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This is to certify that the
dissertation entitled

DYNAMICS OF EMBEDDED FLUOROPHORES IN MODEL
BILAYER LIPID MEMBRANES

presented by .

AARON JAY GREINER

has been accepted towards fulfillment
of the requirements for the

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DYNAMICS OF EMBEDDED FLUOROPHORES IN MODEL
BILAYER LIPID MEMBRANES

By

AARON JAY GREINER

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Chemical Engineering

2009

ABSTRACT
DYNAMICS OF EMBEDDED FLUOROPHORES IN MODEL BILAYER LIPID
MEMBRANES
By

AARON JAY GREINER

Membrane fluidity refers to the rate of translational, rotational, and trans-leaflet
lipid diffusion in bilayer lipid membranes (BLMS). It is an important physical property of
the BLM that can affect the rate at which molecules diffuse across the membrane, the
Signaling and communication between cells, and the activity and function of membrane
proteins. Membrane fluidity often depends on the interactions between constituent lipids
in the BLM or between lipids and other membrane-bound molecules or macromolecules.

This dissertation focuses on four studies that involved the use of optical
techniques to measure the dynamics of fluorescently-tagged lipids in synthetic model
BLM systems that mimic the behavior of cell membranes. The primary tools used for
characterization were fluorescence recovery after pattern photobleaching (FRAPP),
which measures translational diffusion in two-dimensional supported bilayer lipid
membranes (sBLMs), and time-correlated single photon counting (TCSPC), which
measures rotational diffusion in three-dimensional spherical liposomes in solution. Our
results showed that the diffusion of lipid fluorophores in model BLMS can significantly
increase or decrease upon the addition of molecules or macromolecules to the bilayer. For
example, both sonicated and extruded 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC)

liposomes were less fluid upon incorporation of cholesterol, likely to the result of

cholesterol molecules interacting with the hydrophobic acyl chains of the bilayer and
causing the membrane to become more rigid. Addition of the lipid 1,2-dioleoyl-sn-
g1ycero-3-[phospho—rac-(l-glycerol)] (DOPG) to DOPC sBLMs again resulted in a
decrease in membrane fluidity. In this case, however, the reduced fluidity likely resulted
from hydrogen bonding between constituent lipid head groups in the membrane. When
single acyl chain lysophospholipids were added to DOPC sBLMs, the membrane fluidity
increased. This is likely due to a reduction in van der Waals interactions between
hydrophobic acyl chains. However, the fluidity decreased when a fraction of the
lysophospholipids was converted to fatty acids by enzymatic activity of NEST @TE
e_sterase domain), the catalytic domain of neuropathy target esterase (NTE). The observed
decrease in fluidity is attributed to the enhanced packing of fatty acids, relative to
lysophospholipids, in the hydrophobic region of the bilayer. A maximum NEST protein
concentration in fluid sBLMs formed from proteoliposome reconstitution was estimated
and it was demonstrated qualitatively that microsomal membrane proteins at sufficiently
high concentrations can decrease the fluidity of sBLMs reconstituted from rnicrosomes.
The results of these studies give a fundamental understanding of some of the
important interactions that influence the fluidity of model BLM interfaces. The results
may be useful in the design of BLM-based biosensor devices, the performance of which
may depend upon BLM properties such as fluidity. They may also provide insight into

the interactions that affect fluidity in cell membranes.

C0pyn'ght by
AARON JAY GREINER

2009

To my mom, dad, family, and all of the teachers and professors who have been

outstanding mentors throughout my academic career.

ACKNOWLEDGEMENTS

I would like to thank my committee members, Dr. R. Mark Worden, Dr. Robert
Y. Ofoli, Dr. Ilsoon Lee, and Dr. Gary J. Blanchard for being excellent mentors and for
teaching me to focus on novel, important, and fundamental research throughout my
doctoral work at MSU. I would also like to thank Dr. Rudy J. Richardson for donating the
NEST protein plasmid and helping in the interpretation of data obtained with NEST, Dr.
Nichole Hein and Lee Alexander for their help purifying NEST, and Dr. Monique
Lapinski, Angelines Castro-Forero, Dr. Sachin Jadhav, Heather Pillman, Dr. Brian
Hassler, Dr. Neeraj Kohli, and Dr. Sachin Vaidya for their thoughtfiil scientific

discussions and collaborative efforts in research.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................... x
LIST OF FIGURES ............................................................................................................ xi
NOMENCLATURE .......................................................................................................... xv
LIST OF ACRONYMS .................................................................................................. xvii
1. INTRODUCTION ........................................................................................................... 1
1.1. Overview and Significance of the Problem ............................................................ 1
1.2. Background ............................................................................................................ 4
1.2.1. Structure and Function of Biological Membranes ........................................ 4
1.2.2. Model Membranes: Liposomes, Supported Bilayer Membranes, and
Tethered Bilayer Membranes .................................................................................. 5
1.3. Outline of this Dissertation ..................................................................................... 7
1.3.1. Comparison of Liposomes Formed by Sonication and Extrusion: Rotational
and Translational Diffusion of an Embedded Fluorophore ..................................... 7
1.3.2. Effect of Hydrogen Bonding on Rotational and Translational Dynamics of
a_Head group-bound Fluorophore in Bilayer Membranes ...................................... 8
1.3.3. Influence of Lysophospholipid Hydrolysis by the Catalytic Domain of
Neuropathy Target Esterase on Bilayer Lipid Membrane Fluidity ......................... 9

1.3.4. Effect of Membrane Protein Concentration on the Translational Dynamics
of an Embedded Fluorophore in Supported Bilayer Membranes Constituted from

Proteoliposomes .................................................................................................... 10

2. EXPERIMENTAL METHODS .................................................................................... 12
2.1. Overview .............................................................................................................. 13
2.2. Fluorescence Recovery After Pattern Photobleaching ......................................... 13
2.3. Time Correlated Single Photon Counting ............................................................ 15

3. COMPARISON OF LIPOSOMES FORMED BY SONICATION AND EXTRUSION:
ROTATIONAL AND TRANSLATIONAL DIFFUSION OF AN EMBEDDED

FLUOROPHORE .............................................................................................................. 21
3.1. Abstract ................................................................................................................ 21
3.2. Introduction .......................................................................................................... 21
3.3. Materials and Methods ......................................................................................... 23

3.3.1. Vesicle Preparation by Extrusion ................................................................ 23
3.3.2. Vesicle Preparation by Sonication .............................................................. 24
3.3.3. Transmission Electron Microscopy Imaging .............................................. 25
3.3.4. Dynamic Light Scattering ........................................................................... 25
3.3.5. Excitation and Emission Spectra ................................................................. 25

vii

3.3.6. Time-Correlated Single Photon Counting ................................................... 26

3.3.7. Deposition of Supported Lipid Bilayers ..................................................... 26
3.3.8. Fluorescence Recovery After Pattern Photobleaching ................................ 26
3.3.9. Statistical Analysis ...................................................................................... 27
3.4 Results and Discussion .......................................................................................... 27
3.4.1. Liposome Mean Diameter and Size Distribution ........................................ 28
3.4.2. Fluorescence Lifetimes and Rotational Diffusion in Liposomes ................ 29
3.4.3. Translational Diffusion in Supported Bilayer Membranes ......................... 34
3.5. Conclusions .......................................................................................................... 35
3.6. Recommendations for Future Work ..................................................................... 36

4. EFFECT OF HYDROGEN BONDING ON THE ROTATIONAL AND
TRANSLATIONAL DYNAMICS OF A HEAD GROUP-BOUND F LUOROPHORE IN

BILAYER MEMBRANES ................................................................................................ 47
4.1. Abstract ................................................................................................................ 47
4.2. Introduction .......................................................................................................... 47
4.3. Materials and Methods ......................................................................................... 51

4.3.1. Vesicle Preparation ..................................................................................... 51
4.3.2. Time-Correlated Single Photon Counting ................................................... 52
4.3.3. Deposition of Supported Lipid Bilayers ..................................................... 52
4.3.4. Fluorescence Recovery After Pattern Photobleaching ................................ 52
4.3.5. Statistical Analysis ...................................................................................... 53
4.4. Results and Discussion ......................................................................................... 53
4.4.1. Fluorescence Lifetime and Anisotropy Data ............................................... 54
4.4.2. Translational Diffusion in Supported Bilayer Lipid Membranes ............... 59
4.4.3. Head group Hydrogen Bonding in Bilayer Membranes ............................. 60
4.5. Conclusions .......................................................................................................... 61
4.6. Recommendations for Future Work ..................................................................... 62

5. INFLUENCE OF LYSOPHOSPHOLIPID HYDROLYSIS BY THE CATALYTIC
DOMAIN OF NEUROPATHY TARGET ESTERASE ON BILAYER LIPID

MEMBRANE FLUIDITY ................................................................................................. 71
5.1. Abstract ................................................................................................................ 71
5.2. Introduction .......................................................................................................... 72
5.3. Materials and Methods ......................................................................................... 74

5.3.1. Chemicals and Reagents ............................................................................. 74
5.3.2. NEST Expression and Purification ............................................................. 75
5.3.3. NEST Proteoliposome Formation ............................................................... 76
5.3.4. Inhibition Studies ........................................................................................ 76
5.3.5. Determination of Free Fatty Acid Concentration ....................................... 77
5.3.6. Dynamic Light Scattering ........................................................................... 78
5.3.7. Transmission Electron Microscopy ............................................................. 78
5.3.8. Deposition of Supported Lipid Bilayers ..................................................... 79
5.3.9. Fluorescence Recovery After Pattern Photobleaching ................................ 79
5.3.10. Statistical Analysis .................................................................................... 79
5.4. Results and Discussion ......................................................................................... 8O

viii

5.4.1. Transmission Electron Microscopy Images ................................................ 81

5.4.2. Dynamic Light Scattering Data ................................................................... 81
5.4.3. Measurement of Free Fatty Acid Concentration ......................................... 82
5.4.4. Translational Diffusion in Supported Bilayer Lipid Membranes ............... 83
5.4.5. NEST-induced Hydrolysis of P-lysoPC to Palmitic Acid Decreases
Membrane Fluidity ................................................................................................ 85
5.5. Conclusions .......................................................................................................... 87
5.6. Recommendations for Future Work ..................................................................... 88

6. EFFECT OF MEMBRANE PROTEIN CONCENTRATION ON THE
TRANSLATIONAL DYNAMICS OF AN EMBEDDED FLUOROPHORE IN
SUPPORTED BILAYER MEMBRANES CONSTITUTED FROM

PROTEOLIPOSOMES ..................................................................................................... 97
6.1 . Abstract ................................................................................................................ 97
6.2. Introduction .......................................................................................................... 98
6.3. Materials and Methods ......................................................................................... 99

6.3.1. Chemicals and Reagents ............................................................................. 99
6.3.2. NEST Expression and Purification ............................................................. 99
6.3.3. NEST Proteoliposome Formation ............................................................. 100
6.3.4. Preparation of Yeast Membrane Fractions ................................................ 100
6.3.5. Extraction of Lipids From Yeast Cells ..................................................... 101
6.3.6. POPC Liposome Preparation .................................................................... 102
6.3.7. Deposition of Interfaces ............................................................................ 102
6.3.8. Fluorescence Recovery After Pattern Photobleaching .............................. 102
6.3.9. Statistical Analysis .................................................................................... 103
6.4. Results and Discussion ....................................................................................... 103
6.4.1. Interfaces Formed with NEST Proteoliposomes ....................................... 103
6.4.2. Interfaces Formed with Yeast Membrane Fractions ................................. 105
6.4.3. Significance of Lipid to Protein Ratios in the Formation of sBLM
Interfaces ............................................................................................................. 106
6.5. Conclusions ........................................................................................................ 106
6.6. Recommendations for Future Work ................................................................... 107

7. CONCLUSIONS ......................................................................................................... 114

8. APPENDICES ............................................................................................................. 116
8.1. Analysis of Variance (ANOVA) Methodology in Data Analysis ...................... 116
8.2. Non-linear Curve Fitting Analysis ..................................................................... 1 l7

9. REFERENCES ............................................................................................................ 1 18

ix

LIST OF TABLES

Table 3.1. DLS results for extruded and sonicated liposomes of two systems: (1) 98
mol% DOPC and 2 mol% NBD-PC and (2) 68 mol % DOPC, 30 mol% cholesterol, and 2
mol% NBD-PC. Data are means :h SD of three samples. ................................................ 45

Table 3.2. Pre-exponential factor (Ai) ratios and fluorescence lifetimes (ti) of NBD-PC

fluorophores in sonicated and extruded liposomes comprising (1) 98 mol% DOPC and 2
mol% NBD-PC and (2) 68 mol % DOPC, 30 mol% cholesterol, and 2 mol% NBD-
PC ...................................................................................................... 45

Table 3.3. Reorientation times (10R), confined cone semi-angles (60), and wobbling

diffusion coefficients (Dw) of NBD-PC in sonicated and extruded liposomes comprising

(1) 98 mol% DOPC and 2 mol% NBD-PC and (2) 68 mol % DOPC, 30 mol%
cholesterol, and 2 mol% NBD-PC. ............................................................... 46

Table 3.4. Translational diffusion coefficients (DL) and mobile fi'actions for NBD-PC in
sBLMs prepared with sonicated and extruded liposomes comprising (1) 98 mol% DOPC
and 2 mol% NBD-PC and (2) 68 mol % DOPC, 30 mol% cholesterol, and 2 mol% NBD-
PC. .................................................................................................... 46

Table 4.1. Fluorescence lifetimes (1'1 and 1'2) and reorientation times (tom and tom) of
NBD-PE in DOPC, DOPG, and mixed DOPC/DOPG liposomes ............................ 69

Table 4.2. Cartesian components of the rotational diffusion coefficients (Dx and D2) and
the diffusion aspect ratio (Dx/Dz) of NBD-PE in DOPC, DOPG, and mixed
DOPC/DOPG liposomes. Dy = Dz for Type I (prolate) rotors ................................. 69

Table 4.3. Translational diffusion coefficients (DL) and mobile fractions of NBD—PE in
DOPC and mixed DOPC/DOPG sBLMs on fused silica ....................................... 70

Table 5.1. Percent conversion and specific activity of p-lysoPC to palmitic acid for
several systems ....................................................................................... 96

Table 6.1. Translational diffusion coefficients (DL) and mobile fractions for interfaces
formed from systems containing DOPC, NEST, and NBD-PC .............................. 113

Table 6.2. Translational diffusion coefficients (DL) and mobile fractions for interfaces
formed from several systems containing 2 mol% NBD-PC ................................. 113

LIST OF FIGURES

Figure 1.1. Molecular structure of a common lipid in mammalian cell membranes, 1,2-
dioleoyl—sn-glycero-3-phosphocholine (DOPC). The phosphate and choline groups
comprise the hydrophilic head group and the dioleoyl acyl chains constitute the
hydrophobic tail group .............................................................................. 12

Figure 2.1. Schematic diagram of the FRAPP experimental setup. The laser beam is split
into a low intensity monitoring beam (through a filter) and a high intensity
photobleaching beam at optical flat #1. The beams are recombined at optical flat #2 prior
toimpingernentonthesample... ...17

Figure 2.2. Schematic diagram of the FRAPP experimental setup before and after the
laser beam reaches the sample. The dichroic mirror reflects the excitation laser light,
while allowing fluorescent light emitted from the interface to pass through to the detector.
1(_ey: sBLM: supported bilayer lipid membrane; PMT: photomultiplier tube ............... 18

Figure 2.3. A) Fringe pattern in the illuminated area (diameter = 200 pm) obtained using
a 100 lines per inch (25 ,um periodicity) Ronchi ruling. B) Observation area (diameter =
75 pm), restricted by placing an aperture in the camera/photomultiplier tube image
plane ................................................................................................... 19

Figure 2.4. Schematic diagram of the TCSPC experimental setup. Egy: G-T: Glan-
Taylor prism; MCP-PMT: microchannel-plate photomultiplier tube; CFD: constant
fraction discriminator; TAC: time to amplitude converter ..................................... 20

Figure 3.1. Molecular structures of lipids used in this study: (a) 1,2-dioleoyl-sn-glycero-
3-phosphocholine (DOPC), (b) 1-oleoyl-2-[l2-[(7-nitro-2-l,3-benzoxadiazol-4-
yl)amino]dodecanoyl]-sn-glycero-3 -phosphocholine (1 8: 1-12:0 NBD-PC), (c)
cholesterol ............................................................................................ 37

Figure 3.2. Representative excitation and emission spectra of extruded liposomes
containing 98 mol% DOPC and 2 mol% NBD-PC. Both spectra have been normalized for
clarity of presentation ............................................................................... 38

Figure 3.3. TEM images of liposomes comprising 98 mol% DOPC and 2 mol% NBD-PC
produced by (a) extrusion and (b) sonication .................................................... 39

Figure 3.4. Size distribution obtained fi'om DLS, showing percent mass as a function of
diameter for extruded liposomes comprising 98 mol% DOPC and 2 mol% NBD-
PC ...................................................................................................... 40

Figure 3.5. Size distribution obtained fi'om DLS, showing percent mass as a function of
diameter for extruded liposomes comprising 68 mol% DOPC, 30 mol% cholesterol, and 2
mol% NBD-PC41

xi

Figure 3.6. Size distribution obtained from DLS, showing percent mass as a function of
diameter for sonicated liposomes comprising 98 mol% DOPC and 2 mol% NBD-
PC ...................................................................................................... 42

Figure 3.7. Size distribution obtained from DLS, showing percent mass as a function of
diameter for sonicated liposomes comprising 68 mol% DOPC, 30 mol% cholesterol, and
2 mol% NBD-PC .................................................................................... 43

Figure 3.8. Representative FRAPP recovery curve and residual plot (below) for sBLMs
comprising 98 mol% DOPC and 2 mol% NBD-PC. sBLMs were prepared with extruded
liposomes. Similar recovery curves were obtained for sBLMs prepared with sonicated
liposomes. Photobleaching time was 300 ms ................................................... 44

Figure 4.1. Molecular structures of lipids used in this study: (a) 1,2-dioleoyl-sn—glycero-
3-phosphocholine (DOPC), (b) l,2-dioleoyl-sn-glycero-3-[Phospho-rac—(1-glycerol)]
(DOPG), (c) 1,2-dioleoyl-sn glycero-3 -phosphoethanolamine-N—7-nitro-2-l,3-
benzoxadiazol-4-yl (18:1 NBD-PE) .............................................................. 63

Figure 4.2. Representative fluorescence lifetime data and residual plot (below) for NBD-
PE in DOPG/DOPC lipid vesicles. A two-parameter exponential decay, corresponding to
two fluorescence lifetimes (1:1 and 12), gave the best fit of the data. F-test statistics were
used to determine the statistical difference in goodness of fit for higher parameter
exponentials .......................................................................................... 64

Figure 4.3. Schematic diagram of the NBD-PE molecule. The NBD fluorophore exists in
a range of locations in a dielectric gradient, where 8 denotes the dielectric constant in

different regions of the lipid. We interpreted our results in the context that multiple
fluorescence lifetimes arise from NBD diffusing in a dielectric gradient. .................. 65

Figure 4.4. Representative induced orientational anisotropy data and residual plot
(below) for NBD-PE in DOPG/DOPC lipid vesicles. A two-parameter exponential decay,
corresponding to two reorientation times (tom and Tom), provided the best fit of the
data. F -test statistics were used to determine the statistical difference in goodness of fit
for higher parameter exponentials ................................................................. 66

Figure 4.5. Illustrations of (a) Type I (prolate) rotor, and (b) Type II (oblate)
rotor .................................................................................................... 67

Figure 4.6. Representative FRAPP recovery curve for a DOPC/NBD-PE supported
bilayer membrane (sBLM) with residuals (below). Total photobleaching time was 300
Figure 5.1. Molecular structures of the lipids used in this study: (a) 1,2-dioleoyl-sn-
glycero-3-phosphocholine (DOPC), (b) 1-pa1mitoyl-2-hydroxy-sn-glycero-3-
phosphocholine (p-lysoPC), and (c) the fluorophore l-oleoyl-2-[12-[(7-nitro-2-1,3-
benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (N BD—PC). . . . . ...89

xii

Figure 5.2. Representative SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis) processed during NEST purification. Lane assignments are as follows:
Lane 1: marker, lane 2: 3"d column wash, lanes 3 through 8: 1St through 6th column
elutions, lane 9: sample after 48-hour dialysis using 4th elution .............................. 90

Figure 5.3. (a) TEM image showing representative 69:29:2 liposomes after dialysis. (b)
TEM image showing a representative 69:29:2/NEST proteoliposome after dialysis. Such
unilamellar liposomes and proteoliposomes were used to form sBLM structures on
silica ................................................................................................... 91

Figure 5.4. DLS results showing the CHAPS/69:29:2/NEST micelle to proteoliposome
transition during dialysis. Data represent means i SD of three separate dialysis runs. A
Boltzmann sigmoid function, d = d2+((d1-d2)/(l+exp((t-to)/dt))), was fitted to the data. In
this function, d is the time-dependent mean particle diameter, t is time, d1 is the initial
particle diameter, d2 is the final particle diameter, to is the center time value, and dt is the
time constant. The fitted sigmoid had a correlation coefficient of 0.999. Fitted parameters
were as follows: (11 = 1.7 :t 0.6 nm, d2 = 43.0 :I: 0.5 nm, to = 12.9 :I: 0.4 h, and dt = 2.7 :1:
0.2 h ................................................................................................... 92

Figure 5.5. Time-course of conversion of p-lysoPC to palmitic acid during dialysis. A
100% conversion of p-lysoPC to palmitic acid was assigned to 69:29:2 liposomes
containing uninhibited NEST after 48-h dialysis. Data are means :1: SD of two separate
assays. A line was fitted to the data using linear regression analysis. The fitted line had a
correlation coefficient of 0.990 .................................................................... 93

Figure 5.6. DL of NBD-PC in sBLMs for several systems identified by the following
system numbers': (1) 98:2 (98 mol% DOPC, 2 mol% NBD-PC), (2) 98:2 (w/NEST), (3)
69:29:2 (69 mol% DOPC, 29 mol% p-lysoPC, and 2 mol% NBD-PC), (4) 69:29:2
(w/DFP), (5) 69:29:2 (w/NEST+DFP), (6) 69:29:2 (w/NEST), (7) 69:20:9:2 (69 mol%
DOPC, 20 mol% p-lysoPC, 9 mol% palmitic acid, and 2 mol% NBD-PC. Data are means
i SD fiom three different bilayers. Systems with statistically identical DL are labeled by a
common letter (ANOVA, Tukey post-hoc test, p < 0.05). D1, for 69:29:2 bilayers treated
with PMSF and 69:29:2 bilayers with PMSF-inhibited NEST (data not shown) were
statistically identical to 69:29:2 lipid systems with DFP in place of PMSF ................. 94

Figure 5.7. Mobile fractions of NBD-PC for several systems identified by the following
system numbers‘: (1) 98:2 (98 mol% DOPC, 2 mol% NBD-PC), (2) 98:2 (w/NEST), (3)
69:29:2 (69 mol% DOPC, 29 mol% p—lysoPC, and 2 mol% NBD-PC), (4) 69:29:2
(w/DFP), (5) 69:29:2 (w/NEST+DFP), (6) 69:29:2 (w/NEST), (7) 69:20:9:2 (69 mol%
DOPC, 20 mol% p-lysoPC, 9 mol% palmitic acid, and 2 mol% NBD-PC. Data are means
:1: SD from three different bilayers. The mobile fi'actions of all systems are statistically
identical (ANOVA, Tukey post-hoc test, p < 0.05), indicating that neither lipid
composition nor the presence of protein and/or inhibitors influenced liposome
rupture ................................................................................................ 95

xiii

Figure 6.1. Molecular structures of the synthetic lipids used in this study:
dioleoylphosphatidylcholine (DOPC), palmitoyl-oleoyl phosphatidylcholine (POPC), and
the fluorophore NBD-phosphatidylcholine (N BD-PC) ....................................... 108

Figure 6.2. FRAPP recovery curve for an interface formed from proteoliposomes at an
80:1 (w/w) lipid to protein ratio .................................................................. 109

Figure 6.3. FRAPP recovery curve with residuals (below) for an interface formed fi'om
membrane fractions of Saccharomyces pombe cells .......................................... 110

Figure 6.4. FRAPP recovery curve with residuals (below) for an interface formed from
the lipid fi'action of Saccharomyces pombe cell membrane ................................. 111

Figure 6.5. Representative FRAPP recovery curve with residuals (below) for a POPC
sBLM ................................................................................................ 112

xiv

r(0)

NOMENCLATURE

time

fluorescence lifetime

reorientation time

average diffusion rate

diffusion rate with respect to axis 1'

ith pre-exponential factor

wobbling diffusion coefficient

time-dependent light emission intensity polarized parallel to incident beam
time-dependent light emission intensity polarized perpendicular to incident beam
time-dependent anisotropy function

infinite-time anisotropy

initial, zero-time anisotropy

solvent viscosity

number of data points in each data set in ANOVA

semi-angle of the fluorophore confining cone volume

Boltzmann’s constant

total data sets compared in ANOVA
time-dependent post bleach fluorescence
average translational diffusion coefficient
Ronchi ruling stripe periodicity

mobile fraction

XV

parameter used in ANOVA analysis, found from table of critical values for F
distributions

studentized degrees of freedom

value corresponding to the confidence level in ANOVA analysis
spherical volume

temperature

Tukey parameter

statistical p-value

number of model parameters

xvi

 

AN OVA
BLM
DF P
DLS
DOPC
DOPG
ER

FFA

FRAPP
ICso
p-lysoPC
MD

MSE

NBD-PC

NEE-PE

NEST
NTE
PC
PG

PMSF

LIST OF ACRONYMS

analysis of variance

bilayer lipid membrane
diisopropylphoSphorofluoridate

dynamic light scattering

1 -palmitoyl-2-hydroxy-sn-glycero-3 -phosphocholine
1 ,2-dioleoyl-sn-glycero-3-[Phospho-rac-(1 -glycerol)]
endoplasmic reticulum

free fatty acid

fluorescence recovery after pattern photobleaching
half-maximal inhibitory concentration

1 —palmitoyl-2-hydroxy-sn- g1 ycero-3 -phosphocholine
molecular dynamics

mean square for error

1-oleoyl-2-[ l 2-[(7-nitro-2-l ,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-
glycero-3-phosphocholine nitro-benzo derivative

1 ,2-dioleoyl-sn glycero-3-phosphoethanolamine-N-7-nitro-2-l ,3-
benzoxadiazol-4-yl

neuropathy target §s_terase catalytic domain
neuropathy target esterase
phosphatidylcholine

phosphatidyl glycerol

phenylmethylsulfonyl fluoride

xvii

RC

SAM

SD

SDS-PAGE

sBLM

tBLM

TCSPC

TEM

TF

photosynthetic reaction center

self—assembled monolayer

standard deviation

sodium dodecyl sulfate polyacrylamide gel electrophoresis
supported bilayer lipid membrane

tethered bilayer lipid membrane

time-correlated single photon counting

transmission electron microscopy

tissue factor

xviii

1. INTRODUCTION

1.1. Overview and Significance of the Problem

This dissertation outlines four fundamental investigations that involved the
measurement of the diffusion of fluorescently tagged lipid molecules embedded in model
bilayer lipid membranes (BLMS) of different compositions. Model BLMS mimic
biological cell membranes and allow fundamental membrane properties to be measured
in a well-defined, reproducible system. Model BLMS were formed with molecules such
as lipids, cholesterol, and fatty acids, and macromolecules such as membrane-associated
proteins. Lipid fluorophore diffusion in model BLMS was measured optically by
monitoring fluorescence emission upon excitation with a laser beam.

The overall goal of the studies was to obtain a fundamental understanding of the
effect of incorporated molecules and macromolecules on the rotational and/or
translational diffusion of lipid fluorophores in model BLMS. The measurement of such
fluorophore diffusion is useful in assessing membrane fluidity,1 which plays important
roles in cell signaling, cell adhesion, and membrane protein functionality.” In response
to environmental stresses such as temperature, salt concentration, and presence of
solvents, cells adapt by increasing or decreasing membrane fluidity in an effort to
minimize cellular damage or prevent cell death.2 The results of this work may be used to
design bilayer interfaces for BLM-based biosensor and bioelectronic devices and may

provide insight into the important interactions that influence fluidity in cell membranes.

Liposomes are stable, spherically shaped model bilayers that enclose aqueous
cores. They are precursors to the formation of model supported bilayer lipid membranes
(sBLMs) when they adsorb to a hydrophilic interface, fuse together, and rupture. Such
sBLMs are two-dimensional and have a thin film (~l A) of water that separates the
hydrophilic head of the lower leaflet of the bilayer from the substrate.4 Liposomes can
also be used in the formation of model tethered bilayer lipid membranes (tBLMs) in
which the bilayer is physically elevated above the surface, typically by several
nanometers. The formation of sBLMs and tBLMs is often monitored with techniques
such as surface plasmon resonance, electrochemical impedance spectroscopy, atomic
force microscopy, and quartz crystal rrricrogravimetry.5'8

Sonication and extrusion are two common methods of liposome preparation.
Since the mechanism of forming liposomes by sonication (acoustic energy) is vastly
different than extrusion (pressure drop across a polycarbonate membrane), there is some
degree of uncertainty as to whether the diffusion transport properties in model
membranes prepared by the two techniques can be directly compared. In the first study of
this dissertation, we examined whether the rotational and translational diffusion
coefficients of lipid fluorophores in model membranes prepared by sonication and
extrusion can be directly compared. We also assessed the effect of the two preparation
techniques on the mean size and size distribution of liposomes.

The second study was conducted to gain a fundamental understanding of the
effect of lipid head group intermolecular interactions on membrane fluidity. We
measured rotational and translational diffusion coefficients of fluorophores in

phosphatidylglycerol (PG) model BLMS in which the head groups have a tendency to

form relatively strong hydrogen bonds, and in phosphatidylcholine (PC) model BLMS in
which head groups interact primarily via relatively weak van der Waals and screened
electrostatic forces.9’”

In the third study, we incorporated NEST (NTE esterase domain), the catalytic
domain of the membrane protein neuropathy target esterase (N TE), into model BLMS to
study the effect of free fatty acids (F FA) from enzyrne-induced lysophospholipid
hydrolysis on membrane dynamics. This study was motivated by the hypothesis that NTE
may have a physiological role of regulating the physicochernical properties of
membranes, including their fluidity.

Unlike tBLM interfaces, sBLMs are often unsuitable for studies involving
membrane proteins because interactions between the underlying surface and such
proteins have the potential to cause conformational changes that inhibit activity.”’13
However, it has been shown that certain trans-membrane proteins retain proper
functionality in fluid sBLMs even when the protein itself is immobile.”15 Results of the
fourth study show that, at sufficiently large lipid to protein ratios, fluid sBLMs can be
formed following the adsorption and rupture of yeast cell membrane fragments called
microsomes and synthetic membrane-associated protein-containing liposomes termed
proteoliposomes. We assessed the fluidity of sBLMs produced by reconstituting
microsomes with relatively high and low concentrations of membrane protein on silica,
and also determined a range of concentrations of NEST (used here as a model membrane-
associated protein) that can be used in proteoliposomes to form sBLMs.

These studies are important for several reasons. The first study demonstrated that,

while the physical characteristics of liposomes depend on the method of preparation, the

dynamics of fluorophores in BLMS constituted from such systems are independent of the
method of liposome formation. This finding is significant because it establishes that
results from studies that utilize the two liposome preparation methods can be directly
compared. In the second, third, and fourth studies, lipids, fatty acids, and macromolecules
were shown to measurably influence model BLM fluidity. This research provides a
deeper understanding of how constituent molecules influence model BLM fluidity and
may provide insight into the molecules that affect fluidity of cell membranes. Given that
the functionality of certain membrane proteins has been shown to depend on membrane
fluidity,IMO the performance of BLM-based devices that incorporate membrane proteins
may rely on the ability to manipulate membrane fluidity. Understanding the molecules
that influence model membrane fluidity, the mechanisms that underlie these effects, and
how the activity of membrane proteins changes with increasing or decreasing fluidity will
allow for better design of bilayer interfaces for BLM-based devices. The results. of our
work with sBLMs and liposomes may be used to design BLM-based biosensors and

bioelectronic devices

1.2. Background

1.2.1. Structure and Function of Biological Membranes

The membrane is one of the most important components of living cells. It
separates the cell cytoplasm from its surroundings, and is generally depicted as a fluid
heterogeneous BLM comprising mainly phospholipids, cholesterol, carbohydrates, and

proteins. One of the primary functions of BLMS is to maintain a homeostatic state within

a cell by acting as a semi-permeable barrier preventing entry of toxic molecules, most
polar molecules, ions, and macromolecules, while allowing the passage of non-polar and
small polar molecules such as water.21 Another primary function of the BLM is to
provide an environment for membrane proteins to function.22 Unlike prokaryotic cells,
many processes that occur in eukaryotic cells involve organelles that incorporate BLMS.21
These processes include aerobic respiration in mitochondria, protein synthesis in the
Golgi apparatus, and protein transport and folding in the endoplasmic reticulum.”

The basic building blocks of most BLMS are phospholipids, which are
amphipathic molecules composed of one glycerol moiety bonded to a phosphate-based
hydrophilic head group and one or two hydrophobic fatty acid tails attached to the
glycerol base via ester bonds (Figure 1.1). BLM stability arises primarily from van der
Waals and hydrophobic interactions between the acyl chains of the lipid.21 A wide range
of cationic, anionic, and zwitterionic molecules constitute the hydrophilic head group of
phospholipids. Fatty acid chains of different lengths and degrees of saturation make up
the hydrophobic portion of lipids. The various combinations of hydrophilic head groups
and hydrophobic tail groups allow for a multitude of lipids, each with unique physical
properties such as phase transition temperature, solubility, and diffusivity in a membrane

structurezz’23

1.2.2. Model Membranes: Liposomes, Supported Bilayer Membranes, and Tethered

Bilayer Membranes

Mimicking of in vivo characteristics of biological membranes with artificial

model membranes in vitro has led to a more comprehensive understanding of, for

example, membrane protein functionality, lipid-lipid interactions in membranes, and
physical properties of membranes. Three common examples of model membranes are
liposomes (lipid vesicles), sBLMs, and tBLMS. Liposomes are spherically shaped bilayer
structures that form spontaneously when phospholipids such as phosphatidylcholine (PC)
are added to water. Liposomes can be unilamellar or multilamellar, with sizes typically
ranging from tens of nanometers to several microns depending, on the preparation
technique. Liposomes are commonly used in drug delivery due to their unique ability to
store hydrophilic species in the aqueous core and hydrophobic compounds in the acyl
chain region of the BLM.24

McConnell and coworkers were the first to develop artificial, two-dimensional
sBLMs on silica surfaces using molecular self-assembly via liposome fusion.” sBLMs
have also been formed on quartz and mica surfaces in the same manner.”’26 There are
three fundamental mechanisms by which sBLMs are formed from liposomes.27 In the
first, vesicles adsorb to a surface and rupture immediately as a result of highly favorable
lipid-surface interactions, forming individual bilayer patches that associate over time to
form a uniform sBLM. This is usually the case when liposomes comprising a positively
charged lipid such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and a
zwitterionic lipid such as 1,2-dioleoyl-sn—glycero-3-phosphocholine (DOPC) associate
with a negatively charged quartz or silica surface via strong electrostatic interactions. In
the second, vesicles adsorb to the surface and do not undergo fusion and subsequent
rupture until a critical surface coverage is reached. This occurs with the DOTAP/DOPC
system at a moderate salt concentration. The salt screens lipid/surface electrostatic

attraction. The third way involves the adsorption of a supported lipid monolayer on a

surface by Langmuir-Blodgett deposition, followed by formation of the upper leaflet by
the fusion of liposomes.28

tBLMs are two-dimensional model membranes that are physically elevated above
the underlying substrate by tethering molecules or a polymer cushion.29 tBLM systems
are generally used for studies involving membrane proteins.”34 The elevation above the
underlying substrate allows room for proper protein or channel conformation and
prevents undesirable interactions between the protein and the substrate. Construction of
tBLMs typically involves first the formation of a self-assembled monolayer (SAM) that
acts as the lower bilayer leaflet followed by the adsorption of liposomes that rupture to
form the upper bilayer leaflet. Many biosensor and bioelectronic devices that incorporate
membrane proteins and ion channels in model membranes utilize tBLM systems. In

addition, tBLMs can be formed on conductive substrates such as gold, making it possible

to characterize the interface electrochemically.

1.3. Outline of this Dissertation

1.3.1. Comparison of Liposomes Formed by Sonication and Extrusion: Rotational

and Translational Diffusion of an Embedded Fluorophore

Chapter 3 of this dissertation describes collaborative work with Dr. Gary J.
Blanchard and Dr. Monique M. Lapinski of the Department of Chemistry and Angelines
Castro-Forero of the Department of Chemical Engineering and Materials Science.35 Each

of the collaborators made equal contributions to the work.

Liposomes used as model BLMS are typically prepared by sonication or extrusion.
The basic mechanism for breaking down large multilamellar vesicles to relatively small
unilamellar vesicles varies greatly with each process. Sonication involves exposure of the
sample to acoustic energy, while extrusion involves the use of mechanical energy to force
the lipids through a porous polycarbonate membrane via pressure driven flow. Since
these processes differ greatly, it is conceivable that the physical and molecular scale
properties of liposomes prepared by the two techniques would be different, making it
difficult to directly compare model BLM systems reconstituted from sonication and
extrusion. The goal of this study was to explore potential differences by using
fluorescence recovery after pattern photobleaching (FRAPP) to measure translational
diffusion coefficients of fluorophores in sBLMs, time-correlated single photon counting
(TCSPC) to measure fluorescence lifetimes and rotational diffusion coefficients of
fluorophores in liposomes, and dynamic light scattering (DLS) and transmission electron
microscopy (TEM) to assess physical characteristics such as mean liposome sizes and

size distributions.

1.3.2. Effect of Hydrogen Bonding on Rotational and Translational Dynamics of a

Head Group-bound Fluorophore in Bilayer Membranes

Over the past ten to fifteen years, molecular dynamics (MD) simulations have
provided much insight into the molecular level lipid-lipid and lipid-protein interactions in
BLMS, with the most commonly modeled lipids being zwitterionic PC and
phosphatidylethanolamine.10 Results of MD simulations have recently been published

that involve anionic lipids such as phosphatidylserine and PG.11 PG lipids have a glycerol

moiety in the head group, which radial distribution functions from simulations have
shown interact strongly with phosphate oxygen atoms and carbonyl groups of
neighboring lipids in the BLM structure via hydrogen bonding.10 MD simulations also
predict that water molecules form “water bridges” between phosphate oxygen atoms cf
adjacent phospholipids via hydrogen bonding. For zwitterionic PC membranes, radial
distribution functions show interaction between the positively charged nitrogen of the
choline head group and the negatively charged phosphate of neighboring lipids in
addition to the aforementioned “water bridges.” The interactions between head groups of
PG lipids in a membrane structure involve mainly hydrogen bonds, while screened
electrostatic interactions are more prevalent between head groups of PC lipids.

We hypothesized that the rotational and translational diffusion of head-group-
tethered fluorophores in BLMS would be affected by intermolecular lipid head group
interactions. Chapter 4 of this dissertation describes this work, which used FRAPP and
TCSPC measurements to gain insight into the rotational and translational diffusion of
fluorophores in liposomes and sBLMs, respectively, consisting of the two structurally
different lipids, PC and PG.” This work was done in collaboration with Dr. Gary J.
Blanchard and Heather A. Pillman of the Department of Chemistry, with equal

contributions from collaborators.

1.3.3. Influence of Lysophospholipid Hydrolysis by the Catalytic Domain of
Neuropathy Target Esterase on Bilayer Lipid Membrane Fluidity

Neuropathy target esterase (NTE) is an important medically relevant membrane

protein found in human neurons and other cell types. NTE inhibition by organophosphate

compounds has been shown to be directly linked to the onset of a type of paralysis known
as organophosphorous induced delayed neuropathy (OPIDN) as well as motor neuron
disease.“38 Recent studies suggest that NEST selectively hydrolyzes a specific class of
lipid molecules called lysophospholipids.39 The hydrolysis of one lysophospholipid yields
one fatty acid molecule and one hydrophilic glycerophosphocholine head group.
Although the physiological role of NTE is poorly understood, published reports indicate
that the cellular function of NTE may involve the control of cytotoxic accumulation of
lysophopspholipids.4°'41 This work involved use of an assay for measuring fi'ee fatty acid
(F FA) concentrations, in conjunction with FRAPP, DLS, and TEM to study the effect of
NEST-induced lysophospholipid hydrolysis on the dynamics of fluorophores in model
sBLMs. By extension of our work done with NEST and model membranes, we postulate
that NTE may play a role in influencing the fluidity of cell membranes by means of
lysophospholipid hydrolysis. This work is described in Chapter 5 of this dissertation and
involved collaboration with Professor Rudy J. Richardson of the School of Public Health,

Department of Neurology at the University of Michigan.

1.3.4. Effect of Membrane Protein Concentration on the Translational Dynamics of
an Embedded Fluorophore in Supported Bilayer Membranes Constituted from
Proteoliposomes

tBLM interfaces are generally considered suitable platforms for the
characterization of the functionality of membrane proteins. sBLM interfaces have also

been used to study the activity and functionality of trans-membrane proteins.15 However,

10

 

the effect of membrane protein concentration on the rupture of proteoliposomes to form
fluid sBLMs is not well-understood.

In this study, we used FRAPP to determine whether fluid sBLMs with
incorporated proteins can be formed on silica substrates over a range of lipid to protein
ratios. We formed interfaces on silica using yeast microsomal membrane fragments as
well as proteoliposomes comprising synthetic DOPC lipids and NEST, which was used
here as a model membrane-associated protein. We qualitatively assessed the effect of
membrane proteins on sBLM reconstitution with microsomal membrane fragments and
quantitatively determined a range of protein concentrations that would lead to sBLM
formation with DOPC and NEST proteoliposomes. This work is described in Chapter 6
of this dissertation and involved collaboration with Dr. Sachin R. Jadhav of the
Department of Chemical Engineering and Materials Science, with equal contributions
from each collaborator. We acknowledge Dr. R. Michael Garavito of the Department of

Biochemistry and Molecular Biology for supplying the microsome samples.

11

 

Figure 1.1. Molecular structure of a common lipid in mammalian cell membranes, 1,2-
dioleoyl-sn-glycero-3 -phosphocholine (DOPC). The phosphate and choline groups
comprise the hydrophilic head group and the dioleoyl acyl chains constitute the
hydrophobic tail group.

12

2. EXPERIMENTAL METHODS

2.1. Overview

The two fluorescence techniques used to monitor the dynamics of lipid
fluorophores in this dissertation are FRAPP, to measure translational diffusion in two-
dimensional sBLMs, and TCSPC, to measure rotational diffusion in spherical liposomes.
Detailed explanations as well as diagrams of the experimental setups for each technique

are included in this chapter.

2.2. Fluorescence Recovery After Pattern Photobleaching

FRAPP is generally used to measure mobile fiactions and lateral diffusion
coefficients of fluorescently tagged lipids in sBLMs on glass, quartz, silica, and mica
surfaces. This is done by selectively photobleaching sections of the sample and recording
the fluorescence recovery due to the diffusion of fluorophores from the non-
photobleached fiinges. Since it is difficult to determine the translational diffusion of
fluorophores embedded within liposomes, it is common to reconstitute liposomes in the
form of sBLMs on substrates such as those mentioned above. The transformation of
liposomes to sBLMs is accomplished through a series of steps that begin with vesicle
adsorption, transition into vesicle-surface and vesicle-vesicle interactions, and end with
vesicle fusion and/or rupture and subsequent bilayer formation.26’27’42‘43 Translational
diffusion coefficients and mobile fractions of fluorophores in sBLMs can be extracted

from the F RAPP data using a model developed by Starr and Thompson.44

13

The experimental setup (Figures 2.1 and 2.2) consisted of a double syringe pump
used to simultaneously infuse and withdraw solutions from a custom-made 1 mL flow
cell, an inverted microscope with a 32x objective lens, an argon ion laser, a side-on
photomultiplier tube (PMT), a photon counter, and a fast preamplifier. The 488 nm line
of the laser was directed through a 5x beam expander connected to a pair of optical flats
used to toggle between a low intensity (20 ,uW) monitoring beam for continuous
fluorescence detection and a high intensity (500 mW) beam for photobleaching
fluorophores. A Ronchi ruling of 25 ,um periodicity was placed in the back image plane
of the microscope to create a fiinge pattern on the sample. An aperture was placed in
front of the PMT to restrict the observation area, to prevent unbleached fluorophores
from diffusing from outside the bleached area to the observation zone during
measurements of the fluorescence recovery. The photobleached area was approximately
200 ,um in diameter, while the observed area was 75 pm, as shown in Figure 2.3.

To conduct these measurements, a selected region of the sBLM was exposed to an
intense laser beam, causing an irreversible photodegradation or bleaching of fluorophores
in the illuminated regions of the fringe pattern. After photobleaching, fluorescence
recovery in the monitored region occurred at a rate determined by the translational
diffusion of unbleached fluorophores behind the dark fringes at the time of
photobleaching. Thus, the fluorescence recovery data enables estimation of the

translational diffusion coefficient of the fluorophores.25

14

2.3. Time Correlated Single Photon Counting

Fluorescence lifetime and time-resolved orientational anisotropy data of
fluorophores in liposomes can be measured with TCSPC, which is useful for detecting
low intensity light signals with picosecond time resolution.45 In our work, fluorescence
lifetime data were used to assess differences in the local environment of fluorophores,
while anisotropy data were used to estimate the confined volume in which a fluorophore
rotates as well as the rate of rotation within that volume, using a hindered rotor model

developed by Lipari and Kinosita‘m’47

or the free rate of rotation of the fluorophore as a
Type I (prolate) or Type II (oblate) rotor using a free rotor model by Chuang and
Eisenthal.48 Detailed discussions of the hindered rotor and free. rotor models are given in
Chapters 3 and 4, respectively.

The TCSPC experimental apparatus (Figure 2.4) has been described
elsewhere.”50 The essential features include a 7 W mode-locked Nd:YAG CW laser that
produces 1064 nm pulses for 100 ps at a frequency of 76 MHz. Usually, the second or
third harmonic of this light is used to excite a cavity dumped dye laser, which typically
has outputs of ~ 50 mW with ~ 5 ps pulses at a ~ 4 MHz repetition rate. The beam is
then split into a reference beam and a sample beam, which can be frequency-doubled
depending on the excitation wavelength of the fluorophore in the sample. The sample
beam then travels through a set of neutral density filters into a vertical polarizer before
contacting the sample solution. Emitted light is collected at 90° from the incident
excitation beam, and a Glan-Taylor (G-T) prism is used to polarize the emitted light

54.7° with respect to the excitation polarization for fluorescence lifetime measurements,

and either parallel (0°) or perpendicular (90°) to the excitation polarization for anisotropy

15

measurements. The transient fluorescence signals are directed through a monochromator
and subsequently detected with a microchannel plate photomultiplier tube (MCP PMT).
The electronics used to temporally resolve the fluorescence transients includes a constant
fi'action discriminator (CFD) to determine the maximum signal, and a time-to-amplitude

converter (TAC) biased amplifier.

16

  
 

f To Microscope Laser

Optical Iris

\.

<9 0 e

Optical Flat #1

Beam Expande

 

 

 

 

 

 

Optical Flat #2

Figure 2.1. Schematic diagram of the FRAPP experimental setup. The laser beam is split
into a low intensity monitoring beam (through a filter) and a high intensity
photobleaching beam at optical flat #1 . The beams are recombined at optical flat #2 prior

to impingement on the sample.

17

SBLM

Objective !

   

Dichroic Mirror

I E }. LaserLight
5?- s (488 nm)

Filter Ronchi Ruling

v

Filter -

TOPMT

Figure 2.2. Schematic diagram of the FRAPP experimental setup before and after the
laser beam reaches the sample. The dichroic mirror reflects the excitation laser light,
while allowing fluorescent light emitted from the interface to pass through to the detector.
Iggy: sBLM: supported bilayer lipid membrane; PMT: photomultiplier tube.

18

 

(A) (B)

Figure 2.3. A) Fringe pattern in the illuminated area (diameter = 200 pm) obtained using
a 100 lines per inch (25 ,um periodicity) Ronchi ruling. B) Observation area (diameter =
75 ,um), restricted by placing an aperture in the camera/photomultiplier tube image plane.

19

 

mode-locked YAG laser

    
     
    
    

pulse
diagnostics
2(1)

 

cavity dumped dye laser

A fiber optic

...

560 nm - 900 nm ;.—-: switchable
3 delay
olarization . ' _
p control it dIOdc CFD delay [:1

scrambler
raterneter

CFD

 

if

monochromator MCP PMT

Figure 2.4. Schematic diagram of the TCSPC experimental setup. Key: G-T: Glan-
Taylor prism; MCP-PMT: microchannel-plate photomultiplier tube; CFD: constant
fraction discriminator; TAC: time to amplitude converter.

20

3. COMPARISON OF LIPOSOMES FORMED BY SONICATION AND
EXTRUSION: ROTATIONAL AND TRANSLATIONAL DIFFUSION OF AN
EMBEDDED FLUOROPHORE

3.1. Abstract

We report on the mean diameter and size distribution of liposomes formed by
extrusion and sonication, two widely used methods for liposome preparation. We
determined the morphology of liposomes by TEM and the mean diameter and size
distribution by DLS. We also address the issue of whether the diffusion transport
properties of model bilayers prepared by the two techniques differ at the molecular and
mesosc0pic levels. We used the phospholipid DOPC, with and without cholesterol, to
form liposomes, incorporating 18:1-12:0 NBD-PC as a lipid fluorophore to probe
dynamics. The rotational and translational diffusion of l8:1-12:O NBD-PC was
characterized by TCSPC and FRAPP, respectively. We found that, despite the apparent
differences in mean diameter and size distribution, both methods of preparation produced

liposomes that exhibited the same molecular and mesosc0pic scale environment.

3.2. Introduction

BLMS are important components of all cellular systems, and a much research has
been conducted to understand how to use model membranes as a means of
comprehending biological systems. One effective way to create model BLMS in solution
is to form liposome structures by the self-assembly of phospholipids in an aqueous

environment. An additional way is to induce the rupture of liposomes onto a planar

21

surface to form sBLMs. Liposomes can be created by a number of methods5 1'54 with their
size ranging from tens of nanometers to microns depending on the technique used to
create them. Two of the most common techniques used to prepare liposomes are

extrusions“so

and bath sonication}51 with each method yielding different mean diameters
and size distributions.
The extrusion method is used to produce monodisperse, unilamellar liposomes of

55-60.62 Typically, a lipid suspension is forced through a

a predetermined size.
polycarbonate membrane with a well-defined pore size to produce vesicles with a
characteristic diameter near the pore size of the membrane used to prepare them. One
significant advantage of extrusion is that the resulting mean liposome diameter and size
distribution are fairly reproducible from batch-to-batch.

Sonication is also used widely to prepare of liposome samples. With this method,
lipid solutions are agitated using acoustic energy from either a bath or a probe tip
sonicator. The induced pressure breaks up large multilamellar liposomes into relatively
small liposomes that may be either unilamellar or multilamellar in composition.61 The
time of sonication ultimately determines the liposome size, with the smallest radius being
in the range of 10.25 i 0.55 nm.63 An advantage of sonication over extrusion is that it is
less time-consuming; however, the resulting liposome batch-to-batch mean diameter and
size distribution are not as reproducible as those prepared by extrusion.

Because the basic mechanism of breaking down large liposomes to relatively
small liposomes differs greatly with sonication and extrusion, it is conceivable that the

properties of liposomes prepared by the two techniques would be different. This is an

important issue because it bears on whether or not experimental data acquired for model

22

BLMS formed by the two different techniques can be compared directly. The goal of this
study was to evaluate the mean diameter, size distribution, and rotational and
translational dynamics of small unilamellar liposomes prepared by bath sonication and by
extrusion, to determine if the method of preparation has any influence on these selected
bilayer properties. We explored the potential differences by using FRAPP to measure
translational diffusion coefficients and mobile fractions in sBLMs, TCSPC to measure
fluorescence lifetimes and rotational diffusion coefficients in liposomes, and dynamic
light scattering (DLS) and transmission electron microscopy (TEM) to assess mean

liposome diameters and size distributions.

3.3. Materials and Methods

3.3.1. Vesicle Preparation by Extrusion

Phospholipids DOPC, 18:1-12:0 NBD-PC, and cholesterol were purchased from
Avanti Polar Lipids Inc. (Alabaster, AL) and used as received. Figure 3.1 shows the
molecular structures of the lipids used in this study. DOPC was chosen as the primary
lipid in our studies because of its low transition temperature (-20°C),(’4 ensuring that the
resulting lipid bilayers would be fluid at room temperature (23 i 2°C). The fluorophore
1821—12:0 NBD-PC, which will be referred to as NBD-PC throughout the remainder of
this dissertation, was chosen to ensure structural similarity to the phospholipid to which it
is tethered (DOPC, 18:1). In addition, there is a significant body of knowledge on the

65-67

properties of this fluorophore, making it an attractive choice for probing BLM

structures. For each sample, 1 mM lipid solutions containing DOPC/NBD-PC (98:2,

23

mol/mol) or DOPC/cholesterol/NBD-PC (68:30:2, mol/mol/mol) were prepared. The
chloroform solvent was evaporated from the lipid mixture and the dry lipid film was
hydrated using HEPES® buffer (Sigrna-Aldrich, St. Louis, MO) containing NaCl (Fisher
Scientific, Fair Lawn, NJ). The buffer (10 mM HEPES®, 150 mM NaCl, pH 7.4) was
prepared using purified water from a Milli-Q Plus water purification system (Millipore,
Bedford, MA). The mixtures were processed five times through a freeze-thaw-vortex
cycle to ensure complete mixing of the sample constituents. Each cycle consisted of
immersion in liquid nitrogen for 5 min, followed by immersion in a 60°C water bath for 5
min and vortexing for 2 min. After the freeze-thaw-vortex cycle, the solutions were
extruded through two 400 nm pore diameter polycarbonate membrane filters using a
mini-extruder (Avanti Polar Lipids Inc., Alabaster, AL). The pre-extruded vesicle
suspension was then extruded eleven times through two polycarbonate membranes
(Avanti Polar Lipids Inc., Alabaster, AL) with a nominal pore diameter of 100 um. All

extrusions were performed at room temperature (23 :i: 2°C).

3.3.2. Vesicle Preparation by Sonication

Lipid mixtures with the same compositions as those used for the extrusion
experiments were used to form liposomes by sonication. The chloroform-solvated lipid
mixtures were dried under nitrogen and placed under vacuum at —45°C for 3 h. The dried
lipid mixtures were then hydrated in buffer (10 mM HEPES®, 150 mM NaCl, pH 7.4),
and sonicated for 20 min using a bath ultrasonic cleaner (Branson 1510, Branson
Ultrasonic Corporation, Danbury, CT). Fresh liposome solutions were prepared prior to

each experiment.

24

3.3.3. Transmission Electron Microscopy Imaging

A 10 pL aliquot of each sample was fixed on a F ormvar® nickel-coated grid using
a solution containing 2% uranyl acetate stain in water. Images were acquired using a
transmission electron microscope (TEM, JEOL 100CX) operated at an accelerating

voltage of 100 kV.

3.3.4. Dynamic Light Scattering

DLS was performed with a Protein Solutions DynaPro—MS/X system (Wyatt
Technology Corporation, Santa Barbara, CA) on DOPC/NBD-PC liposome solutions,
with and without cholesterol, to determine mean liposome diameters and size
distributions. Liposome solutions were diluted by a factor of ten with buffer and placed
in a polyethylene cuvette for measurement. A total of three measurements were made for

each lipid composition.

3.3.5. Excitation and Emission Spectra

Steady state excitation and emission spectra (Figure 3.2) were acquired for vesicle
samples, for the purpose of characterizing the NBD fluorophore band positions. We used
an emission spectrometer (Spex Fluorolog 3, Edison, NJ) for all measurements, set to a

spectral bandpass of 3 nm for both excitation and emission monochromators.

25

Ewarm

3.3.6. Time-Correlated Single Photon Counting

Fluorescence lifetimes and reorientation times of fluorophores in liposomes were
measured with TCSPC. This information was used to assess the rotational diffusion
coefficients and the local environment of fluorophores. The experimental TCSPC setup is
described in Section 2.3 of this dissertation. For each lipid system considered in this

work, three separate liposome solutions were interrogated with TCSPC.

3.3.7. Deposition of Supported Lipid Bilayers

Fused silica microscope slides (75 mm x 25 mm x 1 mm) were purchased from
Technical Glass Products, Inc. (Painesville, OH). They were cleaned by bath sonication
(Branson 1510, Branson Ultrasonic Corporation, Danbury, CT) in detergent solution for
20 min, rinsed with DI water, baked at 160°C for 4 h and plasma treated (Harrick Plasma,
Ithaca, NY) with oxygen under vacuum (200 mTorr) for 10 min immediately before
bilayer deposition. sBLMs were deposited by vesicle fusion in a custom made flow cell.68
The flow cell was initially washed with buffer, followed by a 1 h incubation with the

liposome solution and a final buffer wash to remove any unadsorbed liposomes. All

experiments were performed at room temperature (23 fl: 2°C).

3.3.8. Fluorescence Recovery After Pattern Photobleaching

FRAPP was used to determine translational diffusion coefficients and mobile
fractions in sBLMs. The experimental FRAPP setup is described in Section 2.2 of this

dissertation. For each lipid system considered in this work, three separate sBLM

26

interfaces were formed on silica slides and each interface was interrogated with FRAPP

at five different locations of the bilayer.

3.3.9. Statistical Analysis

Data fitting was accomplished using OriginPro 7.5 (OriginLab
Corporation, Northampton MA) which uses the Levenberg-Marquardt algorithm for non-
linear least squares fitting. An F-test of comparison was conducted to determine whether
an exponential model using n parameters provided a statistically significant improvement

in the goodness of the fit of fluorescence lifetime and fluorescence anisotropy data than

an exponential model using n-l parameters. Rotational diffusion coefficients (Dw),

translational diffusion coefficients (D1,), and particle diameters are reported as means i:

standard deviation (SD). Multiple comparison ANOVA with a post-hoc Tukey test (p <

0.05) was used to determine if differences among the means were statistically significant.

3.4 Results and Discussion

The purpose of this work was to compare two methods of liposome preparation,
extrusion and bath sonication, to determine if the method of preparation affects the size
and dynamical properties of the resulting liposomes and sBLMs constituted from the
liposomes.

We were primarily concerned with three issues in this work: the physical
attributes (mean diameter and size distribution) of liposomes formed by the two methods,

and the rotational and translational dynamics of fluorophores in liposomes and sBLMs,

27

respectively. We also studied how the incorporation of cholesterol in liposomes affected
each of these issues. We expected that there would be a marked change in the
organization of BLMS with the addition of this component. Each of the issues described

above are considered individually.

3.4.1. Liposome Mean Diameter and Size Distribution

We used TEM and DLS to evaluate the mean diameter and size distribution of
liposomes formed by extrusion and sonication. TEM images (Figure 3.3) show a single
shell surrounding each liposome, suggesting that liposomes formed by the two methods
are both unilamellar in structure. However, the images provide little utility in evaluating
absolute sizes because of potentially large distortions induced by vacuum. Also, the high-
energy electron beam used in imaging could, in principle, damage liposome structures.
Such distortions may appear in the form of non-spherical structures. While appealing
from a visual perspective, TEM images do not lend themselves readily to the evaluation
of the size distribution of the liposomes. To address these issues, we also characterized
the liposomes by DLS, a technique that measures the hydrodynamic diameter of particles
suspended in solution. Because DLS collects scattering information on an ensemble of
particles, it is a relatively simple matter to obtain information on the particle size
tiistt-ihtttion.69'72

The mean diameters of vesicles formed by extrusion were 112 :I: 6 nm with
(Figure 3.4) and without (Figure 3.5) cholesterol. Thus, it appears that the presence of

cholesterol has no apparent influence on the size of liposomes formed by extrusion. This

is not surprising, since we expected that the extruded vesicles would be close to 100 nm

28

in diameter, with or without cholesterol, based on the size of the pores in the
polycarbonate membranes used to prepare them.55’56 On the other hand, liposomes
formed by sonication had diameters of 64 i 3 nm with cholesterol (Figure 3.6) and 28 i- 2
nm without cholesterol (Figure 3.7), showing a significant influence of cholesterol on
size. The mean liposomes sizes of the four aforementioned systems are summarized in
Table 3.1.

The size of sonicated liposomes, although smaller than those made by extrusion in
this study, can be controlled by the duration of sonication.61 We also note that liposomes
formed by sonication had a bimodal size distribution, with and without cholesterol, while
liposomes formed by extrusion only had a distinct bimodal size distribution when they
contained cholesterol. It is not clear if there is a compositional difference between

cholesterol-containing vesicles in the two distribution zones.

3.4.2. Fluorescence Lifetimes and Rotational Diffusion in Liposomes

We measured the fluorescence lifetime and induced orientational anisotropy of
tethered NBD fluorophores using TCSPC. The data provided by lifetime and anisotropy
measurements are different but complementary. For all of the liposome systems studied
here (sonicated and extruded, with and without cholesterol), we observed a fluorescence
population decay (Table 3.2) that can be fitted to a two-component exponential model
using F-statistic analysis. This behavior has been reported for NBD tethered to
phospholipids in a variety of environments.65’73'76 The fluorescence lifetime of NBD is
sensitive to the polarity of its immediate environment, where the shorter lifetime

corresponds to the NBD fluorophore existing in a more polar environment.77 For NBD

29

--I“-_ __ --.—

fluorophores tethered to the acyl chain of a phospholipid, it has been established that the
fluorophore can “loop back” to position itself in close proximity to the polar head group
region of the bilayer in which it resides.65‘67’78 The longer lifetime in this model
corresponds to the NBD fluorophore residing closer to the nonpolar region of the lipid
bilayer. Based on our observation of two fluorescence lifetimes, it is possible that the
fluorophore exists in two discrete populations during the timescale of the measurement.
However, the fact that we observe two lifetime values that differ by more than 4.5 ns
while the proposed environments for NBD are only separated by a few angstroms is
consistent with the fluorophore existing in a dielectric gradient that changes substantially
over the length scale of the fluorophore itself.77’79 We therefore believe that there is
effectively only one environment, hence one fluorescence lifetime, that the fluorophore
experiences, and the fact that we measure two fluorescence lifetimes experimentally is a
result of the fluorophore moving in a dielectric gradient. ,

For all of the liposome systems we studied, the two fluorescence lifetimes are
essentially the same with 1:1 2 2 ns and 11 z 6 ns (Table 3.2). We note that the relative

contributions of the two lifetime components depend to some degree on liposome
composition for the sonicated liposomes (~ 0.05 in the absence of cholesterol versus 0.3
with cholesterol, as opposed to ~ 0.3 for both extruded liposomes). This likely led to the
large differences in the ratio of pre-exponential factors [A 1/(A1+A2) and Az/(A1+A2)]
obtained for the sonicated liposomes, particularly in the absence of cholesterol. However,
owing to the structural complexity of these liposomes and the comparatively limited

information contained in the lifetime data, it would be Speculative to suggest that either

30

the structure of the liposome or the positional distribution of the NBD fluorophores
accounts for the observed differences in prefactors.

The fundamental principle in TCSPC measurements is that fluorophores with
absorption dipoles oriented parallel to the incident polarization are preferentially excited,
creating anisotropy in the system. For the anisotropy decay measurements reported here,
the chemical information contained in these experiments is in the form of the decay

functionality of r(t):

I||(t)—Il(t)

r“) = I”(t)+ 21,0) ‘3'”

 

where I“(t) and I r(t) are the emission intensities polarized parallel and perpendicular to
the vertically polarized incident excitation pulse, respectively.

Hypothetically, if a fluorophore is placed in solution, it will assume all possible
orientations with equal probability, hence the infinite-time anisotropy will decay to zero.
However, if the same fluorophore is incorporated into a BLM, it will likely not be able to
assume all orientations with equal probability due to its confinement, and as a result, r(oo)
will decay to a non-zero value. A fluorophore that is associated in a BLM typically does
not have the structural freedom to fully re-randomize its orientation within its
environment following excitation, giving rise to an infinite-time anisotropy. The zero-
time parameter r(O) is determined by the angle between the excited and emitted transition
dipole moments of the fluorophore, and r(oo) serves as a gauge for the orientational

confinement that the fluorophore experiences.47

31

 

In our experiments, the NBD fluorophore was tethered to the tail group of a
membrane bilayer constituent, and the anisotropy decay for this system was treated in the
context of a hindered rotor model.46’30'82 The tethered NBD fluorophore does not have the
structural freedom to orientationally re-randonrize fully within its hydrophobic
environment in the BLM following excitation, which gives rise to an infinite time

anisotropy, r(oo). For such systems, the function r(t) is governed by

r(t) = r(°<>)+ (( r(0)- r(0°) ) eXp(-t/ Ten) (32)

where r(oo) is the infinite-time anisotropy and r(O) is the zero-time anisotropy. The

parameter 70R is the reorientation time. This is related to the orientational relaxation time

of the fluorophore within its confining volume, and is approximated by"’ 82

7602
240W

 

TOR = (3.3)

where Dw is the “wobbling” diffusion coefficient and 60 is the semi-angle of the

confining cone volume of the fluorophore, which is related to the zero- and infinite-time

anisotropies by47

 

2:; = 0.5cos 60 (1+ cos 6,) (3.1)

32

The values of 60 for the extruded and sonicated vesicles, both with and without

cholesterol, are given in Table 3.3.

For each of the systems tested, the cone angles are the same within experimental
uncertainty, regardless of the method of liposomes preparation. We note that, for the
systems containing cholesterol, the recovered cone angles are measurably smaller (ca.
60° vs. ca. 40°) for both the extruded and sonicated liposomes in comparison to the
systems containing only the phospholipid and the fluorophore. This finding is consistent
with literature reports indicating that the addition of cholesterol to a bilayer serves to

83-87
(1.

make the bilayer less flui Our data point to not only a slightly longer 70R for the

cholesterol-containing system, but also a more restricted volume (60). From these data we
find Dw ~ 100 MHz for the phospholipid bilayer with no cholesterol, and Dw ~ 45 MHz

with the addition of cholesterol. It is interesting to note that there is an apparent
difference in z'OR for the cholesterol-containing liposomes. This difference in
reorientation time, while suggestive of a different environment formed by the two

methods, cannot be taken simply at face value. The cone angles recovered for these two

liposomes are the same within experimental uncertainty, and when the fundamental

quantity Dw is extracted from the 60 and 10R data, we find that there is no statistically

discernible difference for the liposomes formed by extrusion versus those by sonication.
It is important to emphasize that, in all of the anisotropy data, we do not find any

measurable differences between the molecular environments of the liposomes formed by

extrusion and sonication. While there is a slight difference in the fast component of the

NBD fluorescence lifetime for sonicated versus extruded liposomes containing

33

cholesterol, this finding is not reflected in the anisotropy decay dynamics, which are
arguably a more sensitive probe of the local environment of the fluorophore. With this
information, we turn to the translational diffusion behavior of the tethered fluorophore in

sBLMs formed from each type of liposome.

3.4.3. Translational Diffusion in Supported Bilayer Membranes

Measurement of the fluorescence recovery in sBLMs provided information on the
diffusion coefficient and mobile fraction of fluorescent lipids within the membrane.

Equation (3.5) was fit to the photobleaching recovery data,44

 

m 8 4 th 1 36 2D:
f(t):f(0)+3[1—f(0)]{1—(?)exp[- ”02 L ]+§eXp[— _”2_L_]} (3.5)

a

where f(t) is the post-bleach fluorescence (t > 0) normalized with respect to the constant
prebleach fluorescence (t < 0), with t=0 being the time of the bleach pulse, D1, is the
average translational diffusion coefficient of the fluorophores within the bleached area,
and m is the mobile fraction. The parameter a is the stripe periodicity of the Ronchi
ruling in the sample plane. Equation (3.5) assumes a single diffusing species
(fluorophores in the fluid sBLM) and one immobile species (fluorophores residing in
unruptured vesicles attached to the surface).

Equation (3.5) was fitted to the FRAPP data for each of the systems, and the best-
fit values of DL and m were extracted (Table 3.4). These FRAPP data provided two

important pieces of information. The first is that, for a given liposome composition, the

34

fitted values of DL and m were the same for liposomes made by extrusion and sonication.
Thus, differences in size between liposomes prepared by extrusion and sonication did not
seem to affect the translational diffusion of the resulting sBLMs. The second important
finding is that, as expected, the presence of cholesterol in the bilayer served to make the
fluorophore less fluid. This result was fully consistent with the anisotropy decay data
presented earlier, as well as previous findings that the addition of cholesterol to a
phospholipid bilayer decreases the translational diffusion of the lipids.“’88 Figure 3.5
shows a representative fluorescence recovery curve obtained for a sBLM of DOPC

containing NBD-PC as a fluorescent probe.

3.5. Conclusions

The goal of this study was to compare the physical size and dynamic properties of
liposomes prepared by sonication to those prepared by extrusion. Liposomes formed by
the two schemes were characterized in solution and in sBLMs formed via liposome
fusion on a fused silica substrate. The effect of cholesterol on the properties of the
liposomes and sBLMs was also evaluated. Our findings demonstrate that, while the mean
diameter and size distributions of the liposomes do indeed depend on the manner by
which they are prepared, the molecular and mesosc0pic organization of the BLMS, as
sensed through the dynamics of a tethered fluorophore, do not depend on the manner of
liposome formation. The molecular scale organization of the bilayers is determined by
interactions between the constituent species, which themselves do not depend on the

manner in which the bilayer is formed.

35

3.6. Recommendations for Future Work

We have shown that the mean diameter and size distribution of liposomes depend
on the technique used to prepare them. Although bimodal size distributions were
observed in both sonicated and extruded DOPC/cholesterol liposomes, the
lipid/cholesterol composition associated with each peak in the distribution in unknown. It
would be interesting to investigate the effect of liposome preparation methods on the
compositions of the populations in each size grouping of the bimodal distribution.

This study would involve the preparation of extruded and sonicated liposomes
using several lipidzcholesterol mole ratios. DLS would then be used to characterize the
bimodal distributions. The two populations would then be separated using a technique
such as centrifugation and doing a compositional analysis of each population would be
performed using techniques such as high performance liquid chromatography and mass

spectroscopy.

36

 

 

O
/ _ / _
,o=P—o o=P-o
e
H H
“”0
O O 0 O
O O
|
l I
NH
)\O
/
N+
./ \‘o
(a) (b) (c)

Figure 3.1. Molecular structures of lipids used in this study: (a) 1,2-dioleoyl-sn-glycero-
3-phosphocholine (DOPC), (b) l-oleoyl-2-[12-[(7-nitro-2-l,3-benzoxadiazol-4-
yl)amino]dodecanoyl]—sn-glycero-3-phosphocholine (18:1-12:0 NBD-PC), (c)
cholesterol.

37

 

 

1.0 -
. excitation emission
A 0.8 -
=5
3 L
g 0.6 -
t:
8
.5
'8 0.4 -
.E
E
ZCZD 0.2 -
00 r . r . r . r . r J
400 450 500 550 600
Wavelength (nm)

Figure 3.2. Representative excitation and emission spectra of extruded liposomes
containing 98 mol% DOPC and 2 mol% NBD-PC. Both spectra have been normalized for
clarity of presentation.

38

(a)

(b)

 

Figure 3.3. TEM images of liposomes comprising 98 mol% DOPC and 2 mol% NBD-PC
produced by (a) extrusion and (b) sonication.

39

40

 

30-

25-

20%

15*

Percent Mass

10~

 

 

II]

100

IT—TT

Diameter (run)

1000

 

10000

Figure 3.4. Size distribution obtained from DLS, showing percent mass as a function of
diameter for extruded liposomes comprising 98 mol% DOPC and 2 mol% NBD-PC.

40

 

30

 

254

zoi

15-

Percent Mass

10.

 

rrr

 

 

1 r r r rfirr—r

10 100 1000 10000
Diameter (nm)

Figure 3.5. Size distribution obtained from DLS, showing percent mass as a function of

diameter for extruded liposomes comprising 68 mol% DOPC, 30 mol% cholesterol, and 2
mol% NBD-PC.

41

80

 

70—

60-

50-

404

Percent Mass

30‘
20*

10‘

O l r r ITTFI]

10 100 1000

 

 

 

Diameter (nm)

Figure 3.6. Size distribution obtained from DLS, showing percent mass as a function of
diameter for sonicated liposomes comprising 98 mol% DOPC and 2 mol% NBD-PC.

42

45

 

40‘

35‘

30-

25~

20~

Percent Mass

15.
10*

o r r r l r r r I

I r

10 100 1000

 

 

 

Diameter (nm)

Figure 3.7. Size distribution obtained from DLS, showing percent mass as a function of

diameter for sonicated liposomes comprising 68 mol% DOPC, 30 mol% cholesterol, and
2 mol% NBD-PC.

43

 

0.70
0.65 I
0.60 P
0.55
0.50 h
0.45 h
0.40 i
0.35:

I

0.00
-0.05

Normalized Fluorescence Intensity

 

 

 

 

Figure 3.8. Representative FRAPP recovery curve and residual plot (below) for
sBLMs comprising 98 mol% DOPC and 2 mol% NBD-PC. sBLMs were prepared
with extruded liposomes. Similar recovery curves were obtained for sBLMs
prepared with sonicated liposomes. Photobleaching time was 300 ms.

Table 3.1. DLS results for extruded and sonicated liposomes of two systems: (1) 98
mol% DOPC and 2 mol% NBD-PC and (2) 68 mol % DOPC, 30 mol% cholesterol, and 2
mol% NBD-PC. Data are means :1: SD of three samples.

 

 

 

S stem Mean Diameter, Mean Diameter,
y Extruded Vesicles (nm) Sonicated Vesicles (nm)
112i6 28:1:2andl96i10
2 112:1:6and600i30 64:1:3and259i13

 

 

 

 

 

Table 3.2. Pre-exponential factor (A,) ratios and fluorescence lifetimes (1,) of NBD-PC
fluorophores in sonicated and extruded liposomes comprising (1) 98 mol% DOPC and 2

mol% NBD-PC and (2) 68 mol % DOPC, 30 mol% cholesterol, and 2 mol% NBD-PC“.

 

System” xii/(Aims) r. (us) Ass/(Aims) r2 (ns)

 

1 (sonicated) 0.06 :l: 0.01A 1.93 a 0.26E 0.94 e 0.01G 6.10 :l: 0.07J

 

1 (cxtmded) 0.44 e 0.02B 1.85 e 0.10E 0.56 i 0.02H 6.01 e 0.06JK

 

2 (sonicated) 0.31 e 0.03C 2.30 i 0.18F 0.69 e 0.03I 5.91 e 0.10K

 

2 (cxtmded) 0.27 e 0.01D 1.67 a 0.09E 0.73 e 0.01I 6.04 :l: 0.013JK

 

 

 

 

 

 

 

“Data represent means 3: SD of three liposome solutions.

bSystems with statistically identical pre-exponential factor ratios and fluorescence

lifetimes are labeled by common capital letters (ANOVA, Tukey post-hoc test, p < 0.05).

45

 

Table 3.3. Reorientation times (10R), confined cone semi-angles (6’0), and wobbling

diffusion coefficients (DW) of NBD-PC in sonicated and extruded liposomes comprising
(1) 98 mol% DOPC and 2 mol% NBD-PC and (2) 68 mol % DOPC, 30 mol%

cholesterol, and 2 mol% NBD-PC“.

 

 

 

 

 

System” T011015) 601°) 0.. (MHz)
1 (sonicated) 3.03 3. 0.22A 62 at 5C 113 r 27E
1 (extruded) 2.94 r 0.09A 57 s 5C 98 r 21E
2 (sonicated) 3.59 r 0.2813 42 r 3D 44 r 10F
2 (extruded) 3.00 r 0.22A 37 3: 2° 41 r 8F

 

 

 

 

 

 

“Data represent means i SD of three liposome solutions.
bSystems with statistically identical TOR, I90, and Dw are labeled by common capital

letters (AN OVA, Tukey post-hoc test, p < 0.05).

Table 3.4. Translational diffusion coefficients (D1,) and mobile fractions for NBD-PC in
sBLMs prepared with sonicated and extruded liposomes comprising (1) 98 mol% DOPC
and 2 mol% NBD-PC and (2) 68 mol % DOPC, 30 mol% cholesterol, and 2 mol% NBD-

Pc".

 

 

 

 

 

 

 

 

 

Systemb DL (umz/s) Mobile Fraction
1 (extruded) 2.24 r 0.25A 0.78 r 0.18C
1 (sonicated) 2.02 :t 0.15A 0.81 i 0.11C
2 (extruded) 1.65 r 0.22B 0.73 e 0.10C
2 (sonicated) 1.72 d: 0.04B 0.69 d: 0.06C

 

"Data represent means i SD of three liposome solutions.

bSystems with statistically identical DL and mobile fractions are labeled by common

capital letters (ANOVA, Tukey post-hoc test, p < 0.05)

46

4. EFFECT OF HYDROGEN BONDING ON THE ROTATIONAL AND
TRANSLATIONAL DYNAMICS OF A HEAD GROUP-BOUND
FLUOROPHORE IN BILAYER MEMBRANES

4.1. Abstract

We have studied diffusion transport properties of the fluorescently tagged
phospholipid l ,2-dioleoyl-sn glycero-3 -phosphoethanolamine-N-7-nitro-2-l ,3-
benzoxadiazol-4-yl (18:1 NBD-PE) embedded in the head group region of bilayer lipid
membranes comprising DOPC and 1,2-dioleoyl-sn-glycero-3-[Phospho-rac-(l-glycerol)]
(DOPG). The dynamics of the fluorophore were examined using TCSPC to measure
rotational diffusion in lipid vesicles, and FRAPP to determine translational diffusion
coefficients and mobile fractions in sBLMs. TCSPC data revealed that fluorophore
rotational diffusion coefficients in DOPG vesicles were not statistically different from
those in DOPC and mixed DOPC/DOPG vesicles, suggesting that the NBD-PE
fluorophore does not interact strongly with the head group region of these bilayers.
However, FRAPP experiments showed that lateral diffusion is statistically lower in
mixed DOPC/DOPG supported bilayers than in DOPC supported bilayers. These results
suggest that bilayers containing DOPG likely undergo inter-lipid head group hydrogen

bonding interactions that suppress translational diffusion.

4.2. Introduction

Model BLMS in the form of liposomes in solution,89 sBLMs on underlying

8,90

substrates,22 and tBLMs on polymer cushions or monolayers can be used to mimic the

47

properties and dynamics of cell membranes.”35 Membrane fluidity is an important
property of the bilayer that influences cellular functions such as signaling,91 protein
activity,92 and response to stimuli.2 For example, transbilayer diffusion of nitric oxide, an
important signaling molecule that transmits information between mammalian cells, has
been shown to decrease with decreasing membrane fluidity.93 An increase in the fluidity
of mammalian endoplasmic reticulum (ER) membranes resulted in the activation of
certain membrane-bound transcription factors, while activation of transcription factors in
ER yeast membranes was triggered by a decrease in fluidity.94

One of several ways that cells may control membrane fluidity is by adjusting the
concentration of the membrane constituents. For example, incorporation of
lysophosphatidylcholine lipids into egg-phosphatidylcholine (egg-PC) sBLMs has been
shown to increase the translational diffusion coefficient due to fewer van der Waals
interactions between hydrophobic tailgroups. On the other hand, addition of egg-
phosphatidylethanolarnine into egg-PC sBLMs resulted in pronounced head group
hydrogen bonding, thus enhancing van der Waals interactions due to tighter packing and
lowering of the translational diffusion coefficient.22 Bacterial cells have been shown to
isomerize unsaturated membrane fatty acids from cis to trans conformations in the
presence of solvents or toxins in order to decrease membrane fluidity and prevent entry
into the cell.95 The addition of cholesterol to membranes has been well-documented to
decrease fluidity.”96

In the present study, we investigated the effect of phospholipid head group

hydrogen bonding on the translational and rotational dynamics of the chromophore 18:1

NBD-PE embedded in BLMS comprising DOPC and DOPG. The molecular structures of

48

these lipids are shown in Figure 4.1. DOPC and DOPG were chosen as the primary lipids
in this study for two reasons. First, these lipids have low transition temperatures of -20°C
for (DOPC)97 and -18°C for (DOPG),23 which ensures that bilayers reconstituted from
these species would exist in a disordered fluid phase at room temperature (23 d: 2°C).
Second, the two lipids have identical acyl chain lengths but different head group
frmctionalities, so differences in fluorophore dynamics can be attributed primarily to head
group interactions.

The DOPG glycerol head group contains two hydroxyl moieties that can function
as either hydrogen bond donors or acceptors. Hydrogen bonds are comparatively strong
intermolecular interactions (10 to ca. 30 kJ/m012'). However, the DOPC head group does
not contain any functionalities for hydrogen bonding and, as such, should not participate
in head group-head group interactions in a manner analogous to DOPG.

Although zwitterionic PC lipids such as DOPC comprise the majority of lipids
found in biological membranes, anionic lipids such as DOPG also play significant roles
in regulating membrane function.98 PG lipids are believed to be responsible for
maintaining membrane lipid surface charge density, a property that not only affects
membrane permeability to ions and charged metabolites, but also the activity of certain
membrane proteins.99 MD simulations of PC lipid bilayers have generated radial
distribution functions showing hydrogen bonding interactions between the oxygen atoms
of adjacent phosphate molecules through water bridges, as well as screened electrostatic
interactions between the negatively charged phosphate oxygen and the positively charged
choline nitrogen!”103 PG lipids, in contrast, are rich in head group functionalities that

can form hydrogen bonds with other membrane constituents.l°4’105 Hydrogen-bonded

49

10 11 106
9 ) and

water bridges have also been predicted in MD simulations of PG bilayers,
there is the additional capability of forming hydrogen bonds between the glycerol moiety
and phosphate oxygen of neighboring PG lipids?"107 Hydrogen bonds within BLMS may
be measured directly using infrared spectroscopy108 or indirectly by using fluorescence
spectroscopy to monitor the dynamics of fluorophore—tagged lipids in the BLM. In this
study, we investigated interactions between 18:1 NBD-PE, which will be referred to as
NBD-PE throughout the remainder of this chapter, and head group regions of
neighboring lipids in liposomes and sBLMs. We measured translational diffusion
coefficients and mobile fractions with FRAPP, and fluorescence lifetimes and rotational
diffusion coefficients with TCSPC.

We reasoned that a lipid with a head group-bound fluorophore was a better choice
for this study than a fluorescent lipid which has the fluorophore located at the terminus of
a lipid acyl chain. The fluorescently-tagged lipid used in this study (NBD-PE) has the
NBD fluorophore attached to its polar head group, allowing the influence of head group
interactions to be investigated. In addition, the two bulk lipids used to form the BLM,
DOPC and DOPG, have identical hydrophobic tailgroup structures, but differ in head
group functionalities. Thus, using NBD-PE as the probe allowed us to examine molecular
issues related primarily to the lipid head group interactions while maintaining
comparatively constant acyl chain interactions. We note that we can make intact
liposomes composed of 98 mol% DOPG, an anionic lipid. However, no direct
comparison of fluorophore dynamics between purely anionic liposomes and purely

anionic sBLMs can be made because, for sBLM structures on surfaces, only limited

concentrations (up to 39 mol%) of anionic lipids such as DOPG can produce fluid bilayer

50

structures.109 Regardless of this limitation, it is possible to compare mixed lipid systems
in both liposome and sBLM structures, and FRAPP and TCSPC data provide useful

insight into the dynamical properties of the NBD-PE contained within them.

4.3. Materials and Methods

4.3.1. Vesicle Preparation

Phospholipids DOPC, DOPG, and NBD-PE were purchased from Avanti Polar
Lipids Inc. (Alabaster, AL) and used without further purification. 1 mM lipid solutions of
DOPC/NBD-PE (98 mol%, 2 mol%), DOPG/NBD-PE (98 mol%, 2 mol%), or
DOPC/DOPG/NBD-PE (59 mol%, 39 mol%, 2 mol%) were prepared by evaporating the
chloroform solvent from the lipid mixture and hydrating the dry lipid film for 30 min
with Tris®-HC1 buffer (Sigma-Aldrich, St. Louis, MO) containing NaCl (Fisher
Scientific, Fair Lawn, NJ) prior to extrusion. The buffer (10 mM Tris®, 150 mM NaCl,
pH 8) was prepared using water from a Milli-Q Plus water purification system (Millipore,
Bedford, MA). The mixtures were processed five times through a freeze-thaw-vortex
cycle to ensure complete mixing of the sample constituents. Each cycle consisted of
immersion in liquid nitrogen for 5 min, followed by immersion in a 60°C water bath for 5
min, and then vortexing the thawed sample for 2 min. The hydrated mixtures were
extruded 11 times through a polycarbonate membrane with nominal pore diameters of
100 nm and 2000 nm (Avanti Polar Lipids Inc., Alabaster, AL). All extrusions were

performed at room temperature (23 2t 2°C).

51

4.3.2. Time-Correlated Single Photon Counting

Time-resolved fluorescence anisotropy and lifetime data were measured using
TCSPC. The details of this technique can be found in Section 2.3 of this dissertation. For
each lipid system considered in this work, three separate liposome solutions were

interrogated with TCSPC.

4.3.3. Deposition of Supported Lipid Bilayers

Fused silica microscope slides (75 mm x 25 mm x 1 mm) were purchased from
Technical Glass Products, Inc. (Painesville, OH). The slides were cleaned by bath
sonication (Branson 1510, Branson Ultrasonic Corporation, Danbury, CT) in detergent
solution for 20 min, rinsed with DI water, baked at 160°C for 4 h, and plasma treated
(Harrick Plasma, Ithaca, NY) with oxygen under vacuum (150 mTorr) for 10 min
immediately before bilayer deposition. sBLMs were deposited by liposome fusion in a
custom-made flow cell described elsewhere.68 The flow cell was initially washed with
buffer, followed by a 3-h incubation with liposome solution and a final buffer wash to

remove unadsorbed liposomes. All experiments were performed at room temperature (23

:h 2°C).

4.3.4. Fluorescence Recovery After Pattern Photobleaching

FRAPP was used to measure the translational diffusion and mobile fractions of
fluorophores embedded in sBLMs formed on fused silica microscope slides. The details

of this technique are in Section 2.2 of this dissertation. For each lipid system considered

52

in this work, three separate sBLM interfaces were formed on silica slides and each

interface was interrogated with F RAPP at five different locations of the bilayer.

4.3.5. Statistical Analysis

Data fitting was accomplished using OriginPro 7.5 (OriginLab Corporation,
Northampton MA), which uses the Levenberg-Marquardt algorithm for non-linear least
squares fitting. An F-test comparison was conducted to determine whether an exponential
model using n parameters provided a statistically significant improvement in the

goodness of the fit of fluorescence lifetime and fluorescence anisotropy data than an

exponential model using n-l parameters. Rotational diffusion coefficients (Dx, Dy , D2)

and translational diffusion coefficients (D1,) are reported as means 3: SD. Multiple

comparison ANOVA with a post-hoe Tukey test (p < 0.05) was used to determine if

differences among the means were statistically significant.

4.4. Results and Discussion

The goal of this work was to investigate the intermolecular lipid-lipid interactions
between head groups in bilayer structures by examining the rotational and translational
diffusion behavior of a fluorophore embedded in liposomes and sBLMs. We consider the

rotational diffusion and fluorescence lifetime data first.

53

4.4.1. Fluorescence Lifetime and Anisotropy Data

Fluorescence lifetime and anisotropy decay data provided information on the
molecular enviromnent(s) of the NBD fluorophore in vesicles of the following
compositions: 1) 98 mol% DOPC and 2 mol% NBD-PE, 2) 98 mol% DOPG and 2 mol%
NBD-PE, and 3) 59 mol% DOPC, 39 mol% DOPG, and 2 mol% NBD-PE. We also
examined vesicles 100 nm and 2000 nm in diameter to assess the influence of membrane
curvature on rotational dynamics. For all measurements, there were no statistical
differences in either the lifetime or the anisotropy data (not shown) for the two vesicle
sizes, suggesting that size is not an important issue.

The F —statistic analysis showed that a two-component exponential decay was the
best fit for the fluorescence lifetime data for NBD-PE in each vesicle system (Figure 4.2,
Table 4.1). There are two possible explanations for this finding. Either the fluorophore
exists in two different environments, with populations that do not exchange significantly
on the timescale of the excited state relaxation, or the fluorophore exists in a dielectric
gradient where slight changes in the location and/or orientation of the fluorophore with
respect to the gradient give rise to a distribution of fluorescence lifetimes (Figure 4.3).
This latter condition has been previously reported for the NBD fluorophore.65’73 While it
may be tempting to ascribe the different lifetime components to specific chemical
environments, the dependence of fluorescence lifetime on intramolecular and
intermolecular properties is sufficiently complex that it is not possible to make such
assignments in the absence of additional information. Fluorescence anisotropy decay data
(Figure 4.4, Table 4.1), in contrast, can be interpreted within the context of well

established theoretical models.47'48’82’l 1° The anisotropy decay function, r(t), is formulated

54

as the normalized transient difference in emission intensity parallel, I”(t), and
perpendicular, I ((t), to the polarization of the incident excitation pulse as depicted in
Equation (3.1).

The functional form of r(t) contains chemically relevant information. In
particular, there are two possible choices for the interpretation of our fluorescence
anisotropy data because the fluorophore is attached to the head group of the phospholipid.
The functional form of the anisotropy decay allows us to determine whether this vesicle-
incorporated fluorophore behaves as a free or hindered rotor.

A free rotor, such as a fluorophore in solution, is characterized by a zero infinite-
time anisotropy because the fluorophore can assume all orientations with equal
probability. It is important to note that if the fluorophore is free to reorient within a
hemisphere, it will behave as a free rotor because of the inherent polarization symmetry
of the incident electric field. By contrast, for a fluorophore tethered to a lipid (as NBD is
in NBD-PE), under the condition that the probe is not free to access all possible
orientations, a non-zero infinite-time anisotropy, r(oo), results.”82 Based on an F-statistic
analysis, we determined that our experimental data were characterized by a two-
component exponential anisotropy decay for each of the three lipid systems used in this
study and, for each, the infinite-time anisotropy, r(oo), was zero within the experimental
uncertainty. The absence of an infinite-time anisotropy component in the data suggests
that the fluorophore is able to reorient within a hemispherical volume and, as such, it can

be treated as a free rotor.

55

The simplest way to model a fluorophore is to assume that it sweeps out a
spherical volume as it rotates. For this case, we can directly relate the reorientation time

to the average diffusion rate, D, through the Debye-Stokes-Einstein equation1 I 1:

 

Ton - - '6— (4.1)

Here, n is the solvent viscosity, V is the spherical volume, k3 is the Boltzmann constant,
and T is the absolute temperature.

Most fluorophores are not circular in shape, but rather elliptical. Therefore, the
volumes they sweep out as they rotate are more ellipsoidal than spherical. A model

148 for fluorophores with full motional freedom in

developed by Chuang and Eisentha
solution treats fluorophores as either Type I (prolate) or Type II (oblate) ellipsoidal rotors
(Figure 4.5) and relates the anisotropy function, r(t), to diffusion coefficients about each
of the Cartesian axes. We interpret our anisotropy decay data in the context of this
Chuang and Eisenthal model.48 The same essential physics underlying the Chuang and
Eisenthal model must also apply to the dynamics of a tethered fluorophore, especially
when the fluorophore is free to relax in a hemispherical volume (1'. e. no confining cone).
In this model, the Cartesian axes are typically assigned so that the z-axis is perpendicular
to the n—system plane and the absorbing and emitting transition dipole moments lie along
either the x-axis (long axis) or y-axis (short axis) of the molecular plane.”2 The two

limiting cases of this model treat the fluorophore as either a Type I or a Type II rotor,

thereby significantly simplifying the mathematics. The excited and emitting transition

56

dipoles lie coincident with and perpendicular to the unique rotational axis for Type I (Dx

315 Dy = Dz ) and Type II (Dx = Dy 75 Dz) rotors, respectively. The reorientation decay time

constants are related to the Cartesian components of the rotational diffusion coefficients

through the anisotropy decay function as given in Equation (4.2) to Equation (4.5)

below: 1 '3

For a transition dipole oriented along the long x-axis:

Typel: r(t): [1 75-]6XP( —z6Dt) (4.2)

men, r(t)- (— We.“ (21>,+4D,)t)+[.l%]exp(—6D,r) (4.3,

For a transition dipole oriented along the short y-axis:

3
Type I: r(t)=(—10)exp(—(2Dz + 4Dx ) t) + [a] exp (—6th) (4.4)

TypeII: r(t): (1—4 O—JCXN (2Dz+4Dx)t) (4.5)

In this model, the resolution of two reorientation time constants indicates that the
NBD fluorophore behaves as either an x-axis polarized Type H rotor or a y—axis polarized
Type I rotor, according to Equation (4.3) and Equation (4.4). Previous studies of the

solution phase NBD fluorophore using one and two-photon excitation methods suggest

57

that the S1 <— 30 transition of this fluor0phore is short-axis polarized, and a two-
component anisotropy decay would be consistent with NBD reorienting as a Type I rotor
(Equation (4.4)).77 We will use this assignment to evaluate the Cartesian components of
the rotational diffusion coefficient for NBD-PE.

There are no statistically significant differences between the Cartesian
components of the diffusion coefficients (Table 4.2) of tethered NBD in each of the

liposome systems. The data all indicate that the tethered NBD fluorophore is a

substantially anisotropic rotor, with an aspect ratio of Dx/Dz ~ 8. While it may be

tempting to consider the subtle differences between the Dz and Dx values and the ratio of

these coefficients, the uncertainty in the data is sufficiently large that it is not possible to
draw any system-dependent conclusions without other information. The fact that NBD
sweeps out an ellipsoidal volume that is more anisotropic than its axial ratio (see NBD-
PE structure in Figure 4.1) points to the importance of the tethering bond, even in
situations where motion is not constrained to a conic volume.

Rotational diffusion measurements can provide information on the molecular-
scale environment of the reorienting fluorophore, and the information we obtain for
NBD-PE on the molecular length scale suggests that the dynamics of the lipid head group
are determined to a significant extent by the layer of fluid in immediate contact with the
lipid bilayer. This finding is consistent with our observation of a two-component
fluorescence lifetime, which can be accounted for in the context of the fluorophore
residing in a dielectric gradient. The molecular-scale information does not provide a
complete picture, however, of the fluid properties of the bilayer lipid membranes. We

also need to consider molecular motion over longer length scales, and we accomplish this

58

task through the use of translational diffusion measurements of NBD-PE in sBLM

systems.

4.4.2. Translational Diffusion in Supported Bilayer Lipid Membranes

Translational diffusion coefficients and mobile fractions were measured with
FRAPP to provide information complementary to our TCSPC fluorescence lifetime and
anisotropy decay data. Because DOPG vesicles did not adsorb on fused silica and rupture
to form functional bilayers, FRAPP studies were limited to two systems: 1) 98 mol%
DOPC and 2 mol% NBD-PE, and 2) 59 mol% DOPC, 39 mol% DOPG, and 2 mol%
NBD-PE. We determined that 39 mol% DOPG was the maximum concentration of the
anionic lipid that would lead to the reconstitution of stable fluid sBLMs. This finding is
22,114

consistent with previous studies under similar buffer conditions.

A typical FRAPP recovery curve for a DOPC/NBD-PE bilayer is shown in Figure
4.6, and calculated DL values and mobile fractions are given in Table 4.3. Equation (3.5)

was used to extract both diffusion coefficients and mobile fractions. We determined that
differences between the mobile fractions were not statistically significant for bilayers
formed with both lipid compositions. However, differences between DL values were
statistically significant. The diffusion coefficients were 2.91 :I: 0.31 me/s for DOPC

bilayers and 1.82 d: 0.24 pmz/s for mixed DOPC/DOPG bilayers, indicating that addition

of DOPG retards the translational diffusion of NBD-PE.

59

4.4.3. Head group Hydrogen Bonding in Bilayer Membranes

Despite the fact that NBD and DOPG have the capacity to interact via hydrogen
bonds, we observe no effect on the rotational diffusion measurements upon addition of
DOPG. This suggests that the NBD-PE fluorophore does not have a measurable
preference for interacting with either DOPG or DOPC lipid head groups. NBD-PE
translational diffusion in DOPC/DOPG sBLMs does, however, seem to be hindered
relative to that in pure DOPC sBLMs, suggesting that the intermolecular interactions in
the head group region of the lipids may mediate the translational motion of the bilayer
constituents. This finding is consistent with recent MD simulations involving PC and PG
bilayers that suggest the intramolecular and intermolecular hydrogen bonding capability
of PG in POPG bilayers reduces fluidity relative to l-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC)bi1ayers.9

In addition to head group hydrogen bonding interactions, changes in translational
bilayer fluidity may also be attributed to variations in bilayer thickness. MD simulations
have reported the thickness of pure l-palmitoyl-2-oleoyl-sn—glycero-3-phospho—(1'-sn-
glycerol) (POPG) bilayers as ~ 43 A and that of pure POPC bilayers as ~ 37 A,

suggesting that POPG lipids are organized more rigidly and therefore diffuse more
slowly. Therefore, the lower DL for the DOPC/DOPG system could also be attributed, in

part, to a thicker bilayer membrane.

We now turn to a consideration of how these two bodies of data can be
reconciled. The rotational diffusion data indicate that, at the molecular level, the
rotational motion of fluorophores localized in the headgroup region of the bilayer is not

mediated significantly by the headgroup structure or organization of the lipids. This

60

situation is primarily because of the presence of bulk solvent in close contact with this
region of the lipid bilayer structure. The fact that the translational diffusion of these
fluorophores in the bilayers does depend on lipid headgroup identity could be because the
measurement involves the translational motion of the entire lipid molecule, and the fact
that translational motion can depend significantly on any structural heterogeneity that is
present in the bilayer. In our translational diffusion data, there appear to be regions of the
supported bilayer structure that are comparatively less mobile than others. The notion of
immobile regions is not surprising for the DOPC/DOPG mixed systems in light of the
large amount of literature extant relating to the formation of phase-separated structures.
The basis for such phase segregation is understood from a thermodynamic point of view
for multicomponent systems,l 15-122 but for single-component systems, it is fair to question
the origin of such features. One possibility is that there exist interactions between the .
sBLM bottom leaflet and the supporting substrate that give rise to comparatively fewer

mobile regions.

4.5. Conclusions

We have investigated the dynamics of a tethered fluorophore (NBD-PE) in
unilamellar DOPC, DOPG, and mixed DOPC/DOPG vesicles, and in DOPC and mixed
DOPC/DOPG supported bilayer lipid membranes. We used both PC and PG to evaluate
the role of head group hydrogen bonding interactions in mediating the fluidity of lipid
bilayer structures. Within the experimental uncertainty, we obtained the same rotational
diffusion results for tethered NBD in each system for vesicles sizes of both 100 nm and

2000 nm diameter. Our data showed that the molecular-scale rotational motion of the

61

NBD fluorophore does not depend on the type of lipid in the bilayer. We also measured
the translational diffusion of NBD-PE in sBLMs consisting of pure DOPC and a mixture
of DOPC and DOPG. The data indicate a slower rate of translational diffusion in sBLMs
containing DOPG. We believe this is due to hydrogen bonding interactions in the
headgroup region of the constituent lipids, even though the effect on rotational diffusion

appears to be much less pronounced.

4.6. Recommendations for Future Work

A lipid such as DOPG, with a head group functionality capable of hydrogen
bonding, was shown in this study to significantly influence the translational motion of
NBD-PE in sBLMs, but not the rotational motion of NBD-PE in a liposomes. Performing
the same FRAPP and TCSPC experiments with an NBD tailgroup-bound fluorophore
such as NBD-PC (instead of head group-bound fluorophore like NBD-PE) in DOPC and
DOPG model BLMS would complement the work done with NBD-PE. A tailgroup-bound
NBD-PC fluorophore is likely more sensitive to the hydrophobic region of the bilayer
and should provide a more complete picture of the dynamics of lipid mixtures such as PC
and PG. An additional suggestion is to use other lipids that are prone to hydrogen
bonding interactions, such as phosphatidylserine, in BLMS and compare those dynamics

results with those measured for PG systems.

62

   

(a) (b) (c)

Figure 4.1. Molecular structures of lipids used in this study: (a) 1,2-dioleoyl-sn-glycero-
3-phosphocholine (DOPC), (b) l,2-dioleoyl-sn-glycero-3-[Phospho-rac-(l-glycerol)]
(DOPG), (c) l ,2-dioleoyl—sn glycero-3-phosphoethanolamine-N-7-nitro-2-l ,3-
benzoxadiazol-4-yl (18:1 NBD-PE).

63

3000-

 

 

  

 

 

’7 2500 -
a
m I
v
2,5 2000 -
":3
a .
a)
E
__, 1500 -
a)
o I
G
8 1000 -
8
H I
O
.E‘. 500 -
LL.
0 - fllnmimralflt.
U r I I U I I
0 5000 10000 15000 20000

Time (ps)

Figure 4.2. Representative fluorescence lifetime data and residual plot (below) for NBD-
PE in DOPG/DOPC lipid vesicles. A two-parameter exponential decay, corresponding to
two fluorescence lifetimes (1:1 and 1:2), gave the best fit of the data. F-test statistics were
used to determine the statistical difference in goodness of fit for higher parameter
exponentials.

} a = 6-80 (Water)

  
 

Phosphate
8 = 4-10 (Head groups)

8 = 1-3 (Acyl chains)

\

Acyl chains }

 

 

Figure 4.3. Schematic diagram of the NBD-PE molecule. The NBD fluorophore exists in
a range of locations in a dielectric gradient, where 8 denotes the dielectric constant in
different regions of the lipid. We interpreted our results in the context that multiple
fluorescence lifetimes arise from NBD diffusing in a dielectric gradient.

65

0.35 -

0.30 d

0.25 -

0.20 -

r(t)

0.15d

0.10-

0.05 -

0.00 -

 

 

—0.05

 

Figure 4.4. Representative induced orientational anisotropy data and residual plot
(below) for NBD-PE in DOPG/DOPC lipid vesicles. A two-parameter exponential decay,
corresponding to two reorientation times (tom and Tom), provided the best fit of the
data. F-test statistics were used to determine the statistical difference in goodness of fit
for higher parameter exponentials.

66

(a)

 

(b)

    

1
v

Figure 4.5. Illustrations of (a) Type I (prolate) rotor, and (b) Type II (oblate) rotor.

67

.699
it“?

0.45 l
0.40 .
0.35 l
0.30 -'
0.25 -'
0.20 i
0.15 J
0.10 3
0.05 3
0.00 3
-0.05 -‘

 

 

Normalized Fluorescence Intensity

 

Time (s)

Figure 4.6. Representative FRAPP recovery curve for a DOPC/NBD-PE supported
bilayer membrane (sBLM) with residuals (below). The total photobleaching time was 300
ms.

68

Table 4.1. Fluorescence lifetimes (t1 and 12) and reorientation times (tom and tom) of
NBD-PE in DOPC, DOPG, and mixed DOPC/DOPG liposomesa.

 

 

 

 

 

Systemb 1’1 (115) T2 (n5) T0111 (n8) t0112018)

DOPC 1.92 :1: 0.12A 7.83 r 0.21B 0.65 e 0.05C 3.67 :1: 0.10D

DOPG 2.13 :1: 0.07 A 7.70 r 0.11B 0.49 a 0.20C 3.20 3: 0.52”
DOPC/DOPG 1.89 :1: 0.19 A 7.48 r 0.14B 0.77 r 0.52C 3.93 r 0.055

 

 

 

 

 

 

“Data represent means :1: SD of three liposome solutions.

bSystems with statistically identical fluorescence lifetimes and reorientation times are

labeled by common capital letters (AN OVA, Tukey post-hoe test, p < 0.05).

Table 4.2. Cartesian components of the rotational diffusion coefficients (0,, and Dz) and
the diffusion aspect ratio (Dx/Dz) of NBD-PE in DOPC, DOPG, and mixed
DOPC/DOPG liposomes“. Dy = Dz for Type I (prolate) rotors.

 

 

 

 

System” Dx (MHz) Dz (MHz) 19.0)z

DOPC 362 .1. 27A 45 it 2'3 7.97 r 0.82D

DOPG 484 s. 148A 52 1 7B 9.30 it 4.22D
DOPC/DOPG 3035131A 42s1B 7165:1181)

 

 

 

 

 

 

“Data represent means i SD of three liposome solutions.

bSystems with statistically identical rotational diffusion coefficients and diffusion aspect

ratios are labeled by common capital letters (AN OVA, Tukey post-hoe test, p < 0.05).

69

Table 4.3. Translational diffusion coefficients (DL) and mobile fractions of NBD-PE in
DOPC and mixed DOPC/DOPG sBLMs on fused silica“.

 

 

 

Systemb DL (umz/s) Mobile Fraction
DOPC 2.91 e 0.31A 0.79 e 0.11C
DOPC/DOPG 1.82 r 0.24B 0.61 r 0.08C

 

 

 

 

 

“Data represent means i SD of three liposome solutions.

bSystems with statistically identical DL and mobile fiactions are labeled by common

capital letters (ANOVA, Tukey post-hoc test, p < 0.05).

70

5. INFLUENCE OF LYSOPHOSPHOLIPID HYDROLYSIS BY THE
CATALYTIC DOMAIN OF NEUROPATHY TARGET ESTERASE ON
BILAYER LIPID MEMBRANE FLUIDITY

5.1. Abstract

NTE is an integral membrane protein localized in the endoplasmic reticulum in
neurons and in organelles of other cell types. Irreversible inhibition of NTE by certain
organophosphorus compounds produces a type of paralysis known as organophosphorus
compound-induced delayed neuropathy. NTE mutations are also associated with motor
neuron disease. NTE has been identified as a phospholipase/lysophospholipase that
hydrolyzes single-chain lysophospholipids in preference to dual-chain phospholipids. The
physiological role of NTE is poorly understood, although recent studies suggest that it
may control the cytotoxic accumulation of lysophospholipids in membranes. The NTE
catalytic domain (NEST) was used in this work to hydrolyze palmitoyl-2-hydroxy-sn-
glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes composed of
DOPC and the lipid fluorophore NBD-PC. Translational diffusion coefficients and
mobile fiactions were measured in sBLMs by FRAPP. The average D1, (mean 1 SD,
,umzs"l) for membranes that did not contain NEST was 2.44 i 0.09; the average D1, for
membranes containing NEST as well as its inhibitor by diisopropylphosphorofluoridate
was nearly identical at 2.45 i 0.08. By contrast, membranes containing NEST and p-
lysoPC gave an average DL of 2.28 i 0.07. This value is significantly different from the
system with no p-lysoPC, and is due to NEST hydrolysis. By comparison, the system

without NEST which contained the amount of palmitic acid that would have been

71

produced by NEST-catalyzed hydrolysis of p-lysoPC gave a statistically identical average
D1, of 2.26 i 0.06. These results indicate that NEST hydrolysis induces a statistically
significant decrease in the fluidity of sBLMs and that fluidity may be used as a measure

of NTE hydrolysis of lysophospholipids in mammalian cells.

5.2. Introduction

NTE is a 147 kDa serine esterase located in endoplasmic reticulum membranes of
neurons as well as other cell types such as lymphocytes.37"23'126 NTE activity has
traditionally been measured in vitro via hydrolysis of its non-physiological substrate,
phenyl valerate.127 More recently, NTE activity has been measured by hydrolysis of its
more physiologically relevant lysophospholipid substrates, which are distinguished from
other phospholipids by their single hydrophobic acyl chainw’m'130 Aging of NTE
activity by irreversible binding of certain organophosphorus (OP) compounds to the
serine in the active site results in a type of paralysis known as OP compound-induced
delayed neuropathy (OPIDN), for which there is no effective treatment.13 1'13 3 Only those
OP compounds that are involved in the aging reaction in which a negative charge is
created on the phosphyl group of NTEm’135 have been associated with OPHDN.

Mutations in the gene encoding NTE have also been linked to motor neuron disease.38

136,137

Although NTE plays essential roles in embryonic development and the protection of

axons from degeneration)”139

its physiological function in mammalian cells is poorly
understood.

Lysophospholipids in healthy mammalian cell membranes constitute between 0.5

and 6% of the total lipid weight. However, at elevated concentrations, these lipids have

72

been linked to a number of disease states including atherosclerosis, inflammation, and
hyperlipidemia.85’140 In addition, these lipids form micelles that generally have a high
positive average intrinsic curvature because their projected head group area is much
larger than that of their tail groupm

Lysophospholipids act as neurotoxic agents in vitro when added to cell types such
as cerebellar granular neurons and rat spinal cord motor neurons at micromolar
concentrationsm’143 Microscopy images have shown the formation of bulges and bead-
like structures in the aforementioned cells as a result of an imbalance between exocytosis
and endocytosis.142 Lysophospholipids aid in the pore-forming exocytosis event that
releases neurotransmitters from presynaptic vesicles, but inhibit the subsequent
endocytosis and vesicle fission events.143 Channel-independent influx of calcium ions
into cells from the extracellular enviromnent, a supplementary effect of lysophospholipid
addition, has been shown to aid in presynaptic vesicle exocytosis.144 A possible
mechanism of this influx is the spontaneous formation of pore structures in the lipid
membrane.144

Lysophospholipases help control lysophospholipid levels in biological
membranesm’ ‘45 A recent study demonstrated that the ICso for lysophosphatidylcholine
exposed to Neuro-2a cells increased nearly three-fold upon the addition of recombinant
NTE, suggesting that NTE may control the cytotoxic lysophospholipid concentration.129
Other studies also indicate that NTE enzymatic activity helps control lysophospholipid
concentrations.128’146

Because of inherent difficulties in expressing the full length NTE,147 we used the

catalytically active domain of NTE called NEST in this work. NEST is a 55 kDa

73

membrane-associated protein containing amino acids 727-1216 of the full length 1327-
amino-acid NTE protein.39 NEST is used as a surrogate for NTE because it reacts with
ester substrates and inhibitors in the same manner as NTE and is readily produced
heterologously.39‘148 In the present work, we assessed the influence of NEST hydrolysis
of lysophospholipids on the fluidity of bilayer lipid membranes. Membrane fluidity was
assessed by measuring the translational diffusion coefficients and mobile fractions of

fluorescently-tagged lipids in several systems using FRAPP.

5.3. Materials and Methods

5.3.1. Chemicals and Reagents

Phospholipids DOPC, p-lysoPC, and NBD-PC were purchased from Avanti Polar
Lipids Inc. (Alabaster, AL) and used without further purification. The structures of the
phospholipids used in this work are shown in Figure 5.1. The NBD fluorescent moiety of
the NBD-PC is located in the tail region of the lipid. A reasonable assumption of the
result of NEST hydrolysis of a p-lysoPC molecule is that the hydrophilic head group of
the lipid will diffuse into the aqueous mass outside the bilayer while the hydrophobic tail
will remain inside the bilayer. Thus, we expect that much of the change in lipid dynamics
due to NEST hydrolysis will be manifested in the hydrophobic region of the membrane.

CHAPS detergent (purity Z 99% by high performance liquid chromatography)
was purchased from Anatrace (Maumee, OH). Palmitic acid, imidazole,

phenylmethylsulfonylfluoride (PMSF, purity 2 98.5% by gas chromatography (GC)), and

74

diisopropylphosphorofluoridate (DFP, 98% pure by GC) were purchased from Sigma

Aldrich (St. Louis, MO).

5.3.2. NEST Expression and Purification

A gene that encodes human brain NEST was cloned into a pET-21b vector
containing a C-terrninal His6etag and an N-terminal T7 sequence. The expression vector
was subsequently transformed into E. coli BL2l(DE3). A 50 mL culture of transformed
E. coli was used to inoculate a 5 L solution of terrific broth (TB) media containing
ampicillin (0.1 mg/mL) in a fermentation vessel. Cells were grown for 12 h, with
intermittent additions of glucose and ammonium phosphate, until the optical density of
the culture reached ~l .0. The culture was then induced with isopropyl ,B-D-l-
thiogalactopyranoside (1 mM) and allowed to grow for an additional 4 h before
harvesting. Five g of harvested cell pellet were then suspended in 50 mL PEN buffer (50
mM sodium phosphate, 300 mM NaCl, 0.5 mM EDTA, pH 7.4) containing 3% (w/v)
CHAPS detergent. This solution was tip sonicated five times on ice (15 s per time) at a
power of approximately 80 W and centrifuged at 14,900g for 30 min at 4°C. The
supernatant was incubated with 6 mL of nickel-nitrilotriacetic acid (N i-NTA) resin
(Qiagen, Valencia, CA) for l h at 4°C. Two minicolumns (3 mL per column) were
prepared by centrifugation of the protein-bound Ni-NTA resin and the minicolumns were
washed six times with PEN buffer containing 0.3% (w/v) CHAPS. NEST was collected
in elution fractions using PEN buffer containing 0.3% (w/v) CHAPS and 300 mM
imidazole. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was

used to confirm protein expression (Figure 5.2). Protein concentration was estimated by

75

measuring the absorbance at 280 nm in a spectrophotometer. NEST protein was then

incorporated into liposomes to form proteoliposomes as described below.

5.3.3. NEST Proteoliposome Formation

2 mM solutions of DOPC/NBD-PC (98:2, mol/mol) and DOPC/p-lysoPC/NBD-
PC (69:29:2 mol/mol/mol), referred to as 98:2 and 69:29:2, respectively, throughout the
remainder of this chapter, were prepared by evaporating chloroform solvent from the
lipid mixture with nitrogen gas, lyophilizing the lipid film under vacuum for 1 h, and
hydrating the dry lipid film with PEN buffer containing 10% (w/v) CHAPS. An upper
limit p-lysoPC concentration of 29 mol% was chosen because it is not possible to form
bilayer structures at lysophospholipid concentrations approaching 40 mol%.149 The NEST
protein solution (0.1 mg/mL) was added to lipid/CHAPS solutions in a 880:1
lipidzprotein (w/w) ratio to a final volume of 5 mL. The CHAPS/lipid/NEST solutions
were then dialyzed against three 1 L exchanges of PEN buffer for a total of 48 h at 4°C
using snake skin dialysis tubing with a 10,000 MWCO (Therrno Fisher Scientific,
Rockford, H.). Dialysis was performed at 4°C to minimize NEST denaturation. After 48

h, DFP was added at 50 11M to stop the NEST-induced hydrolysis reaction.

5.3.4. Inhibition Studies

Stock solutions of 10 mM DFP in acetone and 200 mM PMSF in 2-propanol were
prepared and stored at 4°C. DFP at 50 ,uM or PMSF at 1 mM was preincubated with 0.1

mg/mL NEST in PEN buffer at room temperature (23 :t 2°C) for 20 min before addition

76

to lipid solutions containing 10% (w/v) CHAPS. To ensure a high level of enzyme
inactivation, inhibitor concentrations used in our experiments were relatively large
compared to reported 20 min ICso values for DFP (~ 0.96 M) and PMSF (~ 0.15 mM)
against NEST activity at 37°C.148 Subsequent steps were repeated as listed above.
NEST/DFP or NEST/PMSF solutions (0.1 mg protein/mL) were added to lipid/CHAPS
solutions in an 880:1 lipid:protein (w/w) ratio to a final volume of 5 mL. The
CHAPS/lipid/NEST solutions were then dialyzed against three 1 L exchanges of PEN

buffer for a total of 48 h at 4°C.

5.3.5. Determination of Free Fatty Acid Concentration

An FFA assay kit from Roche Diagnostics (Penzberg, Germany) was used to
measure the concentration of fatty acids incorporated into liposomes and
proteoliposomes. The assay is based on a reaction scheme developed by Shimizu.150
Briefly, FFA reacts with coenzyme A (CoA) and adenosine 5'-triphosphate in the
presence of acyl-CoA synthetase to form acyl-CoA, adenosine 5'-monophosphate, and
pyrophosphate. Acyl-CoA then forms 2,3-enoyl CoA and hydrogen peroxide in the
presence of acyl-CoA oxidase. Upon reaction with 2,4,6-tribromo-3-hydroxy—benzoic
acid and 4-aminoantipyrine in the presence of peroxidase, hydrogen peroxide forms a red
dye, the absorbance of which is measured with a spectrophotometer.

To ensure that the assay was suitable for this study, chloroform-solubilized
palmitic acid was added to chloroform-solubilized lipids and a lipid/palmitic acid film

was formed by drying with nitrogen gas prior to lyophilization and hydration with PEN

buffer containing 10% (w/v) CHAPS. After liposome formation by dialysis, an additional

77

10% (w/v) CHAPS was added to the sample and the concentration of FFA in the
resulting mixed micelle structures (size measured with DLS) was verified using the kit.
Following dialysis, the concentration of FFA in liposomes and in NEST
proteoliposomes was measured after the addition of 10% (w/v) CHAPS to 1 mL samples
to form mixed micelle structures. A steady-state condition was verified by measuring the
FFA concentration 3 h after the DFP addition. The partition coefficient of palmitic acid
into l,2-dipalmitoyl—sn-glycero-3-phosphocholine (DPPC) unilamellar liposomes at 23°C
is approximately 5.23 x 106.151 Although we provide no direct evidence that palmitic acid
is present within the membrane phase of unilamellar liposomes, this large partition
coefficient for palmitic acid into DPPC suggests that most of the palmitic acid would be

associated with the hydrophobic region of lipid bilayer rather than the aqueous phase.

5.3.6. Dynamic Light Scattering

The mean particle sizes and size distributions of liposomes and NEST
proteoliposomes in solution were measured with a Brookhaven Instruments 90 Plus/BI-

MAS/ZetaPlus machine (Holtsville, NY).

5.3.7. Transmission Electron Microscopy

A 10 11L aliquot of 69:29:2 liposomes, with and without NEST, was diluted 100-
fold with PEN buffer and fixed on a copper-coated grid using a 2% uranyl acetate stain.
Images were obtained with a JEOL 100CX TEM using an accelerating voltage of 100

kV.

78

5.3.8. Deposition of Supported Lipid Bilayers

Fused silica microscope slides (75 mm x 25 mm x 1 mm) were purchased from
Technical Glass Products, Inc. (Painesville, OH). The slides were cleaned by bath
sonication (Branson 1510, Branson Ultrasonic Corporation, Danbury, CT) in detergent
solution for 20 min, rinsed with deionized water, baked at 160°C for 4 h, and plasma
treated (Harrick Plasma, Ithaca, NY) with oxygen under vacuum (150 mTorr) for 10 min
immediately before bilayer deposition. sBLMs were deposited by liposome or
proteoliposome fusion in a custom-made flow cell described elsewhere.68 The flow cell
was initially washed with buffer, followed by a 1-h incubation with liposome or NEST
proteoliposome solution and a final buffer wash to remove unadsorbed particles. All

experiments were performed at room temperature (23 :l: 2°C).

5.3.9. Fluorescence Recovery After Pattern Photobleaching

FRAPP was used to measure the translational diffusion coefficients and mobile
fractions of fluorophores in sBLMs formed on fused silica microscope slides. The details
of this technique are discussed in Section 2.2 of this dissertation. For each lipid system
considered in this work, three separate sBLM interfaces were formed on silica slides and

each interface was interrogated with FRAPP at five different locations of the bilayer.

5.3.10. Statistical Analysis

Data were fit using OriginPro 7.5 (OriginLab Corporation, Northampton MA)

which uses the Levenberg-Marquardt algorithm for non-linear least squares fitting.

79

Mobile fractions, translational diffusion coefficients, and particle diameters are reported
as means :t SD. Multiple comparison ANOVA with a post-hoe Tukey test (p < 0.05) was

used to determine if differences among the means were statistically significant.

5.4. Results and Discussion

We interrogated seven systems of reconstituted BLMs. To aid in interpretation of

the data, the seven systems were divided among three groups (A,B,C) based on the

 

identity of the lipids and molecules present:

0 Group A (Systems 1 and 2 - BLMs containing DOPC and NBD-PC): System 1 and
system 2 consisted of 98 mol% DOPC and 2 mol% NBD-PC reconstituted without
NEST and with NEST, respectively. System 1 is the basic BLM formulation used
in our lab and serves as an important control to assess the effect of incorporating

' NEST into the membrane.

 

0 Group B (Systems 3, 4, and 5 - BLMS containing DOPC, p-lysoPC, and NBD-PC):
BLMS in this group consisted of 69 mol% DOPC, 29 mol% p-lysoPC, and 2 mol%
NBD-PC, with the following variations: no additional constituents (system 3), with
the NEST inhibitor DFP incorporated (system 4), and with both DFP and NEST
incorporated (system 5).

0 Group C (Systems 6 and 7 - BLMs containing DOPC, p-lysoPC, palmitic acid, and
NBD-PC): System 6 had a composition of 69 mol% DOPC, 29 mol% p-lysoPC,
and 2 mol% NBD-PC, with NEST incorporated to measure the effect of

lysophospholipid hydrolysis. System 7 was 69 mol% DOPC, 20 mol% p-lysoPC, 9

 

mol% palmitic acid, and 2 mol% NBD-PC. The fraction of palmitic acid in system

...-a“ tv. -.

80

 

7 is equivalent to the amount of FFA that would be expected from NEST

hydrolysis in system 6, and was used to provide comparative data.

System compositions are summarized in Table 5.1. We used FRAPP to assess the
translational diffusion coefficient and mobile fraction of fluorophores embedded in the

hydrophobic region of each of the seven systems above.

5.4.]. Transmission Electron Microscopy Images

TEM images (Figure 5.3) showed that the 69:29:2 liposomes and 69:29:2/NEST
proteoliposomes formed after dialysis were unilamellar, ensuring that sBLMs formed
from them would also be unilarnellar.l‘""52 Because liposomes and NEST
proteoliposomes were subjected to high vacuum conditions and to a high-energy electron
beam, it is difficult to use TEM images to evaluate the size distribution of liposomes and

proteoliposomes. Thus, size distribution data were obtained by DLS.

5.4.2. Dynamic Light Scattering Data

We used DLS to assess the transition fiom mixed CHAPS/69:29:2 micelles to
liposomes and mixed CHAPS/69:29:2/NEST micelles to proteoliposomes during dialysis.
This was done by measuring the mean particle diameter as a function of time (Figure
5.4). The DLS results show that the mean diameter of NEST-containing micelles before

dialysis was 2.3 :I: 0.2 nm and that the steady state mean diameter of proteoliposomes

81

after a 30—h dialysis was 40.8 :t 3.1 nm. The sigrnoidal natur of our data has been reported
by others for the transition from micelles to liposomes via detergent dialysis.153’154

The mean diameter of 69:29:2 liposomes, without NEST, after dialysis was not
significantly different from proteoliposomes containing NEST, suggesting that, at the
significantly large lipid to protein ratio (880:1 w/w) used- in this study, the incorporation

of NEST has no effect on particle size.

5.4.3. Measurement of Free Fatty Acid Concentration

Negligible conversion of DOPC or NBD-PC to FFA was measured in 98:2
liposomes (system 1) and in 98:2 proteoliposomes with NEST (system 2), in agreement
with previously published results showing that dual acyl chain lipids are not easily
hydrolyzed by NEST 39‘129. There was also negligible conversion of DOPC, p-lysoPC, or
NBD-PC to FFA in 69:29:2 liposomes, without DFP (system 3) and with DFP (system
4), indicating that p-lysoPC did not significantly auto-hydrolyze and that DFP did not
hydrolyze the lipids to form FFA. Results obtained using PMSF in place of DFP in
systems 3 and 4 were not statistically different. When NEST was mixed with DFP
(system 5) prior to dialysis, negligible conversion of DOPC, p-lysoPC, or NBD-PC to
FFA was observed in 69:29:2 proteoliposomes. Significant conversion of the DOPC, p-
. lysoPC, and NBD-PC mixture to FFA was measured in 69:29:2 liposomes containing
NEST (system 6) after 48 h of dialysis (Table 5.1 and Figure 5.5). Control experiments
showed negligible conversion of DOPC and NBD-PC to FFA in system 2 as well as
negligible conversion of DOPC, p-lysoPC, and NBD-PC in system 3. Thus, the

significant levels of FFA observed in system 6 were attributed entirely to the hydrolysis

82

of p-lysoPC to palmitic acid. To aid in comparing data on other systems, the mean
conversion of p-lysoPC to palmitic acid in the 69:29:2 liposomes containing uninhibited
NEST after 48 h of dialysis was assigned a value of 100%, and formed the base value for
all comparisons. We note that when NEST was mixed with PMSF prior to dialysis,
conversion of p-lysoPC to palmitic acid decreased from 100% to 17%. FFA conversion
values (Table 5.1) for p-lysoPC-containing NEST proteoliposomes were calculated as a
ratio of the total moles of palmitic acid measured to the total moles of p-lysoPC present
before the addition of NEST. Specific activities (Table 5.1) were calculated as ymol FFA

produced per mg NEST per hour, after a reaction time of 48 h.

5.4.4. Translational Diffusion in Supported Bilayer Lipid Membranes

Mobile fractions and 0,, of NBD fluorophores in sBLMs on fused silica slides
were measured with FRAPP. There was no statistical difference between the D], values
for group A (systems 1 and 2) of sBLMs, as shown in Figure 5.6. Hence, incorporating
NEST in the sBLM at a sufficiently large lipid to protein ratio (880:1 w/w) does not
significantly affect the translational diffusion of lipids in the absence of enzymatic
activity. In a previous study, proteoliposomes with relatively small lipid to membrane
protein ratios had identical diffusion characteristics as pure lipid membranes, suggesting
that the protein has little effect on membrane dynamics.”5 Also, the differences in the
mobile fiactions of NBD in group A (systems 1 and 2) were not statistically significant
(Figure 5.7), indicating that the protein did not impede proteoliposome rupture and
subsequent sBLM formation at the low protein concentration used. On the other hand, no

fluorescence recovery was measured when proteoliposomes with lipid to protein ratios

83

less than 80:1 (w/w) were adsorbed on silica slides, indicating negligible mobile
fractions. This result is consistent with proteoliposome adsorption without subsequent
rupture and sBLM formation.

The D], for 69:29:2 sBLMs (system 3) was higher than for 98:2 sBLMs (systems 1
and 2). This should be expected based on simple geometric considerations. P-lysoPC has
a head group area that is essentially the same as the dual acyl chain lipids. Therefore,
when the lipid is incorporated into a sBLM, it provides essentially equivalent packing
levels in the head group region of the bilayer. However, its single acyl chain results in
lower packing densities in the hydrophobic regions of the bilayer, thus leading to a more
fluid system. The increase in membrane fluidity upon addition of p-lysoPC to model
membranes has been observed previously, although our data show a more modest
increase in the D1, than was reported by Leu et al.22, who attributed the increase in fluidity
upon addition of p-lysoPC to DOPC bilayers to a decrease in tail group van der Waals
interactions between adjacent lipids. The other two systems in group B (69:29:2 sBLMs
containing DFP (system 4) and 69:29:2 sBLMs containing both NEST and its inhibitor
DFP (system 5)) had statistically similar DL values as those of system 3. Thus, the
incorporation of either DFP or NEST inhibited by DFP did not significantly affect the
fluidity of the membrane. The two inhibitors, DFP and PMSF, are similar in that they
both irreversibly inhibit esterase activity. However, the former does not undergo the
aging reaction the latter does. As has already been discussed, the inhibition of NEST
activity by DFP and PMSF reduced the conversion of p-lysoPC to palmitic acid in

solution by ~83 and ~100%, respectively (Table 5.1).

84

5.4.5. NEST-induced Hydrolysis of P-lysoPC to Palmitic Acid Decreases Membrane
Fluidity

Incorporation of NEST into a 69:29:2 sBLMs led to a statistically lower DL
value, indicating a less fluid membrane. This result, which is attributed to conversion of
p-lysoPC to palmitic acid by NEST hydrolysis, can be explained on purely geometric
considerations. As already noted, upon hydrolysis of p-lysoPC, the head group of the
lipid is assumed to migrate into the aqueous phase while the hydrocarbon tail remains in
the hydrophobic region of the membrane. The net result is that the head group area per
lipid will contract somewhat because of the missing head groups, which would lead to a
denser hydrophobic region in which the diffusion of constituent species will be retarded.
We have demonstrated that hydrolysis of p-lysoPC by NEST decreases D1, in model
bilayer membranes (system 6) and that inhibition of NEST activity by DFP (system 5) or
PMSF negates this effect. Since palmitic acid is formed by NEST-induced hydrolysis of
p-lysoPC, we can determine if the decrease in membrane fluidity is solely the result of
uninhibited NEST. To asses this, we adjusted the lipid composition a priori to correspond
to the 100% p-lysoPC conversion to palmitic acid measured with the FFA assay. The
resulting system contained 69 mol% DOPC, 20 mol% p-lysoPC, 9 mol% palmitic acid,
and 2 mol% NBD-PC as the fluorophore (system 7). The DL measured in sBLMs
reconstituted with this composition was not statistically different than that measured in
69:29:2 sBLMs containing NEST, which is consistent with the reduction in fluidity being
solely due to NEST hydrolysis. In summary, systems in group C (systems 6 and 7) were
less fluid than those in group B (systems 3, 4, and 5), but more fluid than those in group

A (systems 1 and 2).

85

The ability of saturated fatty acids to decrease BLM fluidity has been well
documented.15 6460 For example, addition of palmitic acid to DOPC bilayers has been
shown by digital scanning calorimetry to increase the gel-to-liquid-crystalline phase
transition temperature, which is an indication of a less fluid bilayer.161 A two-dimensional
Voronoi tessellation analysis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) bilayers
showed that, upon addition of palmitic acid, the area per constituent lipid decreased while

161 This suggests that palmitic acid

the area per palmitic acid molecule remained constant.
is incompressible within the bilayer and acts to decrease the void space between lipids,
leading to a decrease in the area per lipid and a decrease in membrane fluidity. Electron
paramagnetic resonance (EPR) measurements also demonstrated that membrane fluidity
decreased in HepG2 cells exposed to palmitic acid. An MD simulation of palrrritic acid in
1,2-dimyristoyl-sn-glycero—3-phosphocholine (DMPC) bilayers also predicted that the
carboxyl carbon atom of the protonated, neutral form of the acid embeds itself deeply
into the membrane, adjacent to the ester group of the DMPC lipids.162 Palmitic acid
molecules at this position in the bilayer induce tighter lateral packing, stretching of
hydrophobic lipid chains, and increased lipid order. Another study involving the addition
of stearic acid to Langmuir monolayers comprising l,2-dipalmitoyl-sn-glycero-3-
phosphocholine (DPPC) and cholesterol showed an increase in monolayer rigidity with
increasing stearic acid concentration.163

The present study may provide some insight into the possible physiological role

of NTE. Cells are known to adjust membrane fluidity by producing phospholipids that

either increase or decrease fluidity based on lipid-lipid intermolecular interactions. These

86

lipids are generally synthesized in the cytosol, mitochondria, and endoplasmic
reticulum.” The results of the present work involving NEST and model membranes are
consistent with a hypothesis that mammalian neurons and other cell types may use NTE-
induced lysophospholipid hydrolysis as a way of adjusting membrane fluidity.
Alternatively, cells may use NTE-induced lysophospholipid hydrolysis to control toxic
lysophospholipid accumulation in cells. In the latter case, the results of the present study
may provide insight into the important effects of the regulation of lysophospholipid

concentration in cells by NTE.

5.5. Conclusions

We have demonstrated that at sufficiently large lipid to protein ratios, NEST
proteoliposomes adsorbed onto silica substrates rupture to form fluid sBLMs. We have
used FRAPP to study seven different bilayer lipid membrane compositions to assess the
effect of p-lysoPC hydrolysis by the catalytic domain of NTE (NEST) on the fluidity of
the systems. There are two important conclusions based on the results of this study. First,
NEST hydrolysis induces a statistically significant decrease in the fluidity of sBLMs
reconstituted on silica. Second, it has been proposed that one physiological role of NTE is
to maintain lysophospholipid homeostasis in mammalian cells. The results of this study
suggest that BLM fluidity may provide a convenient physical measurement that could be

used to assess this function.

87

5.6. Recommendations for Future Work

We used p—lysoPC in this work for primarily two reasons. First, NEST hydrolyzes
p-lysoPC to a greater extent than 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (o-
lysoPC), an unsaturated fatty acid. This increases the probability that a sufficient amount
of p-lysoPC would be hydrolyzed to enable us to observe a measurable difference in
bilayer fluidity. Second, the product of p-lysoPC hydrolysis, palmitic acid, has been
shown to significantly decrease bilayer fluidity. In future studies, it would be interesting
to replace p-lysoPC with o-lysoPC and measure the fluidity in the presence of an

unsaturated fatty acid, which has been reported in some cases to increase bilayer fluidity.

88

 

o o
/ _ / _
O=P—O O=P—O
\ \
0 0
H H
""0 ’OH
O O o O
0 o 0
NH
)\0
/
N
N+
_./ 8
(a) (b) (C)

Figure 5.1. Molecular structures of the lipids used in this study: (a) 1,2-dioleoyl-sn—
glycero-3-phosphocholine (DOPC), (b) 1-palmitoyl-2-hydroxy-sn-glycero-3-
phosphocholine (p-lysoPC), and (c) the fluorophore l-oleoyl-2-[l2-[(7-nitro-2-l,3-
benzoxadiazol—4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (N BD-PC).

89

It'd

 

\i—b
oo—>
\o—>

Figure 5.2. Representative SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis) processed during NEST purification. Lane assignments are as follows:
Lane 1: marker, lane 2: 3rd column wash, lanes 3 through 8: 1St through 6th column
elutions, lane 9: sample after 48-hour dialysis using 4"] elution.

90

(a)

(b)

 

Figure 5.3. (a) TEM image showing representative 69:29:2 liposomes after dialysis. (b)
TEM image showing a representative 69:29:2/NEST proteoliposome after dialysis. Such
unilamellar liposomes and proteoliposomes were used to form sBLM structures on silica.

91

01
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A
E.
d) _.
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2
.2
204
E
a.
t:
8
2 10-
0 ' I ' 1 1 r ' l T 1* ' 1
o 5 1o 15 20 25 30

Time (h)

Figure 5.4. DLS results showing the CHAPS/69:29:2/NEST micelle to proteoliposome
transition during dialysis. Data represent means i SD of three separate dialysis runs. A
Boltzmann sigmoid function, d = d2+((d1-d2)/(1+exp((t-to)/dt))), was fitted to the data. In
this function, d is the time-dependent mean particle diameter, t is time, (11 is the initial
particle diameter, d2 is the final particle diameter, to is the center time value, and (it is the
time constant. The fitted sigmoid had a correlation coefficient of 0.999. Fitted parameters
were as follows: (it = 1.7 :l: 0.6 nm, d2 = 43.0 i 0.5 nm, to = 12.9 i 0.4 h, and (it = 2.7 i
0.2 h.

92

 

 

100 -
80 d
C!
.2 .
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B
> 60 d
1:
O
U
*5 4o -
a.)
8
Si
20 d
0
I ' l ' I ' l ' l ' I fl
0 10 20 30 40 50

Time (h)

Figure 5.5. Time-course of conversion of p-lysoPC to palmitic acid during dialysis. A
100% conversion of p-lysoPC to palmitic acid was assigned to 69:29:2 liposomes
containing uninhibited NEST alter 48-h dialysis. Data are means :1: SD of two separate

assays. A line was fitted to the data using linear regression analysis. The fitted line had a
correlation coefficient of 0.990.

93

    

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95

Table 5.1. Percent conversion and specific activity of p-lysoPC to palmitic acid for
several systems“

 

 

 

 

 

 

 

 

. . g
produced/hr)
1 9822b - - -
2 98:2 (with NEST) - - _
3 69:29:26 0.3 :1: 0.1 - -
4 69:29:2 (with DFP) 0.3 r 0.1 - -
5 69:338.}?“1 100 a 7 - (3.6 :1: 0.24) x 10'2
6 ”iiiggylafl 0.3 e 0.1 99.7 r 7.0 (1.2 r 0.24) x 10“
7 69:20:92" - - -

 

 

 

 

 

 

 

“Data represent means i SD of two separate assays.

b98:2 is 98 mol% DOPC, 2 mol% NBD-PC.

669:2922 is 69 mol% DOPC, 29 mol% p-lysoPC, and 2 mol% NBD-PC.

d69:20:9:2 is 69 mol% DOPC, 20 mol% p-lysoPC, 9 mol% palmitic acid, 2 mol% NBD-
PC

8100% conversion of p-lysoPC to palmitic acid was assigned to the mean conversion
value in 69:29:2 liposomes containing uninhibited NEST after 48-h dialysis.

f Percent reduction in conversion for systems with NEST and either DFP or PMSF,

relative to the mean conversion value in 69:29:2 liposomes containing uninhibited NEST

after 48-h dialysis.
gSpecific activities are reported in terms of FF A produced per mg NEST and after a time

period of 48 h.

96

6. EFFECT OF MEMBRANE PROTEIN CONCENTRATION ON THE
TRANSLATIONAL DYNAMICS OF AN EMBEDDED FLUOROPHORE IN
SUPPORTED BILAYER MEMBRANES CONSTITUTED FROM
PROTEOLIPOSOMES

6.1. Abstract

sBLMs are model membranes commonly formed via self-assembly of liposomes
adsorbed to silica, quartz, or mica surfaces. When membrane proteins are incorporated
into liposomes, they form proteoliposomes, which have also been shown to form sBLMs
via self-assembly on similar surfaces. Although many studies have demonstrated that
sBLMs can serve as an important tool in the study of membrane protein activity and
functionality, the relationship between protein concentration in proteoliposomes and their
ability to form sBLMs via self-assembly is poorly understood. The objective of this work
was to use FRAPP to assess how the concentration of a membrane-associated protein
affects the translational dynamics and mobile fractions of an irnbedded chromophore in a
sBLM reconstituted from proteoliposomes. We used NEST as a model membrane-
associated protein in DOPC/NEST/NBD-PC proteoliposomes self-assembled into sBLMs
on fused silica. At a lipid to protein ratio of 80:1 (w/w), we observed no measurable
fluorescence recovery of NBD-PC in sBLMs, suggesting that the fluorophores resided in
unruptured liposomes on the substrate. The minimum lipid to protein ratio at which we
observed fluid sBLM formation upon proteoliposome deposition on silica was 880:1
(w/w). We also qualitatively assessed the effect of membrane protein concentration on
the dynamics of NBD-PC in bilayer interfaces formed from microsome membrane

fractions of Saccharomyces pombe yeast cells.

97

6.2. Introduction

tBLMs are commonly used to characterize protein-protein interactions,33 ion-
channel activity,32 and membrane protein activity.34 sBLM interfaces are, in contrast to
tBLMs, only separated from the substrate by a 1 A-thick layer of water. sBLMs formed
from liposome self-assembly have been widely used to study liposome rupture and fusion
processes4’42’l64‘l65, lipid-lipid interactions,22 and lipid migration in applied electric
fields.166 One of the major drawbacks of using sBLM interfaces to study membrane
proteins is that the proteins often come into contact and interact with the substrate and
become denatured and/or immobile. In spite of this, many studies have shown that
sBLMs do in fact provide useful tools for characterizing the interactions of trans-

membrane proteins in membranes,“57’168

as well as determining membrane protein
activity and function.14 For example, a peripheral membrane protein acetylcholinesterase
and integral membrane proteins bacteriorhodopsirr and cytochrome oxidase have been
shown to retain their biological activities when placed in PC BLMs reconstituted on
silicon wafers and platinum-coated silica.”’9’170 Trans-membrane photosynthetic reaction
centers (RCs) that were incorporated into fluid sBLMs on glass retained their charge
separation and cytochrome c binding properties even though the RC5 themselves were
immobile.15 Chan et al. demonstrated that not only did glycosyl-phosphatidylinositol
(GPD-anchored membrane receptors remain fluid in sBLMs on glass substrates, but that
the fluid nature of the protein enhanced cell adhesion to the membrane.171 We also
showed in Chapter 5 that sBLMs formed from proteoliposomes containing the

membrane-associated protein NEST and p—lysoPC exhibited a decrease in membrane

fluidity as a result of the enzymatic activity of the protein.

98

In each of these cases, the lipid to protein ratio was selected to ensure that
proteoliposomes adsorbed and ruptured to form fluid sBLMs when exposed to either
glass substrates171 or lipid SAMs formed by the Langmuir—Blodgett technique.170 In the
present work, we estimated a range of lipid to protein ratios that produce fluid sBLMs on
fused silica using proteoliposome self-assembly. We used DOPC as the primary lipid,
NBD-PC as the lipid fluorophore, and NEST as a model membrane-associated protein.
Diffusion coefficients and mobile fractions of fluorophores in the sBLMs were obtained
fiom FRAPP data. We also qualitatively evaluated the effect of membrane proteins on the
fluidity of interfaces formed from microsome membrane fiactions of Saccharomyces

pombe yeast cells.

6.3. Materials and Methods

6.3.1. Chemicals and Reagents

Phospholipids DOPC, POPC, and NBD-PC were purchased from Avanti Polar
Lipids Inc. (Alabaster, AL) and used without further purification. The molecular
structures of these lipids are shown in Figure 6.1. CHAPS detergent (purity Z 99% by
high performance liquid chromatography) was purchased from Anatrace (Maumee, OH).

Irnidazole was purchased from Sigma Aldrich (St. Louis, MO).

6.3.2. NEST Expression and Purification

NEST was expressed and purified according to the protocol in Section 5.3.2 of

this dissertation.

99

6.3.3. NEST Proteoliposome Formation

NEST proteoliposomes were formed by the dialysis method described in Section
5.3.3. NEST protein solution (0.1 mg/mL) was added to 98 mol% DOPC, 2 mol% NBD-
PC, 10 wt% CHAPS solutions in l600:l (w/w) (l l8,000:1 mol/mol), 880:1 (w/w)
(65,000:l mol/mol), 270:1 (w/w) (20,000:l mol/mol), and 80:1 (w/w) (6,000:1 mol/mol)
lipid to protein ratios. In terms of notation, an interface formed from proteoliposomes
with, for example, an 80:1 (w/w) lipid to protein ratio will be referred to as an 80:1 (w/w)
interface throughout this chapter. The presence of liposomes was confirmed by

monitoring the micelle to liposome transition with TEM and DLS (Figures 5.3 and 5.4).

6.3.4. Preparation of Yeast Membrane Fractions

This procedure is described in detail elsewhere.90 Briefly, Saccharomyces pombe
cellsiwere grown in yeast peptone dextrose (YPD) medium (20 g/L yeast peptone, 10 g/L
yeast extract and 2% w/v glucose). Cells were harvested by centrifugation and suspended
in lysis buffer (25 mM Tris buffer, pH 8.0, containing 250 mM sucrose). The cells were
then passed three times through an Emulsiflex-C3 homogenizer and the debris was
centrifuged at 14,900g for 30 min at 4°C. The supernatant was centrifuged and the
membrane pellet was frozen at -80°C. To prepare membrane fi'actions, a portion of the
membrane pellet was suspended in 20 mM Tris-HCl buffer, pH 8.0, containing 200 mM
NaCl and 10% glycerol. The suspension containing the membrane fraction was sonicated
for 5 min, then passed through a 200 nm filter. To incorporate the lipid fluorophore into

membrane fractions, a chloroform solution of NBD-PC was dried under nitrogen and

100

placed under vacuum at -45°C for 3 h. After the lipid film was hydrated with the

membrane fraction suspension, the mixture was sonicated for 20 min.

6.3.5. Extraction of Lipids From Yeast Cells

A mixture of 10 mL methanol, 20 mL chloroform, and 1.5 mL aqueous membrane

fraction suspension were stirred continuously for l h at room temperature. After
separation of the organic and aqueous phases, 0.034% MgClz was added to the organic

phase and the solution was stirred for 10 min. The organic phase was then centrifuged at
3,000 rpm for 5 min after which the upper aqueous phase was aspirated. The organic
phase was then washed with a 10 mL solution of 4:1 (v/v) KCl/methanol, the mixture was
centrifuged, and the upper phase aspirated. The organic phase was then washed with a 10
mL solution of 3:48:47 (v/v/v) chloroform/methanol/water. The mixture was then
centrifuged, washed again with the chloroform/methanol/water solution, and the upper
phase was aspirated. The organic phase was placed in a rotary evaporator at 55°C and
200 mbar until a dry lipid film was formed. The lipid film was dissolved in a 2:1 (v/v)
chloroform:methanol solution and stored at -20°C. Liposomes formed from microsome
lipid fractions were created by adding 2 mol% NBD-PC to the lipid/chloroform/methanol
solution, drying the solution with nitrogen to create a lipid film, placing the film under

vacuum at -45°C for 3 h, and hydrating the film with Tris buffer at pH 8.0.

101

6.3.6. POPC Liposome Preparation

Solutions containing 1 mM POPC/NBD-PC (98:2, mol/mol) in chloroform were
dried under nitrogen and placed under vacuum at -45°C for 3 h. The dried lipid mixtures

were then hydrated in Tris buffer at pH 8.0, and sonicated for 20 min.

6.3.7. Deposition of Interfaces

Fused silica microscope slides (75 mm x 25 mm x 1 mm) were purchased from

Technical Glass Products, Inc. (Painesville, OH). The slides were cleaned by bath
sonication (Branson 1510, Branson Ultrasonic Corporation, Danbury, CT) in detergent
solution for 20 min, rinsed with DI water, baked at 160°C for 4 h, and plasma treated
(Harrick Plasma, Ithaca, NY) with oxygen under vacuum (150 mTorr) for 10 min
immediately before bilayer deposition. Interfaces were deposited by either liposome,
proteoliposome, or membrane fraction adsorption in a custom-made flow cell described
elsewhere.68 The flow cell was initially washed with buffer, followed by a 1 h incubation
with either solutions of POPC synthetic liposomes, NEST proteoliposomes at various
lipid to protein ratios, or yeast membrane fractions. The flow cell was then rinsed with
buffer to remove unadsorbed species. All experiments were performed at room

temperature (23 i 2°C).

6.3.8. Fluorescence Recovery After Pattern Photobleaching

FRAPP was used to measure DL and mobile fractions of fluorophores in the

interfaces formed on fused silica microscope slides. The details of this technique are

102

 

given in Section 2.2 of this dissertation. For each lipid system used in this work, three
separate interfaces were formed on silica slides and each interface was interrogated by

FRAPP at five different locations.

6.3.9. Statistical Analysis

Data fitting was accomplished using OriginPro 7.5 (OriginLab Corporation,

Northampton MA) which uses the Levenberg-Marquardt algorithm for non-linear least

 

squares fitting. Diffusion coefficients and mobile fractions are reported as means i SD.
Multiple comparison ANOVA with a post-hoc Tukey test (p < 0.05) was used to

determine if differences among the means were statistically significant.

6.4. Results and Discussion

6.4.1. Interfaces Formed with NEST Proteoliposomes

Equation 3.5, which assumes one mobile and one immobile species,44 was fit to

the FRAPP data to extract DL values and mobile fractions of the embedded lipid
fluorophores. When proteoliposomes with DOPC lipid to NEST ratios of less than 80:1
(w/w) were deposited on silica slides, no fluorescence recovery was observed, indicating
that the NBD fluorophores were not mobile (Figure 6.2). We interpret the mobile species
as fluorophores in fluid sBLMs and the immobile species as fluorophores in adsorbed but
unruptured liposomes. Therefore, when there is no measurable fluorescence recovery, it

suggests that proteoliposomes adsorbed onto the silica but did not rupture to form

103

sBLMs. At this protein concentration, the transition from adsorbed proteoliposomes to a
fluid sBLM via proteoliposome fusion and rupture is completely inhibited.

Mobile fractions and DL for all DOPC/NEST/NBD-PC interfaces are summarized
in Table 6.1. There was a measurable recovery for the case of proteoliposomes with a
270:1 (w/w) lipid to protein ratio. The DL and mobile fraction of these interfaces were
1.72 i: 0.25 ,umZ/s and 0.75 i 0.13, respectively. Interfaces formed from proteoliposomes
with an 880:1 (w/w) lipid to protein ratio had a D1, of 1.98 :1: 0.09 ,umz/s and a mobile
fraction of 0.79 d: 0.06. AN OVA analysis shows that the DL and mobile fraction for these
interfaces were not statistically different. However, the standard deviations in DL (~5%)
and mobile fraction (~8%) for the 880:1 (w/w) interfaces were less than those for the
270:1 (w/w) interfaces (~15% in DL and ~17% in the mobile fiaction). We believe the
large standard deviations in the DL and mobile fraction for the 270:1 (w/w) interfaces
relative to the 880:1 (w/w) interfaces are the result of a heterogeneous interface in which
some fluorophores diffuse freely in patches of fluid sBLMs, while others remain
immobile in adsorbed but unruptured liposomes. The DL and mobile fraction (2.01 i 0.13
ymz/s and 0.81 d: 0.05, respectively) for 1600:1 (w/w) interfaces were not statistically
different to those for 880:] (w/w) interfaces. In addition, the standard deviations in DL
and mobile fractions were comparable for both interfaces. At an 880:1 (w/w) lipid to
protein ratio, it appears the formation of fluid sBLMs constituted from proteoliposomes is
unimpeded. Therefore, the minimum DOPC lipid to NEST protein ratio at which we
observed fluid sBLM formation upon proteoliposome deposition on silica was 880:]
(w/w). It is conceivable that a fluid interface could be obtained at some other ratio

between 270:1 and 880:1. However, we did not investigate this possibility.

104

6.4.2. Interfaces Formed with Yeast Membrane Fractions

Membrane fractions of Saccharomyces pombe yeast cells with NBD-PC
fluorophores yielded a mobile fraction of 0.24 i 0.06 and a D1, of 1.71 :1: 0.44 ,umZ/s after
deposition on silica (Figure 6.3). The low average mobile fraction shows that the majority
of fluorophores were in adsorbed membrane fractions consisting of lipid and protein that
did not spontaneously form fluid sBLMs. We also speculate that the large standard
deviation in DL values is likely due to mobile fluorophores that experienced hindered
diffusion from proteins and other cell debris in the microsomes. After separation of
membrane proteins from the lipids, the lipid fraction of the yeast membranes produced
sBLMs with a mobile fraction of 0.67 i 0.03 and D1, of 1.90 i 0.14 ymZ/s (Figure 6.4).
We measured comparable DL and mobile fractions in sBLMs reconstituted from POPC
and NBD-PC with no proteins (Figure 6.5). The DL and mobile fractions for interfaces
formed from mixtures of NBD-PC with POPC and with microsome membrane fractions

are summarized in Table 6.2.

The removal of proteins fiom the yeast membrane fractions led to a near 3-fold
increase in the mobile fraction. The 21% reduction in the standard deviation of D1, also
suggests a more uniform bilayer. Although we did not quantify the concentration of
protein or lipid present in the yeast membrane fractions, these results give a qualitative

sense of how membrane protein concentration can significantly influence fluorophore

dynamics in sBLMs.

105

 

6.4.3. Significance of Lipid to Protein Ratios in the Formation of sBLM Interfaces

The minimum ratio of DOPC lipid to NEST protein at which we observed a fluid
sBLMs upon proteoliposome reconstitution on silica was 880:1 (w/w) (65,000 mol/mol).
In studies using a 47 kDa trans-membrane glycoprotein called tissue factor (TF), Contino
et al. used a 100,000:l mol/mol lipid to protein ratio to form sBLMs on the inner surface
of glass microcapillary tubes.172 The TF protein, which forms complexes that initiate
blood coagulation after tissue damage, has an extracellular domain (residues 1-219), a
transmembrane domain (residues 220-242), and a cytoplasmic domain (residues 243-
263).'72 Salafsky et al. incorporated 100 kDa trans-membrane RC’s in proteoliposomes at
a 350:1 mol/mol lipid to protein ratio to form fluid sBLMs on glass surfaces.15 There is
no evidence that the protein concentrations used in the Contino and Salafsky experiments
corresponded to the minimum lipid to protein ratio for fluid sBLM formation. It is

possible that these concentrations were arbitrarily chosen.

6.5. Conclusions

We used four different DOPC lipid to NEST protein ratios to assess the effect of
protein concentration on the dynamics of NBD-PC in interfaces formed by
proteoliposome self-assembly on silica. At a ratio of 80:1 (w/w), proteoliposomes
adsorbed to the silica surface but did not rupture to form a bilayer. At a ratio of 270:1
(w/w), we observed only patches of fluid bilayers, with interspersed regions of
unruptured liposomes. Although the minimum lipid to protein ratio at which we observed

fluid sBLM formation was 880:1 (w/w), previous work suggests that sBLM formation

106

 

from proteoliposomes may be a function of several variables besides than protein
concentration. We also observed that interfaces formed from membrane fractions of
Saccharomyces pombe cells were relatively immobile compared to interfaces formed

from the extracted lipids of these cells.

6.6. Recommendations for Future Work

Based on the above discussion, we could suggest a systematic study with proteins
of different molecular weights and electrostatic charge (adjusted by pH) to understand
how these variables influence sBLM formation. Also, it would be meaningful to assess if
the activity of supported bilayer membrane-bound proteins is preserved. These studies
may be useful in the design of supported BLM-based sensors that incorporate membrane

proteins.

107

O O o O 0 O
O O O
| ' I
NH
24>)
N
N +
o/ \o
(a) (b) (c)

Figure 6.1. Molecular structures of the synthetic lipids, used in this study:
dioleoylphosphatidylcholine (DOPC), palmitoyl-oleoyl phosphatidylcholine (POPC), and
the fluorophore NBD-phosphatidylcholine (NBD-PC).

108

 

 

 

0.40-
:? .
m
r:
9. 0.354
E
8 .
C 9% o. 9*... . ‘00 ..
8 3.. if? 0 '- ° 3.3..
(D 030- 0. O o a. Q~ 0.0..”
e :0 o o ’ O
o "I
.2
u.
'0
“N’ 0.25-
76
E
h
0
Z

0.20 I I I I I I I I I l I ' I I I I I l

-5 o 5 1o 15 20 25 30 35 40

Time (s)

Figure 6.2. Representative FRAPP recovery curve for an interface formed from
proteoliposomes at an 80:] (w/w) lipid to protein ratio.

109

L

.0

N

0|
1

0.20 -

0.15-

0.10-

0.05 d

0.00 -

 

 

 

Normalized Fluorescence Intensity

q

I
01
o d
01

Figure 6.3. Representative FRAPP recovery curve with residuals (below) for an interface
formed from membrane fractions of Saccharomyces pombe cells.

10

15

Time (s)

110

 

0.40 -
0.35-
0.30 ;
0.25 -
0.20 -
0.15 A

0.10-

 

0.05 -

p
.8

 

 

Normalized Fluorescence Intensity

‘0.05 r I r r | r l I 1 I7 j T I v 1

T ' T
-5 0 5 10 15 20 25 30 35 40
Time (s)

Figure 6.4. Representative FRAPP recovery curve with residuals (below) for an interface
formed from the lipid fraction of Saccharomyces pombe cell membrane.

111

 

 

 

.0005 I I I l I I I r I I I I I r I l T I

-5 0 5 10 15 20 25 30 35 40
Time (5)

Normalized Fluorescence Intensity
p
8
l 1

Figure 6.5. Representative FRAPP recovery curve with residuals (below) for a POPC
sBLM.

112

Table 6.1. Translational diffusion coefficients (DL) and mobile fractions for interfaces
formed from systems containing DOPC, NEST, and NBD-PC“.

 

 

 

 

 

Systemb’c DL (pmz/s) Mobile Fraction
160021 2.01 i 0.13A 0.81 :l: 0.05 B
880:] 1.98 d: 0.09A 0.79 a: 0.06B
270:1 1.72 d: 0.25A 0.75 :l: 0.13B
80:1 - -

 

 

 

 

 

“Data represent means i SD of three liposome solutions.

bDOPC lipid to NEST protein ratio (w/w).

cSystems with statistically identical DL values and mobile fractions are labeled by

common capital letters (AN OVA, Tukey post-hoc test, p < 0.05).

Table 6.2. Translational diffusion coefficients (DL) and mobile fractions for interfaces
formed from systems containing 2 mol% NBD-PC“.

 

 

 

 

 

 

 

Systemb DL (umz/s) Mobile Fraction
Yeast microsomes 1.71 35 044A 0.24 :t 0.06B
Yeast microsome lipids 1,90 :1: 0.14A 0.67 d: 0.03C
POPC 2.06 :t 0.20 A 0.76 :l: 0.02”

 

 

”Data represent means i SD of three liposome solutions.

bSystems with statistically identical DL and mobile fractions are labeled by common

capital letters (ANOVA, Tukey post-hoc test, p < 0.05).

113

 

7. CONCLUSIONS

The overall goals of the four studies outlined in this dissertation were to 1)
measure the effect of selected molecules and macromolecules on model BLM fluidity,
and 2) understand on a fundamental level how these molecules and macromolecules
influence membrane fluidity. We accomplished these goals by assessing lipid fluorophore
dynamics in several model BLM systems. Chapter 3 describes the effect of two liposome
preparation methods, sonication and extrusion, on the mean diameter, size distribution,
and rotational and translational dynamics of a fluorophore embedded in DOPC and
DOPC/cholesterol systems. We observed that while the mean diameter and size
distribution of the liposomes depended on the preparation method, the rotati0nal and
translational dynamics of NBD-PC fluorophores embedded in liposomes prepared by
either of the two techniques were not statistically different. The dynamics in
DOPC/cholesterol systems were slower than in DOPC systems due to enhanced van der
Waals interactions in the hydrophobic region of the BLM.

We demonstrated in Chapter 4 that the addition of DOPG to DOPC liposomes had
no statistically significant effect on the rotational dynamics of NBD-PE fluorophores, but
did influence the translational dynamics in sBLMs. Based on this result, we concluded
that the NBD moiety likely rotates as a free rotor and does not interact strongly with
surrounding lipids. However, inter lipid-head group hydrogen bonding involving the
glycerol moiety of DOPG likely retards translational diffusion.

Chapter 5 covers a study on NEST, the catalytic fragment of NTE. We observed
that DOPC bilayer fluidity increased upon addition of p—lysoPC lipids due to fewer van

der Waals interactions in the hydrophobic region of the BLM, but decreased afier NEST

114

hydrolyzed a fraction of the p-lysoPC to form palmitic acid. This latter effect can be
explained on the basis of geometric considerations. The projected area of the head group
of p-lysoPC would decrease as it is hydrolyzed to palmitic acid, resulting in a denser
hydrophobic region. This is an important finding in that it suggests a physical parameter
that may be used in characterizing lysophospholipid control by NTE in mammalian
neuron and other cell types.

In Chapter 6 we studied the effect of a model membrane—associated protein
(NEST) on sBLM formation on silica using proteoliposomes of various lipid to protein
ratios. The minimum lipid to protein ratio at which we observed fluid sBLMs
reconstituted from proteoliposomes was 880:1 (w/w). We also qualitatively assessed the
effect of membrane protein on bilayer formation using natural yeast membrane
fragments.

We believe the results of the four studies may be useful in understanding the
molecules and macromolecules that mediate membrane fluidity in cell membranes. Also,
these results may suggest molecules and macromolecules that can be used to manipulate
Amembrane fluidity for the development of BLM-based protein biosensors and related

devices.

115

8. APPENDICES

8.1. Analysis of Variance (AN OVA) Methodology in Data Analysis

Multi-comparison ANOVA with a post-hoe Tukey test (p < 0.05) was used
throughout this dissertation to determine if there are statistical differences between the
means of multiple data sets. We used the total number of data sets being compared (k)
and the number of data points 02) in each data set to calculate the studentized degrees of
freedom (v) with Equation (A. 1):

V = k(y-—1) (A.1)

An (2 value of 0.95, corresponding to a 95% confidence limit, was chosen and a q
parameter was determined from a table of critical values for F distributions. The q
parameter is a function of k, v, and a. We calculated the mean square for error (MSE)
fi'om Equation (A.2):

k 2

MSE = —_—‘Zi=l i (A2)

Here, S,- refers to the standard deviation of the ith data set. The Tukey parameter (Tu) was

MSE
T u = q‘i—y— (A.3)

Any means that differed by more than Tu were considered significantly different.

calculated with Equation (A3):

116

8.2. Non-linear Curve Fitting Analysis

Throughout this dissertation, we used mathematical models to interpret
fluorescence lifetime, fluorescence anisotropy, and FRAPP data. In general, a model with
n parameters will fit data at least as well as a model with n-l parameters. An F-test
comparison was conducted to determine whether a model with n parameters provided a
statistically significant improvement in the goodness of fit over a model with n-l

parameters. The F-statistic was defined as:

= (112 —z§)(N-5)

F
2122

 

(A.4)

where N is the number of data points and x12 and x22 are the chi-squared goodness-of-fit
statistics for models with n-1 and n parameters, respectively. If this F statistic exceeded

3.0, the fit using the model with n parameters is considered a significant improvement!“

173

117

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