each coordinate.
The relative coordinates were then transformed into a coordi—

nate system based on the crystal axes.

76

77

The refinement of the coordinates with respect to the
electron density map can be objectively performed by using a
series of elegant computer programs written by R. Diamond of
the Medical Research Council, Cambridge, England. The first
program adjusts the measured or guide coordinates to correspond
to the known geometries of amino acid residues and the second
program Optimizes the fit of the adjusted protein model to the
Observed electron density distribution by least squares pro-
cedures.

The mathematical model building procedure (65) builds a
polypeptide chain structure using the geometries Of individual
amino acids and peptide bonds as determined by X-ray crystal-
lographic studies on individual residues. By keeping inter-
atomic distances fixed, but allowing rotations about single
bonds, the model polypeptide chain is folded in such a manner
that the resulting chain and side chain positions approach the
measured coordinates as closely as possible. By minimizing
the sum of the squares of the distances between the guide and
the model coordinates, rotational and translational shifts
are computed and applied to the model coordinates. Because
second and higher order terms are ignored when the derivatives
for the least squares refinement are computed, several itera-
tions are necessary to attain convergence.

Given the sequence of amino acids in the chain and the
measured guide coordinates, the program starts at one end of
the chain and builds a model from the sequence. After a num—

ber of residues have been built, adjustment of the model

78

coordinates of the individual amino acids to the guide coordi-
nates begins at the first residue and moves sequentially along
the peptide chain. A 'molten zone' is defined which contains
a fixed number of residues. All of the residues are refined
in the zone subject to constraints imposed by the fixed resi-
dues outside the molten zone. The molten zone then moves by
one residue along the sequence, and the refinement of all resi-
dues currently in the zone performed. This sliding process
continues until the zone reaches the other end of the chain.
At this point, a list of the initial and refined coordinates
for the input sequence are listed, along with the various
interbond angles.

The model building procedure for the 241 amino acid resi-
dues in a—CHT was performed in several pieces due to storage
limitations of the computer program. For each piece, the
initial and refined coordinates were scrutinized for gross
errors in the initial coordinates due to measurement or record-
ing errors. In a couple of cases, the presence of a 'd' -
amino acid or the wrong stereochemistry for a threonine or
isoleucine side chain was detected. The necessary corrections
were made to the input coordinates and that section of resi—
dues refined again. No attempt was made to readjust the model
or the model coordinates based on the results of the building
procedure except when a substantial error was clearly indicated.
At the end of the refinement, the root mean square deviation
between the input coordinates and the computed coordinates,

using all atoms including the side chains, was 0.39 Angstrom

79

units. This number compares well with the value of 0.35A
reported by Diamond for the myoglobin structure, which he con-
sidered had extremely good guide coordinates (65). If one
assumes the rms error to be equally distributed between the
three coordinates x, y, z, the rms error for any one coordi-
nate is then approximately 0.21A. This is comparable to the
estimated reproducibility Of 0.25A for the raw coordinates.
During the model building process, the backbone angle T

(C -Ca-N) was allowed to vary, with any backbone dis-

carbonyl
tortions being accommodated by deviations of the angle from
the expected value of 109.6°for tetrahedral carbon atoms.

The average value of tau for the whole molecule was 112:10°.
This value agrees with the average tau of 112° Observed by
Lipscomb for carboxypeptidase (66). The difference from the
tetrahedral value was attributed to the packing of side chains

in a large globular molecule which may require the peptides to

be spread slightly at the alpha carbon atoms.

2. Real Space Refinement

The results from the model building program can then be
input to the real space refinement program Of Diamond (68)
along with the observed electron density map. The model is
then adjusted to fit the electron density by minimizing the

integral
f( - )2 dV (15)
p0 pm

where 00 and pm are the Observed and model electron densities,

respectively. The model electron density is calculated as

80

cub—h
G(ai, r-ri) +d (16)

p(r‘)=zxz.
m i 1

in which r is a general position vector,'?3 is the vector
position of the,;th atom and G(ai,'?#?i) is a normalized,

spherical Gaussian function representing the shape (ai) of an

atom's electron density distribution. The Z1 is the number of }

~. "'9"

electrons in the‘ith atom, K is a local scaling factor for
each residue and d is the correction for the level of the

background of the electron density. The refinement procedure

17‘ I--‘.'-‘.-m I -. .r.

Operates on a 'molten zone' of peptide residues, similar to
that in the model building routine. All the residues within
the zone are refined with respect to the Observedelectron
density. Then the zone slides along the peptide chain by one
residue and all of the residues currently in the zone are
refined again. This process continues until the whole chain
has been refined. The real space refinement program minimizes
equation 15 by allowing K, d, ai, Zi, translational and rota-
tional parameters to be varied. The refinement procedure
preserves bond lengths and maintains chain continuity with
the fixed parts of the molecule outside the molten zone.

At the end of each cycle of refinement of the whole mole-
cule, the calculated electron density or difference density
can be computed from the refined structure. In this way the
correctness of the refinement can be ascertained. In an‘
ideal situation, the difference map should be displayed on a
visual display device along with the computed model or the

observed electron density. One could then easily determine

81

those regions for which additional refinement is needed or
for which other forms of help are required. In a refined
structure, the difference map could also facilitate the loca—
tion of solvent molecules which are binding to the protein
molecule or could indicate the possibility Of alternate con-
formations for side chains. In practice, comparison of a ti
difference or calculated map to the Observed map or model in
a manual way is a very laborious task but should be done.

Because the real space refinement program is relatively

rl‘I-VuJ: Mu "" ‘ n.» '

new and detailed results from other protein structure refine-
ment attempts are not yet available, it was necessary to
experiment with the program to determine what types of refine—
ment were best and to become familiar with its Operation. A
short section Of the peptide chain from VAL 23 to GLN 30 was
chosen for experimentation. The electron density correspond-
ing to this chain was well resolved and included two tryptophan
residues, 27 and 29. Initially, the overall scale factor K and
the background parameter d were allowed to vary in addition to
translational and rotational parameters. The radius parameter
ai was fixed at 1.2A, a value used in preliminary work with

the 2.0A resolution myoglobin structure. The root mean square
movement of the atoms in the refinement was 0.44 on the first
cycle. Some larger shifts were Observed for PRO 28, espec-
ially in the z direction. Examination of the Observed electron
density with the model coordinates revealed that the proline
was placed on the edge of the electron density, and that the

observed shifts from the refinement were moving the model to

82

higher electron density. The calculated translational shifts
from a second cycle were in the same direction as in the first
cycle, with an rms movement for all atoms Of 0.27A. The rms
movement of the third cycle was 0.19A with only one of the 68
shifts greater than 0.5A. By the fourth cycle, only one shift
was greater than 0.3A with an overall rms movement of 0.12A.

Alternatively, a cycle of refinement was performed on
this short segment starting from the initial coordinates but
allowing the radius, ai, to vary along with K and d and the
translational-rotational parameters. The shifts Observed
were generally larger than the first cycle where ai was not
refined. The overall rms movement was 0.53A. A comparison
Of some of these shifts to the total movement recorded after
four cycles of the previous calculations indicated that with
ai active convergence could be Obtained with fewer cycles of
least squares. During the refinement of ai, most of the val-
ues increased from 1.2A. Since the resolution of the electron
density map determines the average radius of the Observed
peaks, a value of 1.6 was used to begin refinement Of the full
molecule.

Translational and rotational refinements were performed
after refinement of the overall scale factor and background
level. The atomic radius ai was also allowed to vary. The
root mean square movement recorded for all atoms during the
first cycle was 0.77A; for main chain atoms only (Ca,C,O, and N),
the moVement was 0.57A. Furthermore, it became apparent that

the starting coordinates had a systematic displacement with

u'-:—|._‘—. "rs-'7'“ ‘

83

respect to the electron density along the x direction. The
average shift recorded in the x direction during the first
cycle for all atoms was -0.33A while that for the y and 2
directions was 0.04 and -0.l3A, respectively.

A second cycle was completed starting with the coordi-
nates from the first cycle and allowing the same parameters ?W
to vary as in the first cycle. The rms movement recorded was
0.35A for all atoms and 0.27A for the main chain atoms only.

The movements on the second cycle were generally one half those

PL 3“”; .‘u u

of the first cycle. The average refined radius for all atoms
was 1.57A. The average background level computed for each
amino acid residue was 0.28 eA-3 with extremes from 0.12 to
0.44 eA-3. The background level usually varied smoothly from
residue to residue and did not appear to be a function of
whether the residue was located in the interior of the molecule
or on the exterior.

In general, the refinement program worked well and made
some substantial improvements in the starting coordinates.
In cases where the electron density was not fully resolved,
the side chains sometimes approached one another closer than
expected for vanmder Waals contacts. With several residues,
the carbonyl group of the peptide chain was flipped over when
the electron density was apparently stronger in the new orien-
tation. One such flip occurred for a carbonyl group in the
B-bend section of ILE 48 to TRP 51. The model was built with

the carbonyl of GLU 49 arranged in a type II B-bend (38) but

the bend was actually a type I bend. The refinement program

 

84

changed the orientation of the peptide bond by nearly 180°
on the first cycle into a type I configuration with a supple-
mentary adjustment on the second cycle.

An inherent problem with the real space refinement pro-
cedure is the complete neglect of non-bonded interactions due
to adjacent peptide chains or side groups. As a result, some
orientations Of the side chains and main chain peptide linkages
seem to be in an energetically unfavorable conformation, but
no attempt was made to take these effects into consideration
during the refinement. A program written by Levitt and Lifson
allows a refinement of protein conformation using a macromolec—
ular energy minimization procedure (69). Such a refinement
procedure, alternating with the real space refinement proce—
dure, would probably insure the best fit of the coordinates
of the protein molecule to the electron density. Such a pro—

cedure will be carried out in the future by others.

3. Comparison with the MRC Structure of "Average" a-CHT

A preliminary comparison of the present structure of
a—CHT with that of the average structure of Blow and cowork-
ers at the Medical Research Council has been made using our
partially refined coordinates and their refined coordinates
(70). The comparison has been made between our molecule I
and their averaged molecule, derived by averaging the electron
densities Of the two molecules in the asymmetric unit.

A number of significant differences exist between the two

structures although, in general, the conformations of the two

85

independently determined structures are essentially the same.
Considering the main chain coordinates, the rms deviation Of
atomic positions is approximately 1.3A overall. One region
Of the molecule which shows close correspondence between the
two determinations is the a-helix at the end of the C chain
where the rms deviation is approximately 0.8A. Other short
chain segments throughout the molecule show similarly good
correspondence, while other segments show significant dif-
ferences, the largest of which are in the vicinity of the
dimer interface region where it has already been noted that
significant variations exist between the two molecules in the
asymmetric unit. These regions include the residues GLY 216
to THR 219, TYR 146, ALA 149, ASP 35 and others with rms
deviations which range from 2.6 to 3.5A. The region GLU 70
to SER 77 shows the largest discrepancies between the two
structure determinations; however, these are probably due to
differing interpretations of a somewhat unresolved, diffuse
electron density region. Another distinction between the two
structures is the identification of sulfate ions bound on the
surface of the molecule and in the active site region.
Birktoff and Blow have indicated that only water molecules
are in the active site (70). However, our selenate experi-
ments provide irrevocable evidence for the presence Of a sul—
fate ion hydrogen bonding to the active site serine in the
crystal. Other discrepancies between the two structures prob—
ably arise because of slightly different crystallization con-

ditions. This is especially true in the active site region;

86

Blow and coworkers grew crystals of a-CHT from ammonium sul-
fate solutions containing small amounts Of dioxane, whereas
our crystals were grown without dioxane. There is another
complication in the direct comparison of the active sites Of
the two structures: their 'native' structure is that of the
tosyl-inhibited enzyme rather than that of the active form of
the enzyme used in our work. The fine differences in the
conformation Of the active site and elsewhere in the enzyme
molecule will be revealed upon a closer and more detailed

study Of the two structures.

PART II

THE STRUCTURE OF a-CHYMOTRYPSIN AT pH 6.7

VI . INTRODUCTION

1. Effect of pH on Protein Structure

The physical chemistry of protein molecules is an extremely
complicated subject and is poOrly understood. The structure,
stability, and activity of biological macromolecules are depen-
dent On a large number of variables and attempts to correlate
any one variable with a particular structural feature are almost
certainly doomed to failure except in the best Of cases. As
more and more research is conducted, it becomes obvious that
even the seemingly simplest physical concepts and systems are
understood poorly. One current area of intensive research
is the study of pure water and how its structure is modified
by the presence of folded protein molecules consisting of
hydrophobic cores and hydrophilic surfaces.

One of the experimental variables which plays an impor-
tant role in the prOper functioning of biological macromole-
cules is the hydrogen ion concentration in the surrounding
medium. Of the 20 amino acids which commonly make up protein
molecules, seven have side chains which can ionize in the pH
range of 2 to 13. In addition to the side chains, the amino
and carboxyl groups at either end of a protein chain are also
ionizable. The state Of ionization of these groups is a
strong function of the medium and is sensitive to pH, dielec—

tric constant, ionic strength, and temperature. Table VIII

87

88

lists some of the ionizable groups which commonly occur in

proteins and their respective pK values.

Table VIII. Ionizable Groups in Globular Proteins.*

 

Structure# ‘25
A. Acidic Residues
‘3
Terminal carboxyl N - C - COOH 3.6 - 3.8
H I
R
Asportic Acid R = -CH2COOH 4.1
Glutamic Acid R = -CH2CH2COOH 4.5
Tyrosine R = -CH2 -©- OH 9.5 - 10.5
B. Basic Residues
Histidine R = -CH2 -/C = C\ 6.3 _ 7.8
\ C
‘3
+
Terminal amino - E - C - NH3 7.5 - 8.5
O R
+
Lysine R = -(CH2)4 - NH3 9.6 - 10.4
/NH2
Arginine R = -(CH2)3 - NH — C >13
\ +
NH2

 

*Compiled from references 64 and 71.

#R indicates side chain.

89

A protein molecule is typically composed of a large num-
ber of ionizable groups, a portion of which are positively or
negatively charged at any given pH value. Proteins are often
characterized by their isoelectric point, at which the number
of positive charges equals the number of negative charges.
With so many titratable groups, the titration curves of pro-
tein molecules are extremely complex. Additionally, several
groups in a typical protein molecule will usually display
abnormal characteristics by titrating above or below the
expected values for the groups. This effect can arise from
interactions of various groups with others Of Opposite charge,
ionic groups buried or partially buried within the hydrOphobic
core of the protein, strong hydrogen bonding with ionizable
residues, or the particular electronic environment around a
certain residue. In some cases, there are groups in the pro—
tein that cannot be titrated at all as long as the native con-
formation of the protein is retained; this happens most often
with tyrosine and histidine side chains which can be buried
in the molecule when both groups are uncharged. Such is the
case in hemoglobin, where 22 of 38 imidazole groups can not
be titrated with the molecule in the native state (71).
Similarly, in papain, 6 of 17 tyrosine residues do not titrate
normally. The study of the effect of hydrogen ion concentra-
tion of protein conformation or aetivity must deal with these
considerations and also with the many other electrostatic
forces created by the charged groups and the various inter-

actions which subsequently arise.

90

2. Effect Of pH on a-CHT

The conformation and activity of a-CHT shows a complex
dependence upon the pH of the surrounding medium. There are
some 30 ionizable residues in o-CHT not including the three
terminal a-carboxyl and a-amino groups at the ends Of the
three peptide chains. The interactions of several of these PX
charged groups have been Observed in the dimer interface and
at the uranyl binding site. In addition, the presence Of

both histidine and aspartic acid in the active site suggests

ru_..-C._._ ‘ .'

that the activity of the enzyme might be controlled, to some
extent, by the pH of the medium.

A number of different thermodynamic states representing
various stages of folding or unfolding of the peptide chains
are accessible by changes in pH. The various stages have been
identified by a number Of thermodynamic measurements and are
relatively stable states separated by small free energy dif—
ferences (12). The transition from one state to another is
generally a 'slow' process, but is somewhat dependent upon the
prevailing conditions. The slowness of the transition is
demonstrated by the conversion of o-CHT to the Y form: o-CHT
is stable at acid pH values but slowly converts (hours) to
y-CHT when the pH is raised to alkaline values. The reverse
transition also occurs, but requires a much longer time span
(days).

The activity of a-CHT has been shown to be mainly depen-
dent.upon the ionization states of two amino acids in the

active site region. In the one case, the deprotonation of

91

HIS 57 as the pH is increased coincides with the start of
chymotryptic activity, as measured by the hydrolysis Of neu-
tral substrates such as acetyl-L-tryptophanamide (15). The
rate of hydrolysis becomes a maximum at approximately pH 7.6
to pH 8.0, depending upon the substrate and the conditions of
the solvent. As the pH is increased further, the salt bridge ‘5
between ILE 16 and ASP 194 is destroyed by the deprotonation
of the N-terminal amino group of ILE 16 and enzymatic activ—

ity begins to decrease. Above pH 10, only a small residual

1-_ -—.—---.--l .f :‘2 L.

activity remains. The resultant bell-shaped dependence of
the activity on pH is shown in Figure 15a (14).

Another gross effect that pH causes in the physical prop—
erties of a-CHT concerns the aggregation state of the enzyme
molecules. Under the proper conditions (pHm4, concentrated
protein solutions, high ionic strength), a—CHT exists pri-
marily as dimeric molecules. The participation of charged
electrostatic groups in the dimerization process was proposed
since the extent of dimerization is dependent upon pH, ionic
strength, and salt composition. Figure 15b shows the func-
tional relationship of pH on dimer formation. Egan, et a1.
Observed that the sedimentation constant reached a maximum at
pH 4.0 and that the photo-oxidization of a single histidine
residue caused the disappearance of any tendency for a-CHT
molecules to associate (72). He suggested that a carboxylate
ion and a charged imidazole group were responsible for the
observed behavior. Similar conclusions were drawn by Aune

and Timasheff after analysis of dimerization equilibrium

Figure 15a.

Figure 15b.

92

K (a)

 

 

-_ "'"AW LA. "haw

Typical pH-rate profile for chymotrypsin cata-
lyzed reactions showing rate of reaction K as
a function of pH. (From Hess, et al., ref. 14)

LnK (b)

 

 

Functional relationship of pH on dimer forma—
tion. The natural logarithm of the dimeriza—
tion equilibrium constant versus pH for 0.1 M
NaCl solutions at 25°C. Maximum dimer formation

occurs at pH 4.4. (From Aune and Timasheff,
ref. 73)

93

constants by the method of sedimentation equilibrium as a func-
tion of pH (73). Reference to Figure 10 shows that such an
interaction occurs between TYR' 146 and HIS 57 along with the
reciprocal interaction across the local two-fold axis.

The dimer which occurs in solution may then be the same
dimer as, or very similar to, that which is observed in the
crystalline state at pH 4.2. Support for this possibility
was given by experiments of Gladner and Neurath (74) and
Hexter and Westheimer (75). In the first case, the removal
of TYR 146 by the action of carboxypeptidase resulted in a
molecule which was no longer able to associate to form dimers.
In the latter case, an intermolecular reaction was Observed to
occur between SER 195 and TYR' 146 during photolysis of
diazoacetyl-a-CHT, made by reaction of o-CHT with
p-nitrOphenyldiazoacetate (OZN -<::>- O — g - CHN2) with
release of p-nitrophenol.

The effect Of a change Of pH from 4 to higher values on
the crystals of a-CHT was first Observed in our laboratories
by Mr. R. Timkovich (76) in a flow cell experiment in which
the x-ray diffraction pattern of a crystal was monitored as
various substances are flowed over and through the crystal (21).
Substantial changes in the diffracted intensity of some reflec—
tions were Observed when the pH of the 75% (NH4)ZSO4 soaking
solution was changed from 4 to 7. The Observed intensity
changes suggested that significant protein conformational
changes were occurring in the crystal and that these changes

may be related to the dimerization process or to other

94

conformational changes. To delineate the changes that were
occurring, a full three dimensional X-ray diffraction study

of crystals of a-CHT whose pH was increased to 6.7 was under-

taken.

VII . EXPERIMENTAL

1. Crystal Preparation

Crystals of o-CHT were grown from 50% ammonium sulfate
solutions at pH 4.2 as described earlier. After sufficient
growth had been attained, the solution was replaced with a
75% saturated ammonium sulfate solution adjusted to pH 4.2.
The pH of the solutions was measured by a Beckman Zeromatic
pH Meter with a combination glass electrode. Corrections for
the errors in the hydrogen ion activity due to the high salt
concentration (N3M) were not made; relative pH changes were
considered more important than the absolute pH values.

The hydrogen ion concentration Of the ammonium sulfate
solution was changed by the addition of concentrated ammonium
hydroxide to the test tubes containing crystals. A sudden
change in pH from 4.2 to 6.7 or higher caused all of the
crystals to crack severely. When small amounts of NH4OH were
added periodically over a two-week period, most of the crys-
tals developed cracks, but not as severely as before. However,
a few crystals either did not crack or possessed only minor
cracks; these crystals were used for the diffraction experi-
ments. A similar experiment conducted over the period of a
month produced comparable results. The latter experiment was
performed because it was thought that a slow change of pH

would allow for better accommodation by the crystal lattice of

95

96

any reorganization in the protein structure which might be
occurring.

The diffraction patterns along the principal axial direc-
tions were recorded for a series of crystals at different pH
values during the period of time that the pH was being changed.
Changes in the diffraction pattern for crystals with a pH
between 4.2 and 5.0 were small, although the b axis became
progressively longer as the pH was increased. At pH values
between 5.0 and 5.6, the crystals generally became cracked and
substantial changes in the diffraction pattern occurred. Above
pH 6 until approximately pH 8, only minor additional changes
in the pattern were Observed, with more changes occurring again
above pH 8. Crystals at a pH of 6.7, intermediate between the
two Observed transition pH values, were chosen for three dimen-
sional work. The stability of the diffraction pattern to
X-rays was comparable to that of the native crystals. In addi-
tion, there was no appreciable difference in the X—ray dif-
fracting quality Of the crystals at pH 6.7 as compared to the
native crystals at pH 4.2. Crystals from several different
tubes and crystallization batches showed excellent reproduc-
ibility of the diffraction pattern at pH 6.7.

Typical axial scans along the 9} direction for crystals
at pH 4.2 (native) and pH 6.7 and for an inhibited derivative
of o-CHT, tosyl-a-CHT (p-toluenesulphonyl-aCHT) are shown for
comparison in Figure 16. The diffracted intensities are
plotted on a log scale as a function of the Bragg scattering

angle 20. Each division along the ordinate represents about

97

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4 .
9
3 D
TOSYL ‘ 2 _
7
I4 .
‘ O
20 1 m '0 I .
V
" .. WU
j l I I I I I K1
. ‘ "75°
' ~30
NATIVE
I "O
.. ., we!
20 I. “9°
I
I: L '2 I I V ,30
.. WI
J n a -IO
U I I I I I r I I I o
4 h
. D
1 3'
pu-BJ l
I-
O
M _
2‘ m! 1 __ l I 0
1 2I n "
’ 22 “ RI. .
I 3 I .9 I] .I I 'I ”J
.1 JLJUiel-M- "AIJdUI/l
I 1 r I I 1
36 32 2'. 2'4 20 Is I2 0 4 0
20 - degrees

Figure 16. Axial scans along 9* direction for Tosyl, native
and pH 6.7 crystals of a-CHT. The diffracted
intensity is plotted as a function of 20. The
order of diffraction is indicated above each peak.

98

a factor of two in the diffracted intensity. The three crys—
tals were Of comparable size and diffracting power. An appre-
ciation of the changes that occurred in the diffraction pattern
can be Obtained by comparing the relative intensities Of the
four reflections 5, 6, 7, and 8 in the pH 6.7 crystal to the
corresponding reflections in the native crystal. The magni—
tude of the changes is comparable to that produced by the
inhibition of a-CHT by tosylation. The changes in tosyl dif-
fraction pattern are due to the nearly 100% tosylation of

SER 195 in both molecules in the asymmetric unit and include
various protein movements which have been Observed to occur
upon tosylation. Comparable changes were Observed on the
other axial intensity distributions and in the three dimen-
sional intenSity distribution as compared to that of the
native crystal.

The average cell dimensions measured from four different
pH 6.7 crystals are compared to the native cell dimensions in
Table IX. The standard error of each dimension is given in
parentheses. The a and 9 directions decreased in length by
about 0.1A each, while the b axis length increased very sig-
nificantly by 0.6A. A small increase in the angle (8) between
the a and g axes was also Observed. The net increase in the

cell volume upon change of pH is 1%.

2. Data Collection and Processing
The three dimensional intensity data to 2.8A resolution

were collected in a manner similar to that previously

99

Table IX. Cell Dimensions for pH 6.7 a-CHT Crystals and
Native Crystals.

pH 6.7 Native
a 49.13(5)A 49.24(7)A
b 67.83(7)A 67.20(10)A
c 65.81(7)A 65.94(9)A
B 101.92I6)° 101.79(6)°
Cell Volume 214500 1 700 A3 213400 2 900A3

 

described (24,25) for the heavy atom and inhibited deriva-
tives with the following modifications. The data collection
program was changed to measure only the Observed reflections
which had a figure of merit greater than 0.7 as determined
from the heavy atom phase refinement procedure. The indices
of these Observed reflections (6200) were stored on the DEC
32K Disk File and retrieved sequentially. By this arrange-
ment, and a reduction of the power of the incident Xeray beam,
all the data to 2.8A resolution were collected from a single
crystal in four days time, instead Of the usual five or more
crystals and several weeks of time.

The monitor reflections with high 20 values, measured
after every 100 reflections during the data collection, showed
a total decay of approximately 30% from their initial intensi-
ties after 73 hours Of exposure of the crystal to the x-ray
beam. The decay of the diffracted intensities is a function

of 20, with the greatest decay occurring at the largest 20

100

values. To decrease the effect of decay on the higher order
reflections and the errors associated with its correction,
the reflections from 3.5A to 2.8A resolution were measured
first (2500 reflections with a maximum decay of less than
10%). Measurement of the lower resolution data followed with
an overall decay substantially less than the 30% total decay
recorded for the monitor reflections.

Before conversion to structure amplitudes, the intensi-
ties of the pH 6.7 crystal were corrected for twinning, absorp-
tion, and decay factors as described elsewhere (24). The
resultant structure amplitudes were adjusted to an absolute
scale by fitting the radial distribution curves (average F2
versus 20) for the derivative and the native data. Figure 17
shows the radial distribution for the pH 6.7 crystal (dotted
line) superimposed upon that of the native (solid line) after

prOper scaling of the structure amplitudes.

3. Difference Electron Density and Error Assessment

A 'best' difference electron density map between the
structure at pH 6.7 and that at pH 4.2 was computed using
coefficients

m ( IFPHI - lel ) exp [lap] (17)

where IF and IFPI are the structure amplitudes of the

PHI
higher pH crystal and the native crystal, respectively, m is
the figure of merit of the particular reflection used as a

weight, and up is the ‘best' protein phase. The mean square

error in a difference electron density map of this kind is

.ZOOV

< F">

IOO

Figure 17.

101

 

 

 

X
p—
' X
l
\\ I
\ I
I
X
p-
X
l 1 I g l l
I I I v r
o s l0 IS ao 25 30 29

Radial distribution curves for pH 6.7 and native
crystals. The average value of F2 is plotted
as a function Of 20. Solid curve - native dis-

tribution, dashed curve - pH 6.7 distribution
after scaling.

 

102

given by Henderson and Moffat (77) as

<Ap2> =—- 4:: 2: AF2 (l-mz) + 52 + F2 (18)

V

where AF is the difference in structure amplitudes of the

derivative and native crystals ( IF - IFpI ). The first

PHI
two terms in the summation are the contributions to the rms
error caused by errors in the determination of the phases and
by experimental errors in the AF values, respectively. The
third term gives an estimate of the errors in the assumption
that the phase angles for the native and derivative crystals
are the same when the AF values are computed. The root mean
square error for the ApH difference map was estimated by
equation 18 to be 0.02 eA-3. The difference electron density
map was computed on the same scale as the native electron
density map and was contoured and traced onto the plexiglas
sheets carrying the native map.

Errors due to a lack of isomorphism in the phases and
amplitudes caused by the changes in cell dimensions are dif-
ficult to assess quantitatively. Crick and Magdoff (78) have
shown in a hypothetical case that a 'breathing' motion simi-
lar to that Observed here (increased cell volume) could pro-
duce some significant changes in the diffracted intensities
and phases. Lack of isomorphism errors could add uniformly
to increase the background of the map or could appear as
spurious peaks, distributed randomly throughout the electron

density map. Of the many peaks observed in the difference

density, only a few were unexplainable. If spurious peaks

103

were to be generated, a prOportionate number of peaks should
be Observed in the regions between molecules. The few small
peaks Observed in these solvent regions usually had corres-
ponding peaks in the native electron density. Situated near
the protein molecule, these peaks might represent partially
ordered salt or water molecules which have moved upon change
of pH. The Observed overall background of the difference
electron density is approximately 10.05 eA-3, which is some-

what higher than that predicted by equation 18.

VIII. DISCUSSION OF THE RESULTS

1. General Features of the Difference Map

A substantial number of positive and negative peaks,
representing structural differences between the two structures
at the different pH values, are Observed throughout the dif-
ference electron density map with the majority of the peaks
concentrated near the dimer interface and associated protein
regions. The differences are primarily located on the exterior
of the molecule, especially around residues whose side chains
are polar. However, a few differences are also Observed
within the interior of the molecule and correspond to move-
ments of segments of the polypeptide chain. As was mentioned
earlier, there were essentially no significant difference
density peaks in the solvent region. Furthermore, as might be
expected, there were no significant peaks in or around the
non—polar cavity which was described earlier.

In a number of places in the difference electron density
map, a positive region will be accompanied by a negative
region of approximately the same magnitude located on either
side of a peak in the native electron density map. These
positive-negative contours represent an electron density
gradient or a shift or movement of the atoms or residues which
correspond to the electron density. The direction of the
shift is from the negative position in the native to the

104

105

positive position in the higher pH structure. Consider the
electron density of an atom at a position xl to be represented
by a Gaussian function whose height and width are functions

of the number of electrons, the thermal motion associated
with the atom or residue, and the resolution of the electron
density map (curve pl, Figure 18). If the atom moves to a

new position x2 with electron density 02, the resultant dif-

ference electron density, pz-p can be obtained by a point

1'
by point subtraction of curve 02 from curve pl. The resultant
curve has negative troughs and positive peaks, the depth and
height of which are related to the amount of movement and the
number of electrons involved. The positional shifts can be
estimated by considering the lepe of the difference electron

density and the curvature of the electron density at the

original position,xjj in a manner similar to that Of Booth (79).

slope = _(3Ap/8x)x=x1

 

 

curvature (32p/3x2)x=xl (19)

While this expression was derived for the position of a single
atom, estimates for group movements can be made also. The
largest difference density slopes or gradients were 0.4 to
0.6 eA-4, while the curvatures in the electron density at the
peak positions were on the order of 0.7 to 0.9 eA-S, and cor—
respond to group positional movements on the order of 0.4 to
0.8A.

One final feature Of the difference electron density map

which strengthened confidence in its correctness and its

Figure 18.

106

AX

a f’.

 

(fr- 4)

Gaussian curves showing effect of a small shift
on the difference density.

107

interpretation was the existence of many two-fold related
counterparts for many of the positive and negative peaks. If
spurious peaks were generated by a lack of isomorphism between
the native and pH 6.7 crystals, it seems improbable that
equivalent peaks related by a non-crystallographic symmetry

element would be generated as well. r

2. Histidine 40
One Of the largest structural changes in the pH differ—

ence density is associated with HIS 40. The peptide chain

tn-) 'l1‘ ’0’.

from residues 35 to 41 lies on the surface of the u-CHT mole—
cule in the dimer interface region. While PHE 39 extends into
the interface, HIS 40 points toward the interior of the mole-
cule. The imidazole ring is positioned so that one side of
the ring is relatively accessible to the solvent. The peptide
chain 192 to 194 is found on the other side of the ring, with
the plane of the ring roughly parallel to the direction of
this peptide chain. The side chain Of SER 32 is in the inte-
rior of the molecule with its hydroxyl group directed toward
the imidazole ring. A schematic drawing of the location of
HIS 40 with respect to these residues is shown in Figure 19.
The imidazole ring is positioned so as to allow the for-
mation of a hydrogen bond with the hydroxyl group of SER 32
or to the carbonyl oxygen of GLY 193 through the N62 atom of
the ring, although the latter bond is less likely to occur, due
to the long distance (~4.5A) between the oxygen and nitrogen

atoms involved. The orientation of the ring precludes the

 

Figure 19.

108

'$WW""'
“

III l0!

Schematic drawing of important residues around
HIS 40. Main chain atoms connected by Open
bonds while side chain atoms are connected by
solid bonds. Oxygen atoms - a ; nitrogen atoms
-0; carbon. atoms -0.

109

formation of simultaneous hydrogen bonds through both the N61
and N82 nitrogen atoms. The hydrogen bond formed with either
possibility would probably utilize the N£2 nitrogen atom and
would depend upon whether the ring is flipped around the Ca-
CB bond so that N61 is pointing toward the solvent or toward
GLY 193. In chymotrypsinogen, the inactive precursor of a-CHT,
HIS 40 is found in a hydrogen bond complex with the side chain
Of ASP 194. Upon activation to a-CHT, it has been prOposed
that the ASP 194 side chain swings away from HIS 40 which then
forms a hydrogen bond with SER 32 or GLY 193 (16).

Since histidine residues typically have a pK around
pH 6.5, it is expected that in crystals at pH 4.2 the HIS 40
imidazole ring would be protonated and positively charged,
with both ring nitrogen atoms carrying hydrogen atoms and the
positive charge delocalized over the ring. When the pH of
the crystals is changed to 6.7, the difference electron den-
sity shows a peak of large positive density on the right side
of the electron density corresponding to the ring and negative
density on the left as depicted in Figure 19. These peaks
imply a movement of approximately 0.5A of the imidazole ring
of HIS 40 towards GLY 193 to a position more favorable for
the formation of a hydrogen bond through N62 to the carbonyl
oxygen of 193. The difference electron density also shows a
positive density on and near the carbonyl oxygen of GLY 193,
again indicating the presence Of increased electron density

between the carbonyl group and N22 of HIS 40.

110

Coupled with these Observed changes, a negative region
with no accompanying positive region is observed adjacent to
the N61 position of the HIS 40 ring, corresponding to a small
peak in the native electron density. The loss of a water

molecule bound to the N nitrogen atom at low pH could

61
explain the negative peak. With this assumption, the orienta-
tion of the imidazole ring in the native structure would then I}
be as shown in Figure 19. At pH 4.2, the conformations of L
HIS 40 and HIS' 40 are somewhat different; the pH change
causes slightly different motions of the side chains with the I
final positions Of both imidazole rings in approximately the
same conformation. Figure 20 shows the difference electron
density map superimposed on the electron density map of HIS 40
and neighboring regions Of one molecule.
The aromatic side chain of TRP 141 is located approxi-
mately 5A above the side chain of HIS 40 with the two rings
being nearly coplanar. The fluorescence of a-CHT increases
with increasing pH above pH 6 when an ionizing group of pKNG
Iloses a proton (14). Model studies by Katchalski and coworkers
(80) have shown that the fluorescence of tryptophan is quenched
when a 'complex' is formed by the interaction Of a charged
imidazole ring of a histidine residue with the indole ring of
tryptophan. An increase of fluorescence is Observed when the
imidazole ionizes and disrupts this complex. From the experi-
ments on CHT mentioned above, it is probable that such a

complex is formed between TRP 141 and HIS 40 at low pH values.

 

Figure 20.

Figure 21.

Electron density around HIS 40. Difference
electron density between pH 6.7 and native _3
crystals shown in white; contours at 0.15 eA
starting at 0.15‘3. The ring of HIS 40 is seen
edge on from a position corresponding to GLY 193
in Figure 19. Peak marked P belongs to the
alpha carbon of PHE' 39.

Electron density in vicinity of TYR' 146 and

HIS 57. Difference electron density shown in
white as described in Figure 20. The carboxy-
late oxygen moves from its position, 01, in the
native electron density to position 0 ' when the
pH is changed. The bridging SO4= between TYR' 146
and SER 195 (not shown) is indicated. The
negative—positive density on the HIS 57 ring
indicates a small rotation.

111

 

 

112

When the pH is raised to 6.7, the deprotonation of the HIS 40

ring breaks the complex and increased fluorescence results.

3. Dimer Interactions

Aune and Timasheff have made a careful study of the
dimerization-equilibrium constants by ultracentrifuge experi-
ments (73). From their analysis, they postulated that the {1
electrostatic interaction (at pHm4) Of the protonated ring It
Of HIS 57 and the terminal carboxylate ion of TYR' 146 is I
responsible for the Observed pH dependence of dimerization I
and that the other electrostatic interactions between ASP' 64 - 1”
a-NH3 ALA 149 or LYS' 36 - a-NH3 ALA 149 only serve to increase
the free energy of formation of the dimer.

Because of the number of electrostatic interactions which
occur around the two-fold axis, one might expect to see changes
in this region when the hydrogen ion concentration is changed.
Not surprisingly then, the pH difference map revealed a num-
ber of molecular rearrangements in the interface region between
the two molecules in the dimer. The largest interfacial change
was associated with the carboxylate groups of TYR 146 and
TYR' 146. As mentioned earlier, the carboxylate groups in the
dimer at pH 4.2 were arranged so that they can interact with
the charged imidazole rings of HIS 57 and HIS' 57. When the
pH is raised to 6.7, the imidazole rings become deprotonated
and neutral, and any electrostatic interactions which might

have existed are disrupted.‘ The difference electron density

map showed a large positive peak (No.35 eA-B) near the

113

carboxylate ion on both tyrosine residues in the interface
with a negative peak (-0.35 eA-B) on the carboxylate electron
density. Figure 6 shows the arrangement of residues in the
region of the HIS 57 - TYR' 146 interaction at pH 4.2 and
Figure 21 shows the electron density map around these resi-
dues with the pH difference map superimposed. The net result
of the Observed difference peaks is a rotation Of a carboxy-
late oxygen (01) away from the HIS 57 side chain and corres-
ponds to an out of the page movement, into the solvent. There
is apparently no movement or reorientation of the phenolic
group of the tyrosine residue or in the bridging 804: ion in
either two-fold interaction.

In addition to the large peaks observed in the pH dif-
ference electron density around TYR' 146 there are smaller
positive and negative peaks (20.15 eA-3) associated with the
main chain and side chains of residues ARG 145 to GLY 140.
The magnitude of the difference density peaks decreases from
residue 145 to 140; a large movement at TYR 146 results in a
series of smaller changes in the several residues away from
146, with the changes decreasing as the distance from 146
increases along the chain. The net result of these positive
and negative peaks is to produce a small, complex movement of
the whole chain segment away from HIS 57 and the interface
region. This movement is presumably initiated by the reori—
entation of the carboxylate ion Of TYR' 146. Similar move-

ments are also observed for the two-fold related chain segment.

1.“ _‘-."‘

II

' fa-n— ,.—-T—_—f

114

Other differences, somewhat lesser in magnitude, are
Observed in the dimer interface around residues ALA 149 and
ASP' 64. The N-terminal amino group of ALA 149 forms a salt
bridge with ASP' 64 of the two-fold related molecule and vice
versa. The electron density around the ALA' 149 - ASP 64
interaction is strong and generally resolved. However, the
two-fold related interaction is not so Obvious or distinct.
One reason for this unclarity is the formation Of a compli-

cated arrangement involving the side chains of ASP 35,

4
higher pH, a negative peak in the difference map coincides

ASP' 64, ALA 149 and a 80 ion (see Table VII). At the

with the electron density found to be a sulfate ion; this
correspondence suggests the loss or movement of the sulfate
ion from its original position. A similar change also occurs
in the two-fold related molecule. A detailed interpretation
of all of the observed differences in this region is very dif-
ficult and not without considerable uncertainty. The impor-
tant conclusion is that some protein rearrangements take place

at this interaction site upon the change Of pH.

4. Active Site

A considerable number of changes are also Observed in
the active site region of the molecule and with residues asso-
ciated with the active site. Figure 22 shows the electron
density of residues 194 to 198 of molecule I with the pH dif-
ference electron density superimposed. Peaks in the differ—

ence density in the active site are relatively small (0.1 to

IFigure 22.

 

115

.17.?

.8

”—A“ ‘3. .-

FA~I I.» . I
I

 

m-MO
0

“I.

”I. 07

Electron density map around SER 195. Positive
regions in the pH difference map indicated by
plus signs, negative regions by minus signs.
The arrows indicate direction of movement when
the pH is changed to 6.7.

116

0.15 eA-3) and numerous, but taken as a whole, are significant.
In molecule I, the Observed changes combine to imply a move-
ment of the chain from 194 to 198 towards the dimer two-fold
axis as indicated by the arrows in the figure. The negative—
positive region on the main chain of SER 195 shows this move-

ment clearly; also, the same configuration of peaks on ILE 16

“-11
_,_ea

is observed. Molecule 1' shows an opposite behavior upon
change Of pH around its active site. The negative regions in

molecule I are generally replaced by positive regions and vice

[PP—I-lnqn—z .a-u . u..-..<~
1 1

versa. Again, taken as a whole, the chain from 194 to 198
in molecule 1' appears to move away from the two-fold axis.
The reasons for these movements are rather difficult to
rationalize until the differences between the two independent
molecules at pH 4.2 are considered. At pH 4.2, the difference
map computed between the electron densities of the two mole-
cules in the asymmetric unit showed that the same residues
in molecule I were further from the two—fold axis than the
corresponding residues in molecule I'. The pH changes are in
the direction as to make the active sites of both molecules
more equidistant from the two-fold axis and, presumably, more
alike in the resultant configuration of their active sites.
In addition, molecule I' undergoes other small rearrangements
upon change Of pH. In both molecules, there are a number of
positive and negative peaks around the CYS 191 - CYS 220
disulfide bridges and are probably also associated with the

movements Of the chain from 194 to 198.

117

In addition to the movements of SER 195, there are also
pH induced differences in the other components in the active
site. The imidazole ring of HIS 57 in molecule I undergoes
a small rotation around the Ca - C8 bond which points the
nitrogen N62 toward the oxygen of the SER 195 side chain. In
molecule 1', a different behavior is observed for HIS' 57.
Here, the imidazole ring does not move toward SER' 195 but
moves toward the two-fold axis in the plane Of the ring.

While some pH changes appear to make the two active sites

more similar, other pH changes occur which Oppose the simi-
larity. In any event, the exact stereochemistry of the impor-
tant residues in the active site during catalysis remains
uncertain.

Other pH changes are Observed in the side chain of ASP 102
and in several residues on either side of ASP 102. When the
pH is increased, the side chain carboxyl group exhibits a
slight movement away from the imidazole ring of HIS 57. At
pH 4.2, the carboxylate ion Of ASP 102 is presumably nega-
tively charged and the imidazole ring of HIS 57 positively
charged. Upon a change to more neutral conditions, the
electrostatic interaction between these groups would decrease
as HIS 57 becomes deprotonated and uncharged. A slight repul-
sion of the carboxylate of ASP 102 would then be expected.
Such a movement is implied further by a series of positive and
negative pH differences along the main chain from ASN 101 to
LEU 106, in addition to the differences Observed on the side

chain Of ASP 102 itself.

rW—mm-Ln-LGsrn‘. v
‘fll . l .3. .
I
w..- . -
.- - ..

118

5. Uranyl Interface

As described in section IV—S, the uranyl binding inter-
face appears tO be formed by a pair of carboxyl groups on
different molecules coming together to possibly form a Spe—
cial complex stabilized by proton fluctuations. Such a complex
is strongest at the pH which corresponds to the pK of the
interacting groups. If the pH is changed to a value outside
the pK ranges, disruption of this complex would be expected
and is observed to occur with a-CHT.

As noted earlier, the electron density around the uranyl
binding site is complex; so are the pH differences. The
largest positive-negative peaks are associated with a movement
of the guanidinium side chain (positively charged) of ARG 154
toward the carboxylate group of GLU 21 and a reciprocal move-
ment of GLU 21 toward ARG 154. The result of these movements
is to change the environment around the U02++ binding site

substantially. Based on these observations Of this region,

++
2

if at all, at higher pH values.

it is probable that UO ions would not bind as strongly,

6. Other pH Changes

Upon examination of some of the non-electrostatic inter-
actions which occur in the interface region during dimeriza-
tion, it becomes apparent that the change in pH, by loosening
the salt-like interactions between the two molecules, dis-
turbs the hydrophobic interactions as well. Two of the non—

electrostatic interactions which must undergo some mutual

U1

I‘m-QI‘ALL - 1..." :4".-
D

v
"99“ -

119

accommodation in order to form the dimer (PHE 39 - PHE' 39 and
SER 217 - SER' 217) show some relatively substantial changes.
The pH difference density of the two PHE 39 residues shows
large but differing motions. The main chain Of PHE 39 moves
toward the two-fold axis while the main chain of PHE' 39

moves along the two-fold axis, maintaining somewhat respectable
van der Waals contacts with each other. There are also move-

ments Of the main chain Of SER 217 to SER 218 but ng_observed

 

changes in the two-fold related segment SER' 217 - SER' 218.
It is possible that the movement of these residues is not
related to the change in pH directly. When the dimer is
formed, strong electrostatic interactions might force these
residues to adopt configurations which are of a higher energy
than normal. When the pH is raised, and the molecules tend
to separate, the hydrOphobic residues might relax, possibly,
to lower energy configurations.

Many of the other changes observed when the pH is changed
are observed to be changes in the water or ion structure on
the surface Of the molecule. Most of the Observed peaks occur
near serine or threonine side chains and the charged side
chains, both acidic and basic. For instance, the sulfate mole—
cule bound near ASN 95 moves some 3A away to take a position
near ASN 100. SER 217, SER' 223, ASP 70 and other residues
appear to have attracted a water molecule with pH change,
while SER 186, CYS l, HIS 40, ASP 35, and TYR 94, among others,
seem to show a loss or movement of solvent molecules. For

instance, positive difference density peaks are also Observed

120

near the main chain carbonyl groups of GLY 211, SER 159, and
LEU 243, with negative densities near ILE 99, GLY 211, GLN 18
and TRP' 215.

Apart from an occasional peak of low magnitude which
seems to be spurious, a curious feature was Observed near the
tryptOphan residues. On the edge of each indole ring of five .
tryptOphan residues, TRP 27, 29, 51, 172 and 215, there is a {11
positive difference density contour, occurring in almost I
every case near the six membered end of the indole ring. The
reason for these positive peaks at these positions is unknown; I»I
they do not seem to be due to motions of the tryptophan rings 1'
as there are no associated negative contours. A solvent mole-
cule associating with the non-polar aromatic ring is equally
unlikely. The explanation for these peaks probably awaits
future developments in the understanding of protein physical

chemistry.

7. Final Comments

A summary of the changes in the molecular structure and
their probable interpretations of crystalline a-CHT upon
change of pH Of the surrounding medium from 4.2 to 6.7 is
given in Table X. The Observed pH changes are both inter—
molecular and intramolecular; the former concern the processes
of dissociation of a dimeric molecule and the latter are
probably related to pH induced conformational changes of the
enzyme. A large number of the pH induced intramolecular

changes involve active site components; similar changes may

Table X.

1.

HIS 40 and HIS' 40

TYR 146 - HIS' 57
TYR' 146 - HIS 57

ALA 149 - ASP' 64

Active Site

Uranyl Binding Site

PHE 39 - PHE' 39

SER 217 - SER 218

Summary of Changes

121

of o-CHT Upon Change of pH.

Rotation of ring 0.5A toward
carbonyl of 193 with loss of
H20 Off N61 - breaks complex

with TRP 141.

Movement of carboxyl terminal
away from interaction with HIS
57. Causes movement of residues

141 to 146 in the process.

Complicated set of movements

around salt bridge, displacement

Of interacting SO4 molecule.

*' .hr-sns AJuho-

1 - .' 0 .‘_ q
IV P I'.—l,
".fi‘AV-‘U o

I

Movement Of residues 192-198 in
both molecules to become more
nearly alike. Slight movement of
ASP 102 away from HIS 57. Differ-
ent movements for HIS 57 and

HIS' 57.

Movement Of ARG 154 toward GLU 21,
with subsequent movement of GLU 21.
Movements occur in both molecules
although only molecule I has

uranyl binding site.

Complicated motions occurring
around the hydrophobic interaction

of these two groups.

Movement of main chain in molecule
I but not in mOlecule I'. Residues
are involved in non-electrostatic
interaction with two-fold related

residues.

122
Table X. Continued.

8. Solvent Changes Loss Of H20 from carbonyl of
TRP' 215 and SER 186, CYS 1,
ASP 35, TYR 94 and others. Gain
water at SER 217, SER' 223, ASP
70. Movement of SO = from ASN 95

4
to ASN 100.

 

A 5.: «-12'Aa‘ I 9. K“

. L

W‘s...
-
‘ 50W- - '

also occur in the active site in solution. The ramifications
of all the Observed changes have yet to be unraveled.

For instance, to be studied in detail are the effects
of lowering the pH below 4.2. It has been proposed that the
salt bridge between ILE 16 and ASP 194 will be disrupted below
pH 3.0 due to the protonation of the carboxylate group Of
ASP 194 (12). Preliminary data indicate that the magnitude
of the changes in the diffraction pattern at pH 2.7 are com-
parable to those Observed at pH 6.7. Also to be investigated
in detail is the structural transition which also occurs above
pH 8, again presumably due to the rupture Of the salt bridge
16 — 194 with deprotonation of ILE 16. Preliminary diffrac-
tion data also Suggest that the changes Observed in the transi—
tion from pH 4.2 to 6.7 are reversible when the pH is returned
to 4.2.

The crystalline state possesses many advantages for the
study of the effects of pH and other experimental variables
on the structure of proteins and enzymes. The crystalline

forces prevent excessive movements and changes from occurring,

123

but the large spaces between molecules in protein crystals
and the large prOportion of solvent probably allows the mole-
cule to assume a conformation similar to that in solution.

By careful interpretation and study of the results, X-ray
diffraction can provide meaningful information and give new
insights to be used in unraveling some of the complexities of

protein physical chemistry.

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APPENDIX

I ‘- ,

 

Residue

A Chain
1 CYS
2 GLY
3 VAL
4 PRO
5 ALA
6 ILE
7 GLN
8 PRO
9 VAL
10 LEU
11 SER
12 GLY
13 LEU

B Chain
16 ILE
17 VAL
18 ASN
19 GLY
20 GLU
21 GLU
22 ALA
23 VAL
24 PRO
25 GLY
26 SER
27 TRP
28 PRO
29 TRP
30 GLN

SIDE CHAIN ENVIRONMENT AND CLARITY IN THE
ELECTRON DENSITY OF a-CHT

Environment

 

£40)meth

[11H

(Ami-{Elm

HHHHCD

Good

Weak
Well
Some
Weak
Some
Weak
Weak

Comments*

 

density in bridge to 122

density for side chain
defined density for ring 5
CB density

but defined density
density for side chain
density

rfl_\_‘-‘:L 3*. 2'|‘- -‘ 1

density

No definitive density
for remainder of A chain

Forked side chain density,
salt bridge to ASP 194

No density for CG2
No density beyond CG

Weak

density for side chain

Strong density

Good

density

No density beyond CB

Good

Side
Very
Good
Very
Good

129

density

chain on edge of density
strong density

density

good side chain density
side chain density

130

 

Residue Environment
31 VAL I
32 SER I
33 LEU I
34 GLN S
35 ASP S
36 LYS E
37 THR S
38 GLY

39 PHE E
40 HIS

41 PHE I
42 CYS

43 GLY

44 GLY

45 SER I
46 LEU I
47 ILE I
48 ASN E
49 GLU E
50 ASN E
51 TRP S
52 VAL I
53 VAL I
54 THR I
55 ALA I
56 ALA I
57 HIS S
58 CYS S
59 GLY

60 VAL I

Comments*
Good density

CB on edge of density-—
H bond to HIS 40

for CD1.

side chain density

Weak density
Well defined
Density o.k.
to CD

Strong density for side chain

Weak density

Good side chain density

Partially exposed to solvent
well defined

Good density

P.- #3.-“ ‘1
.\ .
'\'

Strong disulfide bridge to
CYS 58

>C=o on edge of density

Well defined density
Forked density for side chain
Good definition

Good to CB,
chain

less to end of

Strong density for whole side
chain

Complete, defined density
Good density

Slightly forked density

No density past CB

Density for one branch only
CB on edge of density

Well defined

Well resolved density

Good density for bridge to
CYS 42

No density for CG2

131

 

 

Residue Environment Comments*
61 THR S Sufficient density
62 THR E Good density
63 SER B Good density

64 ASP S Salt bridge to ALA' 149
65 VAL S Density not resolved from main
chain
66 VAL I Resolved density F
67 VAL S Side chain on edge of density g
68 ALA S CB in bump on main chain g
69 GLY E f
70 GLU E Poor density for main and side 3
chain k
71 PHE S Good density for ring 1
72 ASP E Hard to fit side chain to
density
73 GLN E Poor density for side chain
74 GLY
75 SER E Side chain in good density
76 SER E Poor density--solvent
'77 SER E Poor density
78 GLU S Good side chain density
79 LYS E Weak side chain density
80 ILE S No density for one branch
81 GLN E Good density for whole side
chain
82 LYS E Good density to CG, less to NZ
83 LEU S Good density for CG
84 LYS S Continuous density for side
chain
85 ILE S No density for CD1
86 ALA 8 CB in bump on main chain
87 LYS E Weak density
88 VAL S Forked density
89 PHE S Density somewhat less than

other phenyls
90 LYS E No density past CE

132

 

 

 

Residue Environment Comments*
91 ASN S Good density for whole side
chain
92 SER E No density for 0G
93 LYS E Complete, resolved density for
side chain
94 TYR 5 Somewhat diffuse, typical of
TYR residues
95 ASN S Good density--H bonding to
SO4=
96 SER E Good density
97 LEU E Different orientations in two
molecules
98 THR S Complete side chain density
99 ‘ILE S Forked density, good
100 ASN S Good density for side chain
101 ASN S Complete side chain density
102 ASP I Good density for carboxyl group
103 ILE I Slightly forked density
104 THR I Side chain on edge of solid
density
105 LEU I Good to CG, weaker density
for rest
106 LEU I Well defined density
107 LYS E Density weak beyond CG
108 LEU I Good density
109 SER S Side chain with H20 molecule
110 THR E Density not resolved from main
chain
111 ALA E No density for CB
112 ALA S Strong CB density
113 SER E Good density, with H20
114 PHE S Funny-shaped density for
phenyl ring
115 SER 8 Density elongated, H20
116 GLN E No density past CA
117 THR S Side Chain density not resolved

from main chain

133

 

 

 

 

Residue Environment Comments*
118 VAL S Density to CB only
119 SER S Good density
120 ALA S Weak density for CB
121 VAL I Weak density past CB
122 CYS S Good bridge density to CYS l
123 LEU I Slightly forked density
124 PRO I Well defined Fm
125 SER 5 Well defined 1
126 ALA S More than enough density for CB 2
127 SER B Side chain good &
128 ASP S No density for CB, good for

C00"
129 ASP E Weak density for carboxylate e
130 PHE S Well defined
131 ALA E No CB density
132 ALA E Weak density for CB

133 GLY Good main chain density

134 THR E No density for CG2

135 THR 8 Good density

136 CYS S Strong density--bridge to
CYS 201

137 VAL S CGl on edge of density

138 THR I Good density

139 THR I Poor density for side chain

140 GLY Main chain density weak

141 TRP I Weak side chain density

142 GLY

143 LEU I Density good to CG

144 THR S Good density with H20

145 ARG E Density good except for CB
and CG

146 TYR E Density somewhat irregular

for ring; carboxyl terminal
density good

Residue
C Chain
149 ALA
150 ASN
151 THR
152 PRO
153 ASP
154 ARG
155 LEU
156 GLN
157 GLN
158 ALA
159 SER
160 LEU
161 PRO
162 LEU
163 LEU
164 SER
165 ASN
166 THR
167 ASN
168 CYS
169 LYS
170 LYS
171 TYR
172 TRP
173 GLY
174 THR
175 LYS
176 ILE

Environment

 

HUIMCDMUJHUJHUJUJUJMHMM

U)

134

Comments*

 

Molecule I density weak,
I' very good

Strong to CB, weaker for rest
of side chain

Diffuse density

Somewhat weaker than for other
prolines

Sufficient density for side
chain

Weak density, not definitive
Fair density
Good density

r___wmm‘dn,nfi§7

Weak but sufficient

CB on edge of density

Good density

Branched density

Good definition

Excellent, forked density
Acceptable density

Fair density

Fair density for side chain
Sufficient density

Good, different orientation
in 1'
Good

Complete, orientation differ—
ent in two molecules

Density for whole side chain

Good, molecule I has H20 but
0
not I

Good, flat density

Density for side chain
No density for only CB
No density for CGZ

135

 

 

 

Residue Environment Comments*
177 LYS S Weak density for NZ
178 ASP E Two possible orientations
179 ALA S CB on edge of density
180 MET I Strong density on sulfur
181 ILE S Density for one branch only
182 CYS I Strong density
183 ALA I Good CB definition ‘E
184 GLY
185 ALA I 1
186 SER E Good density '
187 GLY g
188 VAL 5 cs density only L
189 SER I Sufficient for side chain
190 SER I Not very definitive
191 CYS S Strong
192 MET E Weak on I, strong density on I'
193 GLY
194 ASP I Good, salt bridge to ILE 16
195 SER 8 Well defined
196 GLY
197 GLY
198 PRO I Flattened density
199 LEU I Forked density
200 VAL I Slightly forked
201 CYS 8 Strong density
202 LYS E Good density for whole side
chain
203 LYS E Density extends to CB
204 ASN E o.k.
205 GLY
206 ALA S No CB density
207 TRP S Excellent shape
208 THR 8 Density well resolved
209 LEU I Slightly forked

Residue
210 VAL
211 GLY
212 ILE
213 VAL
214 SER
215 TRP
216 GLY
217 SER
218 SER
219 THR
220 CYS
221 SER
222 THR
223 SER
224 THR
225 PRO
226 GLY
227 VAL
228 TYR
229 ALA
230 ARG
231 VAL
232 THR
233 ALA
234 LEU
235 VAL
236 ASN
237 TRP
238 VAL
239 GLN

Environment

 

I

mmmwmwmm HHHH

(DUDE-ICU (DH CDHHHHU)

U)

136

Comments*

 

Density for one branch only

Good
Weaker density with two branches
Good density

Less density than other TRP
residues

Poor density for side chain
Density fairly good

Density good for one branch
Strong density

Good density--with $04= ion
Little density for side chain
Good density

Complete density—~with SO4
ion of SER 221

Strong density

Branched density
Good definition, somewhat weak

Good density for whole side
chain

Adequate density

Complete density for side
chain, H20

Forked density
No density for CG2

No density for CB, good for
rest of side chain

Excellent density
Good definition
CB on weak density

 

Residue
240 GLN
241 THR
242 LEU
243 ALA
244 ALA
245 ASN

Environment

 

E

MCDEIUIU)

 

137

Comments*

 

Very good definition
Slightly forked
Forked, somewhat weak density

Good density for CB

Strong density at end of side
chain

*IUPAC-IUB conventions for peptide nomenclature (81) are used
with Latin equivalents of the Greek superscripts.

 

(IIIIIUHIH

5
0
4
3
7
7

u
H
H
"
"I'I
“
E “l
H
II
I.“

1293 03

 

)IHINHW