NMR Studies of Lateral Diffusion of a

Transmembrane Peptide in DMPC/DHPC Bicelles

and Morphological Characterization of

DMPC/CHAPSO Bicelles

by

Hannah Hazel Antonio Morales

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Chemistry University of Toronto

© Copyright by Hannah Hazel Antonio Morales (2013)

ii

NMR Studies of Lateral Diffusion of a Transmembrane Peptide in DMPC/DHPC Bicelles and

Morphological Characterization of DMPC/CHAPSO Bicelles

Hannah Hazel Antonio Morales

Master of Science

Department of Chemistry University of Toronto

2013

ABSTRACT

NMR is used as a technique to study diffusion and morphology in bicellar model

membranes. 1H stimulated-echo pulsed-field-gradient NMR was used to study lateral

diffusion of the alpha-helical transmembrane peptide WALP19 with a polyethylene

glycol (PEG)(MW 2000) tag incorporated into magnetically aligned bicelles. Both

peptide and control lipid DMPE-PEG2000 exhibited normal Gaussian diffusion and

followed Arrhenius-type temperature dependence, befitting the free-area model for lateral

diffusion. Activation energy for peptide lateral diffusion (75 kJ/mol) was larger by 25%

than that of the lipid, which is consistent with the triple layer model of the lipid

membrane. The bicelles used had the canonical mixture of 1,2-dimyristoyl-sn-glycero-3-

phosphocholine(DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine(DHPC), and were

also compared with bicelles composed of DMPC and bile salt analogue CHAPSO. As

assessed by 31P-NMR, DMPC/DHPC bicelles exhibit greater order, but CHAPSO-

containing bicelles maintain magnetic alignment at higher temperatures. Surface charge

on DMPC/CHAPSO bicelles dampens their differences with DHPC-containing mixtures

in order and alignment.

iii

ACKNOWLEDGMENTS

I would like to offer my deepest gratitude and appreciation to all those who have been

instrumental in the completion of this work, especially to the following:

My supervisor Prof. Peter M. Macdonald for accepting me in his research lab and

patiently providing support, direction, and focus for my journey as a graduate student. I

owe him my sincerest thanks not only for being an excellent mentor, but also for his

sincere concern towards the well-being and growth of his students.

My collaborators Prof. Mu-Ping Nieh of the University of Connecticut and Dr. David

Tulumello and Prof. Charles Deber of the Division of Molecular Structure and Function

at SickKids Hospital in downtown Toronto. NMR and biophysical chemistry experts

Prof. Scott Prosser and Prof. Voula Kanelis of the University of Toronto Mississauga for

their invaluable input towards the progress of my research projects.

Prof. M. Cynthia Goh, who inspired me to take the leap of faith in graduate studies, for

her continual support after I arrived in Canada from the Philippines and embarked on my

own path of academic and personal growth.

The former and present members of the Macdonald lab who have all contributed to the

expertise and practical knowledge I have acquired for my research projects, especially

Qasim Saleem, Rohan Alvares, and Angel Lai, for also enriching my life through their

valued friendships, insightful conversations, and gracious appeasement of my culinary

curiosities. Special mention also goes to Yuvraj Pannu for his brotherly kindness and

unselfish drive.

To the various friends I have made in Canada, who helped me in my initial year of many

adjustments and shared in the motley of joys and challenges in the years that followed. I

especially thank the first friends I made from the Department of Chemistry: “manong”

Jordan Dinglasan, “manay” Mayrose Salvador, Yoshi Suganuma, and my former

roommate Ruby Sullan. I can always trust and count on your care and support.

iv

Mentors and friends in the Church communities I am blessed to part of, for the

fellowship, moral and spiritual support, and prayers, even from different places of the

world: Quezon City, Philippines; California, USA; and here in Mississauga. Special

mention goes to Dr. J. Hernes Abante for his wise advice and counsel, and for the sincere

care for my overall well-being which was very much felt in spite of the intercontinental

distance.

To the most important people in my life: my family, my beau Christian Simeon, and my

Lord God Almighty. No words can sufficiently describe my gratefulness for your

unwavering love and support. Maraming salamat sa inyong walang-sawang paggabay at

pagmamahal na hindi mapapantayan magpakailanman.

To my present and future families

and to Him from whom all blessings flow

This thesis is lovingly dedicated to.

-Hannah Hazel A. Morales

“Gracious God watches over”

Proverbs 3:5-6

v

TABLE OF CONTENTS

Abstract .........................................................................................................................ii

Acknowledgments ....................................................................................................... iii

Table of Contents .......................................................................................................... v

List of Tables ............................................................................................................... vii

List of Figures.............................................................................................................viii

List of Equations ........................................................................................................... x

List of Symbols ............................................................................................................. xi

List of Abbreviations ..................................................................................................xiii

Chapter 1: Introduction ................................................................................................ 1

1.1. Lipid membranes and aggregates .......................................................................... 2

1.2. Membrane proteins ............................................................................................... 5

1.3. Bicelles as model membrane systems ................................................................... 6

1.4. Diffusion in cell membranes ................................................................................. 9

1.4.1. Models of lateral diffusion in lipid membranes............................................... 9

1.4.2. Fluorescence-based methods to study diffusion ............................................ 10

1.4.3. Diffusion measurements from pulsed field gradient NMR spectroscopy ....... 12

1.5. References .......................................................................................................... 14

Chapter 2: Experimental ............................................................................................ 20

2.1. Materials ............................................................................................................ 20

2.2. PEGylated WALP19 peptide synthesis ............................................................... 20

2.3. Sample preparation ............................................................................................. 22

2.3.1. DMPC/DMPG/DHPC bicelles with transmembrane peptide ........................ 22

2.3.2. DMPC/DMPG/CHAPSO bicelle samples with free PEG .............................. 22

2.4. NMR spectroscopy ............................................................................................. 23

2.5. References .......................................................................................................... 24

vi

Chapter 3: Lateral Diffusion of a Transmembrane -Helical Peptide in DMPC/DHPC Bicelles ............................................................................................ 25

3.1. 31P NMR and magnetic alignment of bicelles with WALP19-PEG2000 .............. 26

3.2. Hydrophobically-anchored PEG diffusion in interbicellar spaces ........................ 29

3.3. Relaxation issues ................................................................................................ 33

3.4. Conclusions ........................................................................................................ 37

3.5. References .......................................................................................................... 38

Chapter 4: Morphological Characterization of DMPC/CHAPSO Bicellar Mixtures via NMR .................................................................................................. 42

4.1. Magnetic alignment of DMPC/DMPG/CHAPSO mixtures from 31P NMR ......... 43

4.2. Water and PEG diffusion in interbicellar spaces ................................................. 48

4.3. Thermal stability of CHAPSO bicelles................................................................ 53

4.4. Conclusions ........................................................................................................ 56

4.5. References .......................................................................................................... 57

Chapter 5: Summary .................................................................................................. 62

vii

LIST OF TABLES

Table 1.1. Lipid types with different molecular shapes and their preferred aggregate structures. ................................................................................................................ 4

Table 3.1. The 31P chemical shifts, , of the bicellar lipid peaks and the effective ratios of planar-to-curved phospholipid populations, q*, with the peptide WALP19-PEG2000 incorporated at 1 mole% with respect to DMPC. ................... 28

Table 3.2. The 31P chemical shifts, , of the bicellar lipid peaks and the effective ratios of planar-to-curved phospholipid populations, q*, with the lipid DMPE-PEG2000 incorporated at 1 mole% with respect to DMPC..................................... 29

Table 4.1. Activation energies for water and PEG-1000 diffusion in various negatively magnetically aligned bicelles. ............................................................... 53

viii

LIST OF FIGURES

Figure 1.1. Updated model of the cell membrane adapted from the original fluid-mosaic model. ..................................................................................................... 3

Figure 1.2. Phase transition of lipid bilayer from the gel phase, where diffusion is restricted, to the highly fluid liquid-disordered phase. .......................................... 3

Figure 1.3. Schematic of structures found in transmembrane proteins: (a) single-spanning and multi-spanning alpha-helices and (b) rolled-up beta sheets forming a beta-barrel. .......................................................................................... 5

Figure 1.4. Bicelle morphology as a function of q, the molar ratio of long-chain lipids to short-chain lipids/detergents. ........................................................................... 7

Figure 1.5. Magnetic alignment of DMPC/DHPC bicelles and corresponding 31P spectra.. ............................................................................................................... 8

Figure 1.6. The basic Stejskal-Tanner pulsed field-gradient NMR pulse sequence producing a spin echo. ....................................................................................... 12

Figure 1.7. The stimulated echo pulsed field-gradient pulse sequence used for diffusion experiments in NMR. .......................................................................... 13

Figure 3.1. Schematic of the transmembrane alpha-helical peptide WALP19 with a 2000Da PEG tag at the N-terminus inserted in bicelles spontaneously aligned in a magnetic field. ............................................................................................ 25

Figure 3.2. 31P NMR spectra acquired at different temperatures of aligned bicelle samples with 20 wt% lipid, composed of q=(DMPC+DMPG)/DHPC=3 with DMPG/DMPC = 0.1 and 1 mol% WALP-PEG2000 with respect to DMPC. ...... 27

Figure 3.3. Semi-logarithmic diffusion decay plots of the normalized intensity of the WALP19-PEG2000 resonance from 1H PFG STE NMR spectra of negatively magnetically-aligned DMPC/DMPG/DHPC, R=0.10, = 20 wt% mixtures as a function of the experimental factor, k = ( g )2·( – /3)/10-10 for different temperatures. ..................................................................................................... 31

Figure 3.4. Temperature dependence of DMPE-PEG2000 versus WALP19-PEG2000 lateral diffusion in negatively magnetically-aligned DMPC/DMPG/DHPC, R=0.10, = 20 wt% mixtures: (A) Diffusion coefficients versus temperature and (B) Arrhenius plot of the natural logarithm of the diffusion coefficients versus reciprocal temperature. ........................................................................... 32

ix

Figure 3.5. Longitudinal and transverse relaxation times T1 and T2 of the DMPE-PEG2000 and KWALP19-PEG2000 1H resonance at 3.55 ppm versus temperature in negatively magnetically-aligned DMPC/DMPG/DHPC, R=0.10, = 20 wt% mixtures. .......................................................................... 34

Figure 3.6. 1H PFG STE NMR spectra at 30°C of (A) DMPE-PEG2000 and (B) WALP-PEG2000 incorporated in negatively magnetically-aligned DMPC/DMPG/CHAPSO, R=0.10, = 20 wt% mixtures at 1 mol% with respect to DMPC. Resonances shown are those associated with the PEG tag.. ... 35

Figure 3.7. 1H NMR spectra at 25°C of a 750 Da methoxy PEG (mPEG) sample in D2O. .................................................................................................................. 35

Figure 4.1. 31P NMR spectra of DMPC/DHPC and DMPC/CHAPSO mixtures, q = 3 and = 25 wt%, with differing DMPG content, at the indicated temperatures. . 44

Figure 4.2. 31P NMR residual chemical shift anisotropy of DMPC as a function of temperature in various negatively magnetically-aligned, q = 3, = 25 wt% bicellar mixtures ................................................................................................ 46

Figure 4.3. 1H PFG STE NMR spectra of negatively magnetically-aligned DMPC/DMPG/CHAPSO, R=0.10, = 25 wt% mixtures at 60°C as a function of increasing gradient pulse amplitude. ................................................ 48

Figure 4.4. Semi-logarithmic plots of the normalized intensity of the water resonance and PEG-1000 resonance from 1H PFG STE NMR spectra of negatively magnetically-aligned DMPC/DMPG/CHAPSO, R=0.10, = 25 wt% mixtures at 60°C as a function of the experimental factor, k = ( g )2·( – /3)/10-10 for different experimental diffusion times................................................................ 50

Figure 4.5. Arrhenius plot of the natural logarithm of the diffusion coefficients of water and PEG-1000 versus reciprocal temperature for various negatively magnetically-aligned q = 3, = 25 wt% bicellar mixtures. ............................... 52

Figure 4.6. Chemical structures of CHAPSO and DHPC. ............................................. 54

x

LIST OF EQUATIONS

Equation 1.1. Stokes-Einstein equation: = ....................................................... 9

Equation 1.2. Displacemement in two-dimensional diffusion: = 4 ..................... 9

Equation 1.3. Free-area model of lateral diffusion: = exp( )

............ 10

Equation 1.4. Hydrodynamic model Of lateral diffusion:

~ ln 0.5772 ...................................................................... 10

Equation 1.5. Stejskal-Tanner equation:

= exp exp exp ( ) ..................................... 13

Equation 3.1. Effective ratio of planar-to-curved phospholipids: = ................. 28

Equation 3.2. Flory equation: = ................................................................... 30

Equation 4.1. The diffusion coefficient relating to the diffusion tensor components:

= + ............................................................................... 49

Equation 4.2. Root-mean-square displacement: = (4 ) ............................ 51

xi

LIST OF SYMBOLS

1H proton 2H deuterium 31P phosphorus-31

A alanine

length of one monomer

molecular cross-sectional area

( ) free area at a given temperature

total lipid concentration

diffusion coefficient

Dzz diffusion coefficient along the z-direction

diffusion coefficient parallel to the bilayer normal

diffusion coefficient perpendicular to the bilayer normal

activation energy of diffusion

gradient pulse amplitude

membrane thickness

lipid hexagonal phase

NMR signal intensity

K lysine

Boltzmann’s constant

L leucine

degree of polymerization

q long-chain lipid to short-chain lipid molar ratio

q* effective ratio of planar-to-curved phospholipids in bicelle

mean square displacement

charged to neutral long-chain lipid molar ratio

xii

radius of the diffusant

Flory radius

hydrodynamic radius

absolute temperature in Kelvin

diffusion time

longitudinal relaxation time

transverse relaxation time

lipid phase transition temperature / melting temperature

W tryptophan

magnetogyric ratio

experimental diffusion time

gradient pulse duration

residual chemical shift anisotropy

observed DMPC chemical shift

isotropic chemical shift

viscosity

viscosity of the membrane

viscosity of the bathing medium

geometric correction factor

chemical shift anisotropy

NMR pulse sequence delay time

angle between the bilayer normal and the direction of the field gradients

chemical shift

xiii

LIST OF ABBREVIATIONS

CHAPSO 3-(cholamidopropyl)dimethyl-ammonio-2-hydroxyl-1-

propanesulfonate

D2O deuterium oxide

DHPC 1,2-dihexanoyl-sn-glycero-3-phosphocholine

DIEA diisopropylethylamine

DMF dimethylformamide

DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine

DMPE 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine

DMPG 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (sodium salt)

FCS Fluorescence Correlation Spectroscopy

Fmoc N-(9-fluorenyl)methoxycarbonyl

FRAP Fluorescence Recovery After Photobleaching

HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]

pyridinium 3-oxid hexafluorophosphate

HDO deuterated water

HFIP hexafluoroisopropanol

HPLC High-Performance Liquid Chromatography

MAS Magic Angle Spinning

mPEG methoxy polyethylene glycol / polyethylene glycol methyl ester

NHS N-hydroxylsuccinimide

NMR Nuclear Magnetic Resonance

PAL Peptide Amide Linker

PEG polyethylene glycol

PEG1000 polyethylene glycol, average molecular weight 1000 Da

PEG2000 polyethylene glycol, average molecular weight 2000 Da

PFG Pulsed-Field Gradient

PS polystyrene

rf radiofrequency

xiv

rms root-mean-square

SANS Small Angle Neutron Scattering

SE Spin Echo

SPT Single Particle Tracking

STE Stimulated Echo

TEM Transmission Electron Microscopy

TFA trifluoroacetic acid

TM transmembrane

UV ultraviolet

WALP19 AWWLALALALALALALWWA peptide

1

Chapter 1: Introduction

Diffusion is a fundamental physical parameter of biomolecules and plays an important

role in the dynamics of reactions and processes in living systems. In the cell membrane,

lateral diffusion of lipids and membrane proteins can be pivotal to their function,

including conformational changes and triggers of catalysis at cell surface and cascades of

events in the integral part of the cell.1–3 Studies of lateral diffusion in the cell membrane

can also provide information about membrane structure and interactions with other

membrane-associated biomolecules.4

The goal of this research is to study the diffusion of membrane proteins in a bicellar lipid

membrane. Bicelles are membrane mimetic systems which consist of a mixture of long-

chain lipids and short-chain amphiphiles, and have been commonly used for the study of

membrane-associated molecules.5–7 Bicelles can spontaneously align in a magnetic field

and form extended lamellae amenable for the study of diffusion via stimulated-echo

pulsed-field gradient (STE PFG) NMR.8,9

Among the first reported bicellar mixtures was that of the phospholipid 1,2-dimyristoyl-

sn-glycero-3-phosphocholine (DMPC) and the bile salt analogue 3-

(cholamidopropyl)dimethyl-ammonio-2-hydroxyl-1-propanesulfonate (CHAPSO) as the

detergent component,10 but most studies on bicelle characterization have focused on the

canonical bicellar mixture of DMPC/DHPC (1,2-dihexanoyl-sn-glycero-3-

phosphocholine) introduced by Sanders and Schwonek.11 However, CHAPSO has been

observed to improve aspects of DMPC bicelles when employed as a replacement of the

short-chain lipid of DHPC, including a wider range of temperature range of magnetic

alignment.12,13 A better understanding of the DMPC/CHAPSO bicellar system is

foundational for its utilization as a membrane mimetic for structural, dynamic, and

diffusion studies of membrane-associated biomolecules.

2

The results discussed in this thesis is divided into two major parts, the first being the

investigation of the feasibility of employing the STE PFG NMR technique to measure the

diffusion of a polyethylene glycol (PEG)-tagged alpha-helical transmembrane peptide

WALP19 incorporated in magnetically aligned DMPC/DHPC bicelles. The second part

of this report focuses on the NMR studies of DMPC/CHAPSO bicelles to compare their

behavior as a function of temperature with that of DMPC/DHPC bicelles.14

Portions of this thesis will be submitted for publication, and the copyright will be

transferred to the publisher at that time.

1.1. Lipid membranes and aggregates

Biological membranes are essential for living cells to be compartmentalized and

separated from the outside environment. However, the membrane is not simply a barrier

from foreign agents or a gateway to essential nutrients or signaling molecules; it is a site

where proteins and various biomolecular complexes reside, both integral and peripheral,

to perform their vital roles in diverse cellular processes.

Figure 1.1 shows a model of the cell membrane, primarily based on the fluid mosaic

model proposed by Singer and Nicholson,15 which depicts the membrane as a matrix of

lipids for randomly dispersed embedded proteins. The model is continually amended and

updated to reflect subsequent discoveries uncovering further complexities.16,17

Nevertheless, the cell membrane will be constantly understood as a dynamic structure,

both at its surface and within the bilayer.18 Movements of its various components include

lateral diffusion within the lipid matrix to provide association, dissociation, and structural

changes important for membrane function.

3

Figure 1.1. Updated model of the cell membrane adapted from the original fluid-mosaic model.15 Figure taken from Ref. 16.

The lipid bilayer is the basic structure of the cell membrane.15 It can exist in different

physical states, the most common being the gel phase and the fluid phase. Each lipid has

a characteristic phase transition temperature, Tm, also called the melting temperature,

where the phase change is induced, as depicted in Figure 1.2. The gel phase, also called

the solid-ordered phase, exists at lower temperatures and is characterized by a compact

lipid network where the lateral diffusion is greatly reduced. In the fluid phase, or liquid-

disordered phase, the lipid acyl chains become more mobile due to thermal kinetic

energy. Diffusion is thus more favored at this phase because of the increased fluidity of

the membrane.17,19

Figure 1.2. Phase transition of lipid bilayer from the gel phase, where diffusion is restricted, to the highly fluid liquid-disordered phase. Figure taken from Ref. 19.

4

Lipids are amphiphilic biomolecules – containing both hydrophilic and hydrophobic

domains – and a bilayer is only one of the possible structures that they form

spontaneously in aqueous solution. Table 1.1 lists the aggregate forms preferred by the

amphiphiles depending on their shape and molecular packing parameters.20 The

hydrophobic effect drives lipid self-assembly in a manner that minimizes contact

between the nonpolar lipid chains and aqueous solution and encourages the energetically

stable interactions between the polar headgroups and water or other polar domains. The

resulting structures form at conditions when the enthalpic cost of self-assembly is offset

by the entropic cost of the water molecules rearranging to form a cage around the

exposed hydrophobic domains.17

Table 1.1. Lipid types with different molecular shapes and their preferred aggregate structures. Adapted from Ref. 20.

Lipid Shape Structures formed Diagram

Single-chained lipids (detergents) with large head-group areas

Cone Spherical micelles

Single-chained lipids with small head-group areas Wedge

Globular or cylindrical micelles

Double-chained lipids with large head groups Single-chained lipids with very small (uncharged) head-groups

Truncated cone Flexible bilayers / vesicles

Double-chained lipids with small head-group areas

Cylinder Planar bilayers

Double-chained lipids with small head-group areas, unsaturated chains, high temperature

Inverted truncated cone

Inverted micelles

5

1.2. Membrane proteins

Membrane proteins are vital for the proper functioning of the cell and are thus targeted

by 60% of modern medicinal drugs.21 These proteins act as enzymes that catalyze diverse

processes; transporters and channels that allow movement of molecules across the lipid

barrier; and receptors that transmit information from the extracellular environment.18

Membrane proteins are generally classified as either extrinsic/peripheral or integral,

depending on how they interact with the lipid membrane. Peripheral membrane proteins

associate with the membrane surface through electrostatic, hydrophobic, and other non-

covalent interactions with lipid head groups or other membrane-bound proteins. On the

other hand, integral proteins are embedded in the membrane and are called

transmembrane (TM) proteins when they span the lipid bilayer.

Figure 1.3. Schematic of structures found in transmembrane proteins: (a) single-spanning and multi-spanning alpha-helices and (b) rolled-up beta sheets forming a beta-barrel. The grey area depicts the lipid bilayer. Adapted from Ref. 22.

Integral membrane proteins are coded by an estimated 20-30% of the genome.23 Figure

1.3 depicts the alpha-helical and beta-barrel structures adapted by integral proteins. The

distribution of the amino acids for transmembrane segments is non-random, as

hydrophobic residues isoleucine, leucine, valine, and alanine are preferentially found

embedded in the membrane. On the other hand, aromatic residues commonly occur at the

lipid-solution interface of the bilayer.24

(a)

(b)

6

Despite their importance, less than 2% of the structures found in the Protein Data Bank

are classified as transmembrane proteins. An explanation for the predominance of water-

soluble proteins in biochemical studies is the challenging nature of experiments involving

membrane proteins due to their extreme hydrophobicity. To remain in their functional

form during studies, membrane proteins must be kept in membrane mimetic

environments, which could introduce further complexities to the traditional biophysical

techniques.

1.3. Bicelles as model membrane systems

Bicelles, or bilayered micelles, are model membranes which consist of mixtures of long-

chain lipids and short-chain amphiphiles,10,25 typically DMPC and DHPC, respectively.

In separate aqueous solutions, DHPC forms micelles by itself while DMPC assembles by

itself into extended lamellar bilayers. DMPC/DHPC mixtures form a planar DMPC-rich

bilayer with a DHPC-rich coating at the edge regions that stabilize the otherwise exposed

hydrophobic chains of the long-chain lipids.9,26

The structures formed by the bicelles depend on the long-chain to short-chain lipid molar

ratio, q, the total lipid concentration, , and temperature.27 Figure 1.4 is a schematic of

the dependence of bicelle morphology on q. At low q, discoidal bicelles form with a

DMPC-rich bilayer and DHPC-rich rim. At higher q, a DMPC-rich extended lamellar

structure is formed with toroidal perforations coated by DHPC. 26

7

Figure 1.4. Bicelle morphology as a function of q, the molar ratio of long-chain lipids to short-chain lipids/detergents. Adapted from Ref. 26.

Bicelles tend to spontaneously align in a magnetic field due to the net magnetic

susceptibility anisotropy of the assembly of lipids.28,29 Magnetic alignment can be

assessed via 31P NMR spectroscopy, as shown in Figure 1.5 for DMPC/DHPC bicelles.

Below the Tm of the long-chain lipid, bicelles exhibit standard discoidal morphology and

remain unaligned.27 The corresponding 31P spectra show an isotropic peak associated

with DHPC overlaying the characteristic powder pattern of DMPC with a chemical shift

anisotropy of = . Above the Tm, negative magnetic alignment of the bicelles

occur, where the normal of the bilayer is perpendicular to the magnetic field. At high q,

bicelles also convert their morphology to the perforated lamellae form. The

corresponding 31P spectra show distinct narrow resonances for both the DMPC and the

DHPC, with the DMPC peak having a chemical shift near .9

q = 0 Micelle

q 1 Discoidal Bicelle

q < 3 Discoidal Bicelle

q > 3 Perforated Lamella

q Lamella

DMPC

DHPC

8

Figure 1.5. Magnetic alignment of DMPC/DHPC bicelles and corresponding 31P spectra. Figure taken from Ref. 9.

Bicelles have been widely used for as model membranes for structural studies of

proteins.5–7 For high-resolution solution state NMR, discoidal isotropic bicelles are more

appropriate than micelles in solubilizing membrane proteins. Bicelles approximate the

lipid membrane environment, as they may avoid the curvature and strain effects common

with micelles.6 Magnetically aligned bicelles, on the other hand, have been used to

measure residual dipolar couplings of soluble proteins by inducing partial alignment of

the protein in the interbicellar space.30,31 Aligned bicelles with perforated lamellar

morphology have been used to study diffusion of lipids using NMR, and have potential

for studies of membrane protein diffusion.8,9

9

1.4. Diffusion in cell membranes

Self-diffusion is a random-walk process induced by thermal motions of the diffusants in

solution. The self-diffusion coefficient, D, characterizes the radial distribution of the

molecules relative to their original position. In a homogenous system without thermal or

concentration gradients, the diffusion reflects a Gaussian distribution around the original

position of the diffusant. Typical self-diffusion coefficients are on the order of 10 -9 m2s-1

to 10 -12 m2s-1 for small molecules and large polymers, respectively, in nonviscous liquids

at room temperature.32

The diffusion coefficient of a sphere with hydrodynamic radius in an isotropic

medium of viscosity is given by the Stokes-Einstein equation:

= 6

where represents Boltzmann’s constant and T is the absolute temperature. For a two-

dimensional system, pertinent to lateral diffusion along a membrane, the diffusion

coefficient relates to the displacement of the diffusant in Equation 1.2:

= 4 (1.2)

where is the mean square displacement of a randomly diffusing particle and is the

diffusion time.33

1.4.1. Models of lateral diffusion in lipid membranes

The commonly used models for lateral diffusion in lipid membranes are the free area

model and the continuum hydrodynamic model.33–35 In the free area model, the diffusant

is comparable to the size of the solvent, i.e., the lipid molecules in the bilayer. The

diffusion coefficient is dependent on whether there is enough kinetic energy for the

diffusant to jump into a free area at a given temperature ( ( )) of comparable size to the

molecule ( ). This relation is described in Equation 1.3:

(1.1)

(1.2)

10

= exp ( )

Where is the ‘unhindered’ diffusion coefficient, is a geometric factor that corrects

for overlap of free-areas, and is the activation energy associated with diffusion. For

this size regime, the diffusion coefficient has an Arrhenius type temperature-dependence

in model lipid membranes.9,26,33 For a lipid bilayer, the activation energy of diffusion

also takes into account the interactions of a diffusant with its neighbors and the bounding

fluid in the bilayer.33

The continuum hydrodynamic model was proposed by Saffman and Delbrück 36 for

diffusants of cylindrical shape inserted vertically in thin viscous fluid sheets such as

membranes. The model is applicable to the diffusion of such particles having a size far

larger than that of the lipids, and is governed by Equation 1.4:

~ 4 ln 0.5772

Where m and w are the viscosities of the membrane and the bathing medium,

respectively, h is the membrane thickness, and is the radius of the membrane

diffusant, assuming a cylindrical form in total. The hydrodynamic model predicts a weak

size dependence of the diffusion coefficient to the size of the membrane diffusant.9,26,34

1.4.2. Fluorescence-based methods to study diffusion

Fluorescence-based methods are popularly used for diffusion measurements in lipid

membranes due to advantages with speed, resolution, and sensitivity. These techniques

include FRAP (fluorescence recovery after photobleaching)4,37,38, FCS (fluorescence

correlation spectroscopy) 39–41, and SPT (single particle tracking)42,43. The drawback to

these methods is the need for a photostable fluorescent moiety chemically attached to the

molecule to be monitored.

(1.3)

(1.4)

11

FRAP is based on flashing an intense light on a discrete region of the sample, thus

photobleaching the fluorescent molecules therein, and observing the recovery of

fluorescence from the inward diffusion of the neighboring unbleached fluorophores. The

diffusion coefficient is extracted from the analysis of the resulting fluorescent recovery

versus time. Also, the mobile fraction of the measured population of the probe molecules

is related to the degree to which the fluorescence was recovered, compared to its pre-

bleached state. To obtain accurate diffusion coefficients from FRAP, the membrane

geometry and the beam profile should be known and the membrane environment should

be uniform and free from intrinsic and photoinduced obstructions. 4,37,38

Translational diffusion can also be probed by fluorescence correlation spectroscopy

(FCS), which involves autocorrelation analysis of a time dependent fluctuation of signal

from the fluorescently tagged molecule of interest. Fluorescence intensity fluctuations on

a timescale of milliseconds to seconds are associated with the lateral diffusion of the

species through the detection volume. The autocorrelation curve reflects the self-

similarity of the signal as a function of time, and is fitted with a model to extract the

diffusion coefficient. FCS requires lower fluorophore concentrations and lower laser

power than FRAP, but needs long measurement times on a stable sample with precise

instrumentation for reliable quantitative results. 39–41

While FRAP and FCS observe the ensemble averages of diffusion within a sample, SPT

tracks the motion of the tracer molecule within the plane of the membrane. The probe is

usually a fluorescent dye or protein, but can also be latex beads or colloidal gold

nanoparticles observed through video microscopy. SPT can determine whether the

diffusion is normal or anomalous and can detect variations of behavior such as directed

and confined motion with the sample. However, being a single-particle technique, the

analysis can be biased by sampling effects. 42,43

12

1.4.3. Diffusion measurements from pulsed field gradient (PFG) NMR spectroscopy

Translational diffusion can also be probed in a non-invasive manner using the pulsed

field gradient technique in NMR spectroscopy.9,32,44,45 The most commonly used pulse

sequences are the spin echo (SE) 46 and the stimulated echo (STE) 47,48 versions. The

experiment applies controlled pulses of a magnetic field with a linear gradient to give a

transient spatial dependence to the nuclear spin resonance of interest.

In the PFG SE technique, a 90°-180° radiofrequency (rf) pulse sequence (Figure 1.6) is

employed to produce a Hahn echo. 49 The magnetic field is essentially homogeneous

except for the short pulses of time when the magnetic field gradient is turned on. The

nuclear spins are dispersed and refocused by the identical field gradient pulses such that

the effect is canceled if the spins have not diffused away from their original position.

However, if translational motion did occur, the attenuation of the intensity of the resultant

signal from the echo is proportional to the displacement of the spatially labelled signal

along the direction of field gradient.32,44,46

Figure 1.6. The basic Stejskal-Tanner pulsed field-gradient NMR pulse sequence producing a spin echo. Adapted from Ref. 44.

The PFG STE technique, on the other hand, exploits the advantages of having three 90°rf

pulses in the pulse sequence (Figure 1.7). The echo after the third pulse, termed by Hahn 50 as a stimulated echo, is not as limited by the intrinsic transverse relaxation time (T2) of

the nuclear spins, as compared to the spin echo. Rather, the relaxation attenuation of the

13

stimulated echo between the second and third rf pulses is dependent on the nuclear spin

longitudinal relaxation time (T1). The PFG STE experiment is thus more advantageous in

observing diffusion where T1 >> T2, such that the effect of relaxation on signal

attenuation is lessened.26,32,47 Systems with slower diffusion can then be probed, as longer

diffusion times can be attained in the experiment, being now limited by T1 instead of

T2.8,51

Figure 1.7. The stimulated echo pulsed field-gradient pulse sequence used for diffusion experiments in NMR. Figure from Ref. 9.

The intensity, I, of the signal from the resulting echo in the PFG STE experiment is

governed by the Stejskal-Tanner equation:

= exp2

exp exp ( )3

Where is the magnetogyric ratio, D is the diffusion coefficient along the direction of the

magnetic field gradient, and is the experimental diffusion time. Either the gradient

pulse amplitude, , or its duration, , is incremented for a series of diffusion experiments.

The diffusion coefficient can then be extracted in a straightforward manner by fitting the

resulting echo intensity decay to the above equation.9,26

(1.5)

14

1.5. References

(1) Petersen, N. O. Diffusion and Aggregation in Biological Membranes. Can. J.

Biochem. Cell Biol. 1984, 62, 1158–1166.

(2) Axelrod, D. Lateral Motion of Membrane Proteins and Biological Function. J.

Membr. Biol. 1983, 75, 1–10.

(3) Ramadurai, S.; Holt, A.; Krasnikov, V.; van den Bogaart, G.; Killian, J. A.;

Poolman, B. Lateral Diffusion of Membrane Proteins. J. Am. Chem. Soc. 2009,

131, 12650–12656.

(4) Jacobson, K.; Ishihara, A.; Inman, R. Lateral Diffusion of Proteins in Membranes.

Annu. Rev. Physiol. 1987, Vol. 49, 163–175.

(5) Sanders, C. R.; Prosser, R. S. Bicelles: a Model Membrane System for All

Seasons? Structure 1998, 6, 1227–1234.

(6) Prosser, R. S.; Evanics, F.; Kitevski, J. L.; Al-Abdul-Wahid, M. S. Current

Applications of Bicelles in NMR Studies of Membrane-Associated Amphiphiles

and Proteins. Biochemistry 2006, 45, 8453–8465.

(7) Dürr, U. H. N.; Gildenberg, M.; Ramamoorthy, A. The Magic of Bicelles Lights

Up Membrane Protein Structure. Chem. Rev. 2012, 112, 6054–6074.

(8) Soong, R.; Macdonald, P. M. Lateral Diffusion of PEG-Lipid in Magnetically

Aligned Bicelles Measured Using Stimulated Echo Pulsed Field Gradient 1H

NMR. Biophys. J. 2005, 88, 255–268.

(9) Macdonald, P. M.; Saleem, Q.; Lai, A.; Morales, H. H. NMR Methods for

Measuring Lateral Diffusion in Membranes. Chem. Phys. Lipids 2013, 166, 31–

44.

15

(10) Sanders, C. R.; Prestegard, J. H. Magnetically Orientable Phospholipid Bilayers

Containing Small Amounts of a Bile Salt Analogue, CHAPSO. Biophys. J. 1990,

58, 447–460.

(11) Sanders, C. R.; Schwonek, J. P. Characterization of Magnetically Orientable

Bilayers in Mixtures of Dihexanoylphosphatidylcholine and

Dimyristoylphosphatidylcholine by Solid-State NMR. Biochemistry 1992, 31,

8898–8905.

(12) Cavagnero, S.; Dyson, H. J.; Wright, P. E. Improved Low pH Bicelle System for

Orienting Macromolecules Over a Wide Temperature Range. J. Biomol. NMR

1999, 13, 387–391.

(13) Wang, H.; Eberstadt, M.; Olejniczak, E. T.; Meadows, R. P.; Fesik, S. W. A

Liquid Crystalline Medium for Measuring Residual Dipolar Couplings Over a

Wide Range of Temperatures. J. Biomol. NMR 1998, 12, 443–446.

(14) Li, M.; Morales, H. H.; Katsaras, J.; Ku erka, N.; Yang, Y.; Macdonald, P. M.;

Nieh, M.-P. Morphological Characterization of DMPC/CHAPSO Bicellar

Mixtures: A Combined SANS and NMR Study. Langmuir 2013, Accepted (DOI:

10.1021/la402799b)

(15) Singer, S. J.; Nicolson, G. L. The Fluid Mosaic Model of the Structure of Cell

Membranes. Science 1972, 175, 720–731.

(16) Engelman, D. M. Membranes Are More Mosaic Than Fluid. Nature 2005, 438,

578–580.

(17) Dowhan, W.; Bogdanov, M.; Mileykovskaya, E. Chapter 1 - Functional Roles of

Lipids in Membranes. In Biochemistry of Lipids, Lipoproteins and Membranes

(Fifth Edition); Dennis E. Vance; Jean E. Vance, Eds.; Elsevier: San Diego, 2008;

pp. 1–I.

(18) Luckey, M. Membrane Structural Biology : with Biochemical and Biophysical

Foundations; Cambridge University Press: New York, 2008.

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(19) Eeman, M.; Deleu, M. From biological membranes to biomimetic model

membranes. Biotechnol. Agron. Soc. Environ. 2010, 14, 719–736.

(20) Israelachvili, J. N.; Mar elja, S.; Horn, R. G. Physical Principles of Membrane

Organization. Q. Rev. Biophys. 1980, 13, 121–200.

(21) Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. How Many Drug Targets Are

There? Nat. Rev. Drug Discov. 2006, 5, 993–996.

(22) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Membrane

Proteins. In Molecular Biology of the Cell; Garland Science: New York, 2002.

(23) Wallin, E.; Heijne, G. V. Genome-wide Analysis of Integral Membrane Proteins

from Eubacterial, Archaean, and Eukaryotic Organisms. Protein Sci. 1998, 7,

1029–1038.

(24) Landolt-Marticorena, C.; Williams, K. A.; Deber, C. M.; Reithmeier, R. A. F.

Non-random Distribution of Amino Acids in the Transmembrane Segments of

Human Type I Single Span Membrane Proteins. J. Mol. Biol. 1993, 229, 602–608.

(25) Ram, P.; Prestegard, J. H. Magnetic Field Induced Ordering of Bile

Salt/phospholipid Micelles: New Media for NMR Structural Investigations.

Biochim. Biophys. Acta - Biomembr. 1988, 940, 289–294.

(26) Macdonald, P. M.; Soong, R. Diffusion NMR and Bicelle Morphology. Can. J.

Chem. 2011, 89, 1021–1035.

(27) Whiles, J. A.; Deems, R.; Vold, R. R.; Dennis, E. A. Bicelles in Structure–

Function Studies of Membrane-Associated Proteins. Bioorganic Chem. 2002, 30,

431–442.

(28) Boroske, E.; Helfrich, W. Magnetic Anisotropy of Egg Lecithin Membranes.

Biophys. J. 1978, 24, 863–868.

17

(29) Scholz, F.; Boroske, E.; Helfrich, W. Magnetic Anisotropy of Lecithin

Membranes. A New Anisotropy Susceptometer. Biophys. J. 1984, 45, 589–592.

(30) Bax, A.; Tjandra, N. High-resolution Heteronuclear NMR of Human Ubiquitin in

an Aqueous Liquid Crystalline Medium. J. Biomol. NMR 1997, 10, 289–292.

(31) Tjandra, N.; Bax, A. Direct Measurement of Distances and Angles in

Biomolecules by NMR in a Dilute Liquid Crystalline Medium. Science 1997, 278,

1111–1114.

(32) Stilbs, P. Fourier Transform Pulsed-gradient Spin-echo Studies of Molecular

Diffusion. Prog. Nucl. Magn. Reson. Spectrosc. 1987, 19, 1–45.

(33) Almeida, P. F. F.; Vaz, W. L. C. Chapter 6 Lateral Diffusion in Membranes. In

Structure and Dynamics of Membranes; Lipowsky, R.; Sackmann, E., Eds.;

Handbook of Biological Physics; North-Holland, 1995; Vol. Volume 1, pp. 305–

357.

(34) Vaz, W. L. C.; Goodsaid-Zalduondo, F.; Jacobson, K. Lateral Diffusion of Lipids

and Proteins in Bilayer Membranes. FEBS Lett. 1984, 174, 199–207.

(35) Vaz, W. L. C.; Clegg, R. M.; Hallmann, D. Translational Diffusion of Lipids in

Liquid Crystalline Phase Phosphatidylcholine Multibilayers. A Comparison of

Experiment with Theory. Biochemistry 1985, 24, 781–786.

(36) Saffman, P. G.; Delbrück, M. Brownian Motion in Biological Membranes. Proc.

Natl. Acad. Sci. 1975, 72, 3111 –3113.

(37) Mullineaux, C. W. FRAP Analysis of Photosynthetic Membranes. J. Exp. Bot.

2004, 55, 1207–1211.

(38) Rayan, G.; Guet, J.-E.; Taulier, N.; Pincet, F.; Urbach, W. Recent Applications of

Fluorescence Recovery after Photobleaching (FRAP) to Membrane Bio-

Macromolecules. Sensors 2010, 10, 5927–5948.

18

(39) Chiantia, S.; Ries, J.; Schwille, P. Fluorescence Correlation Spectroscopy in

Membrane Structure Elucidation. Biochim. Biophys. Acta BBA - Biomembr. 2009,

1788, 225–233.

(40) Machán, R.; Hof, M. Lipid Diffusion in Planar Membranes Investigated by

Fluorescence Correlation Spectroscopy. Biochim. Biophys. Acta BBA - Biomembr.

2010, 1798, 1377–1391.

(41) Machá , R.; Hof, M. Recent Developments in Fluorescence Correlation

Spectroscopy for Diffusion Measurements in Planar Lipid Membranes. Int. J.

Mol. Sci. 2010, 11, 427–457.

(42) Saxton, M. J.; Jacobson, K. SINGLE-PARTICLE TRACKING:Applications to

Membrane Dynamics. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 373–399.

(43) Alcor, D.; Gouzer, G.; Triller, A. Single-particle Tracking Methods for the Study

of Membrane Receptors Dynamics. Eur. J. Neurosci. 2009, 30, 987–997.

(44) Price, W. S. Pulsed-field Gradient Nuclear Magnetic Resonance as a Tool for

Studying Translational Diffusion: Part 1. Basic Theory. Concepts Magn. Reson.

1997, 9, 299–335.

(45) Price, W. S. Pulsed-field Gradient Nuclear Magnetic Resonance as a Tool for

Studying Translational Diffusion: Part II. Experimental Aspects. Concepts Magn.

Reson. 1998, 10, 197–237.

(46) Stejskal, E. O.; Tanner, J. E. Spin Diffusion Measurements: Spin Echoes in the

Presence of a Time-Dependent Field Gradient. J. Chem. Phys. 1965, 42, 288–292.

(47) Tanner, J. E. Use of the Stimulated Echo in NMR Diffusion Studies. J. Chem.

Phys. 1970, 52, 2523.

(48) Tanner, J. E. Erratum: Use of the Stimulated Echo in NMR Diffusion Studies. J.

Chem. Phys. 1972, 57, 3586.

19

(49) Carr, H. Y.; Purcell, E. M. Effects of Diffusion on Free Precession in Nuclear

Magnetic Resonance Experiments. Phys. Rev. 1954, 94, 630.

(50) Hahn, E. L. Spin Echoes. Phys. Rev. 1950, 80, 580–594.

(51) Soong, R.; Macdonald, P. M. PEG Molecular Weight and Lateral Diffusion of

PEG-ylated Lipids in Magnetically Aligned Bicelles. Biochim. Biophys. Acta BBA

- Biomembr. 2007, 1768, 1805–1814.

20

Chapter 2: Experimental

2.1. Materials

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-dihexanoyl-sn-glycero-3-

phosphocholine (DHPC); 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (sodium salt)

(DMPG); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene

glycol)-2000] (DMPE-PEG2000); and 3-[(3-cholamidopropyl)dimethylammonio]-2-

hydroxy-1-propanesulfonate (CHAPSO) were purchased from Avanti Polar Lipids Inc.

(Alabaster, AL). Hexafluoroisopropanol (HFIP), deuterium oxide (D2O), and deuterium-

depleted water were purchased from Sigma-Aldrich (St. Louis, MO). Deuterium oxide

(D2O) was also obtained from Cambridge Isotope Laboratories (Andover, MA). All

solvents used in peptide synthesis and purification were purchased from Caledon

Laboratory Chemicals (Georgetown, ON) in either peptide or HPLC grade. Low load

polyamine linker-polyethylene glycol-polystyrene resin was purchased from Applied

Biosystems (Foster City, CA). Fmoc amino acids (>98%), and HATU (>99%) was

purchased from GL Biochem Ltd. (Shanghai, China). All chemicals were used without

further purification. All water used is of Milli-Q quality.

2.2. Peptide synthesis

The transmembrane peptide WALP19 (AWWLALALALALALALWWA), lysinated at

the C-terminus and labeled at the N-terminus with polyethylene glycol, average

molecular weight 2000 Da, (PEG2000) was produced by Dr. David Tulumello from the

research laboratory of Prof. Charles Deber at the Hospital for Sick Children (Toronto,

ON) using standard solid state synthesis methods. Briefly, the protocol is as follows:

Peptide synthesis was performed using standard Fmoc (N-(9-fluorenyl)

methoxycarbonyl) chemistry on a PS3 peptide synthesizer (Protein technologies, Inc.)

21

using the manufacturer’s protocol. Low load PAL-PEG-PS (4 -aminomethyl-3 ,5 -

dimethoxyphenoxyvaleric acid-poly(ethylene glycol) polystyrene) resin (Applied

Biosystems) was used which produces an amidated C-terminus upon cleavage.

Peptides were PEGylated at the N-termini by incubating the resin-bound peptides with

excess N-hydroxylsuccinimide functionalized polyethylene glycol (NHS-PEG2000) label

under basic conditions. 2 g of resin containing WALP19 was weighed out and afterwards

swelled with 10 mL of dimethylformamide (DMF) for 10 min. NHS-PEG2000 (0.375 g)

was dissolved in 10mL of DMF, with 200 µL of diisopropylethylamine (DIEA). The

resin was combined with label, covered with nitrogen gas, and continuously shaken

overnight. The resin was rinsed and then dried. The same labeling step was repeated with

additional resin using 0.2 g of NHS-PEG2000.

Peptides were cleaved from the resin with 2 hr incubation with 88% trifluoroacetic acid

(TFA), 5% phenol, 5% ultrapure water, and 2% tri-isopropylsilane. Cleaved peptides

were precipitated in cold ether overnight, dried, and redissolved in 50% acetonitrile, 50%

deionized water, 0.1% TFA. Peptides were purified by reverse-phase high pressure liquid

chromatography on a C4 preparative column (Phenomenex), using an acetonitrile/water

gradient (with 0.1% TFA). Mass spectrometry was used to identify the molecular weight

of the purified peptides. The concentration of an initial peptide aliquot was determined

using quantitative amino acid analysis performed by the Advanced Protein Technology

Centre of the Hospital for Sick Children (Toronto, ON). Using the concentration

determined by amino acid analysis, the molar extinction coefficient at 280 nm was

determined to be 23050 M 1 cm 1 in 50% acetonitrile, 50% water, 0.1% TFA. The

concentrations of further peptide purifications were determined by UV absorbance at 280

nm using this molar extinction coefficient. Peptides were lyophilized following

purification and stored in aliquots at –20 °C.

22

2.3. Sample preparation

2.3.1. DMPC/DMPG/DHPC bicelles with transmembrane peptide

Desired amounts of the lyophilized peptide was resolubilized in HFIP, vortexed, and

sonicated for 10 minutes. Solvent was evaporated over nitrogen and the process is

repeated two more times to ensure de-aggregation of the peptide. DHPC micelles were

made in a 50 mM citrate buffer solution, pH 7.4, 99% D2O and used to hydrate the

peptide film by cycling twice on 10 minutes sonication and 3 minutes mixing by vortex.

DMPG and DMPC powders were dispersed in the resulting solution to achieve a total

lipid concentration, , of 20 wt% with a lipid molar ratio q = (DMPC+DMPG)/DHPC

3. The mixture was subjected to at least 5 cycles of heating to 40°C and cooling to 0°C in

an ice bath, spending at least 10 minutes at each temperature. A completely clear and

transparent solution indicated that bicelles were successfully formed. The peptide content

of the bicelle sample was kept to a maximum of 1 mol% with respect to DMPC.

2.3.2. DMPC/DMPG/CHAPSO bicelle samples with free PEG

Mixtures of DMPC/DMPG/CHAPSO were prepared having a constant molar ratio q =

(DMPC+DMPG)/CHAPSO 3, with DMPG present at molar ratios of R =

DMPG/DMPC = 0, 0.01, and 0.10. Also, a mixture of DMPC/DHPC having a molar ratio

q = DMPC/DHPC 3 was prepared, i.e., lacking DMPG. Lipid powders of the desired

composition were dispersed in D2O with 1 mol% H2O to achieve a total lipid

concentration, , of 25 wt%. The lipid/water mixtures were subjected to temperature

cycling between low ( 4°C) and high ( 40-50°C) temperatures in combination with

vortex mixing until the samples became completely transparent, indicating the formation

of bicelles.

Polyethylene glycol, average molecular weight 1000 Da, (PEG-1000), was incorporated

into the non-deuterated bicelle mixtures as a diffusion probe. PEG-1000 was added from

an aqueous stock solution to a final concentration of 0.8 mol% with respect to DMPC.

The addition was performed subsequent to bicelle formation and at a temperature where

23

the bicelle sample was least viscous, i.e., 4°C for DMPC/DHPC and 10-15°C for the

CHAPSO bicelles. At this concentration and this size, the added PEG-1000 exerted no

discernible perturbation of the bicelle properties, including propensity for magnetic

alignment.

2.4. NMR spectroscopy

All NMR spectra were recorded on a Varian Infinity 500 MHz NMR spectrometer using

a Varian 5 mm double resonance liquid probe equipped with gradient coils along the z-

direction. The sample temperature was controlled to ±0.5 K of the desired value, as

calibrated separately using ethylene glycol. 1

The bicelle samples were transferred to an NMR tube between 5-15°C and placed in the

bore of the NMR spectrometer. To encourage magnetic alignment, the bicelles were

annealed by repeatedly cycling the temperature between 25-35°C. The quality of

alignment was assessed via 31P NMR spectroscopy.

31P NMR spectra were recorded at 202.31 MHz using single pulse excitation, quadrature

detection, complete phase cycling of the pulses, and WALTZ proton decoupling during

the signal acquisition with a proton decoupler field strength of 4 kHz. Typical acquisition

parameters are as follows: a 90° pulse length of 16 s, a recycle delay of 1 s, a spectral

width of 100 kHz, and a 32k data size. Spectra were processed with zero filling and an

exponential multiplication equivalent to 100 Hz line broadening prior to Fourier

transformation and were referenced to the isotropic peak of the bicelles, which is set to 0

ppm at temperatures between 10-15°C.

1H NMR diffusion measurements were performed at 499.78 MHz using the stimulated

echo (STE) pulsed field gradient (PFG) sequence, 2,3 with square gradient pulses directed

along the longitudinal (z) axis and having a constant duration (5 ms) with variable

gradient pulse amplitude. Phase cycling of the radio frequency pulses were employed to

remove unwanted echoes.4 Typical acquisition parameters are as follows: a 90° pulse

length of 25 s, a spin echo delay of 5 ms, a stimulated echo delay between 1000 and 100

24

ms, a recycle delay of 1 s, a spectral width of 10 kHz and a 32K data size. Spectra were

processed with zero filling and an exponential multiplication equivalent to 5 Hz line

broadening prior to Fourier transformation. Gradient strength (typically 300 G cm-1) was

calibrated from the known diffusion coefficient of HDO at 25°C. 5

2.5. References

(1) Ammann, C.; Meier, P.; Merbach, A. A Simple Multinuclear NMR Thermometer.

J. Magn. Reson. 1969 1982, 46, 319–321.

(2) Tanner, J. E. Use of the Stimulated Echo in NMR Diffusion Studies. J. Chem.

Phys. 1970, 52, 2523.

(3) Tanner, J. E. Erratum: Use of the Stimulated Echo in NMR Diffusion Studies. J.

Chem. Phys. 1972, 57, 3586.

(4) Fauth, J.-M.; Schweiger, A.; Braunschweiler, L.; Forrer, J.; Ernst, R. R.

Elimination of Unwanted Echoes and Reduction of Dead Time in Three-Pulse

Electron Spin-Echo Spectroscopy. J. Magn. Reson. 1969 1986, 66, 74–85.

(5) Mills, R. Self-Diffusion in Normal and Heavy Water in the Range 1-45°. J. Phys.

Chem. 1973, 77, 685–688.

25

Chapter 3: Lateral Diffusion of a Transmembrane -Helical

Peptide in DMPC/DHPC Bicelles

Diffusion is a fundamental physical parameter of biomolecules. Reactions and

interactions of biological importance are affected by the lateral diffusion of cell

membrane components that take part in the process.1 Stimulated-echo pulsed-field

gradient (STE PFG) NMR has been used for lateral diffusion studies of lipids in

magnetically aligned bicellar mixtures.2,3 The lateral diffusion of the model alpha-helical

transmembrane peptide WALP in magnetically aligned DMPC/DMPG/DHPC bicelles

(Schematic shown in Figure 3.1.) was measured using STE PFG NMR in order to explore

the feasibility of this technique to study the diffusion of various membrane proteins in

their environment.

Modified WALP19 peptide sequence: PEG2000 – AWWLALALALALALALWWA – KKKK

Figure 3.1. Schematic of the transmembrane alpha-helical peptide WALP19 with a 2000Da PEG tag at the N-terminus inserted in bicelles spontaneously aligned in a magnetic field. The bicelles align with the bilayer normal perpendicular to the magnetic field.

The WALP family of peptides has been developed by Killian and coworkers 4 and has

been used since then as a model for alpha-helical intrinsic membrane proteins.5–7 WALP

has alternating leucines and alanines – helix-forming amino acids – with variable length

for its hydrophobic region and is flanked with tryptophan that anchors the peptide termini

at the membrane surface near the lipid carbonyl region.6,8 WALP19 was used in this

26

study as DMPC lipids have been shown to keep its bilayer phase with the 19-residue

peptide incorporated in the membrane.4

The WALP19 peptide was further functionalized with lysines at the C-terminal to make

the extremely hydrophobic peptide more manageable during synthesis and subsequent

handling. Such lysine residue clusters give a high local positive charge, which increases

the water solubility of the peptide and prevents aggregation.9 WALP19 was also labeled

with a 2000-Da polyethylene glycol (PEG) tag at its N-terminus. In addition to being

effective in imparting aqueous solubility to hydrophobic peptides,10,11 essentially all the

protons of PEG are magnetically equivalent, giving a sharp resonance at 3.55 ppm, thus

providing a readily-visible NMR signal.

3.1. 31P NMR and magnetic alignment of bicelles with WALP19-PEG2000

Negatively aligned bicelles have the bilayer normal perpendicular to the static magnetic

field. The bicelles have to be properly aligned in order for the lateral diffusion coefficient

of the PEGylated peptide in the bicelle to be measured directly using PFG NMR. As the

gradients in the spectrometer are directed along the z-axis parallel to the magnetic field,

the measured diffusion coefficient for this case is then precisely equal to the lateral

diffusion along the bilayer.12

31P NMR spectroscopy is well-suited to measure the quality and direction of bicellar

magnetic alignment.13 As seen in Figure 3.2, the sharp, well-resolved resonances are

indicative of bicelles with negative magnetic alignment over the temperature range being

studied. The most intense resonance at the largest absolute chemical shift is DMPC,

while the resonance at the smallest absolute chemical shift is DHPC. The least intense

resonance in the middle is from DMPG, with a ratio of 1:10 with DMPC.

27

Figure 3.2. 31P NMR spectra acquired at different temperatures of aligned bicelle samples with 20 wt% lipid, composed of q = (DMPC+DMPG)/DHPC = 3 with R = DMPG/DMPC = 0.1 and 1 mol% WALP-PEG2000 with respect to DMPC. Chemical shifts are listed in Table 4.1.

Alpha-helical peptides are known to have positive magnetic anisotropy,14 and can thus

counteract the negative magnetic anisotropy of the bicellar lipid assembly. 31P NMR was

used to monitor the bicelle alignment and ensure its stability before and after diffusion

measurements were done. Another possible issue is excessive hydrophobic mismatch

between the lipid bilayer and the transmembrane peptide, which can induce formation of

non-bilayer lipid structures, such as the isotropic phase and the HII hexagonal phase.4

28

However, 31P NMR, being sensitive to lipid polymorphism,15 did not give any indication

of either of these phases being present in the sample.

The chemical shifts for the 31P resonances of the bicellar lipids are shown in table 3.1.

The frequency of the DHPC resonance shifts progressively toward the long chain lipids

DMPC and DMPG. This is interpreted by Triba et al.16 in the “mixed micelle model”

wherein DHPC migrates progressively from the edge region of the bicelle to the DMPC-

rich planar region because of the increased miscibility of DHPC with the DMPC with

increasing temperature. The effective ratio of planar-to-curved phospholipids, q*, can

then be calculated using Equation 3.1:

q =q +1

Where q = ([DMPC]+[DMPG])/[DHPC] and = / and and

are the observed chemical shifts of DHPC and DMPC, respectively. The effect of

q* increasing with temperature has been observed in both negatively aligned and

positively aligned bicelles with lamellar morphology.17 Therefore, the presence of the

WALP19-PEG2000 in the bilayer does not prevent the lipids from behaving as expected

in the mixed bicelle model.

Table 3.1. The 31P chemical shifts, , of the bicellar lipid peaks and the effective ratios of planar-to-curved phospholipid populations, q*, with the peptide WALP19-PEG2000 incorporated at 1 mole% with respect to DMPC.

Temperature (ppm)

(ppm)

(ppm)

= q*

28.0 -11.9 -8.5 -3.6 0.31 4.8 30.0 -12.0 -8.6 -4.3 0.36 5.2 35.0 -12.3 -8.9 -5.6 0.46 6.4 40.0 -12.6 -9.2 -6.9 0.55 7.9

The control sample for this experiment are similar bicelles but with the PEGylated lipid

DMPE-PEG2000 incorporated in the bilayer, which has been successfully used in lateral

diffusion studies of lipids in bicelles using the PGF NMR method.2,17 Table 3.2 shows

(3.1)

29

that the chemical shifts of the bicellar lipids and the effective ratio of planar-to-curved

lipids, q*, has the same trend with temperature as the sample with the PEGylated peptide,

with the values not being very far from each other. Thus, low concentrations of the

transmembrane peptide WALP19-PEG2000 inserted in the bicelle do not compromise the

magnetic alignment of the system. The negatively aligned bicelle behaves more or less

the same as when either DMPE-PEG2000 or WALP19-PEG2000 is incorporated within.

Table 3.2. The 31P chemical shifts, , of the bicellar lipid peaks and the effective ratios of planar-to-curved phospholipid populations, q*, with the lipid DMPE-PEG2000 incorporated at 1 mole% with respect to DMPC.

Temperature (ppm)

(ppm)

(ppm)

= q*

28.0 -10.9 -7.4 -2.6 0.24 4.3 30.0 -11.0 -7.6 -3.2 0.29 4.6 35.0 -11.5 -8.0 -4.5 0.39 5.6 40.0 -11.9 -8.5 -5.8 0.49 6.8

3.2. Hydrophobically-anchored PEG diffusion in interbicellar spaces

A PEG group attached covalently to molecule hydrophobically anchored in a negatively

aligned bicelle has a relatively narrow 1H NMR resonance peak compared to the lipid

resonances which are all broadened out due to significant residual homonuclear dipolar

interactions.2 The diffusion of PEG, as limited by its WALP19 anchor spanning the

viscous lipid membrane, can then be monitored using the 1H STE PFG NMR technique

to report the diffusion coefficient of the transmembrane peptide. This measurement is

valid when interlamellar and intralamellar PEG-PEG interactions do not interfere with

the peptide diffusion along the bilayer.

To eliminate the possibility of interlamellar PEG-PEG interactions, the size of the

PEG2000 relative to the available space has to be considered. A hydrophobically

anchored PEG group is modeled to take a “mushroom” conformation.18 The distance that

it extends from the surface is the radius, , which is defined in the Flory equation19 as

seen in Equation 3.2:

30

=

Where N is the degree of polymerization, i.e., the number of monomers, and a is the

length of one monomer.

PEG has a monomer length of 3.5 Å,20 and so both WALP19-PEG2000 and DMPE-

PEG2000, with 45 ethylene oxide units, would have a Flory radius of 35 Å. The

interlamellar spacing of DMPC/DHPC aligned bicelles with 20 wt% lipid has been

measured to be at least ~140 Å with maximum bilayer thickness of ~37 Å.21 Since the

Flory radius of PEG2000 is much less than the interbicellar space, it is unlikely that the

PEG chains from the different lamellae sheets will overlap and cause inhibited

diffusion.2,17

On the other hand, intralamellar PEG-PEG interactions are likely to occur when the area

covered by the half-sphere “mushroom” PEG at = is comparable to the bilayer

surface area. This is the critical “overlap” concentration, which would be at 1.6 mole%

for DMPE-PEG2000 in a DMPC bilayer, assuming DMPC occupies a footprint of 60Å.22

PEG diffusion is limited by the diffusion of the hydrophobic anchor when the

concentration of the PEGylated molecule is below the overlap concentration of the PEG

tag with respect to the bilayer lipids.2 The concentration used for either DMPE-PEG2000

or WALP19-PEG2000 are both only at 1 mole% with respect to DMPC in the bicelle

samples. Thus, their measured lateral diffusion should not be slowed down due to overlap

and entanglement from intralamellar PEG-PEG interactions.2,17

As a control, the diffusion of the lipid DMPE-PEG2000 was measured in magnetically

aligned bicelles with similar composition to be used for the PEGylated peptide. The

diffusion decays were single exponential (results not shown), indicative of diffusion with

normal Gaussian distribution around the original position of the diffusant. The diffusion

coefficients replicate published data acquired from DMPC/DHPC bicelles with q = 3

using STE PFG NMR.23

The diffusion decays of WALP19-PEG2000, as shown in Figure 3.3, are also single-

exponential, exhibiting normal Gaussian diffusion behaviour. Earlier trials that were

(3.2)

31

unsuccessful in incorporating all of the peptide in the bicelle resulted in double-

exponential diffusion decays of the WALP19-PEG2000, wherein the fast component had

a diffusion coefficient similar to a free PEG confined in interlamellar space. The single-

exponential diffusion decays are then indicative of the absence of any detectable free-

floating peptide not inserted in the membrane.

Figure 3.3. Semi-logarithmic diffusion decay plots of the normalized intensity of the WALP19-PEG2000 resonance from 1H PFG STE NMR spectra of negatively magnetically-aligned DMPC/DMPG/DHPC, R=0.10, = 20 wt% mixtures as a function of the experimental factor, k = ( g )2·( – /3)/10-10 for different temperatures.

The calculated diffusion coefficients of WALP19-PEG2000 with respect to temperature

are graphed in Figure 3.4A. The value at 28°C is within the range of the diffusion

coefficient reported for WALP in liposomes measured at room temperature using FCS.24

0.1

1

0 10 20 30 40 50

PEG

1 H S

igna

l I/I

0

( g )2 ( – /3) 10-10

28°C

30°C

35°C

40°C

32

As expected, faster diffusion occurs with increasing temperature, since the diffusants

acquire greater thermal motion with the influx of kinetic energy. This trend is also

present in the DMPE-PEG2000, as seen in the same figure, only that the peptide

diffusion is 35% - 45% slower relative to that of the lipid in similar bicelles.

Figure 3.4. Temperature dependence of DMPE-PEG2000 versus WALP19-PEG2000 lateral diffusion in negatively magnetically-aligned DMPC/DMPG/DHPC, R=0.10, = 20 wt% mixtures: (A) Diffusion coefficients versus temperature showing WALP19-PEG2000 lateral diffusion is slower than DMPE-PEG2000 by a factor of approximately 2; (B) Arrhenius plot of the natural logarithm of the diffusion coefficients versus reciprocal temperature.

Figure 3.4B shows the Arrhenius-type behaviour of the temperature dependence of the

diffusion coefficients for the lipid and the alpha-helical peptide. This indicates that both

species are in conformity with the predictions of the free-area model for lateral diffusion

in this size range.3,12,25 The calculated activation energies of diffusion, , are 75 kJ/mol

and 58 kJ/mol for WALP19-PEG2000 and DMPE-PEG2000, respectively.

For the free-area model, the interactions of a diffusant with its neighbors and the

bounding fluid in the bilayer are accounted for in the calculated activation energy of

diffusion.25 The WALP19 peptide spans the whole bilayer, whereas DMPE only goes

0

2E-12

4E-12

6E-12

8E-12

1E-11

1.2E-11

1.4E-11

1.6E-11

25 35 45

Diffu

sion

Coe

ffici

ent [

m2/

s]

Temperature [ C]

DMPE-PEG-2k

-27.5

-27

-26.5

-26

-25.5

-25

-24.5

3.15E-03 3.25E-03 3.35E-03

ln (P

EG D

iffus

ion)

1/Temperature (K)

WALP19-PEG-2k

A B

33

through halfway. Liu, et al.26 have put forth a “triple layer” model of a lipid membrane,

where the regions closest to the aqueous interface are highly viscous and more restrictive

to diffusion compared to the more fluid middle region inhabited by the hydrophobic lipid

acyl chains. The measured diffusion coefficients of WALP19-PEG2000 and DMPE-

PEG2000 confirm the prediction that a molecule spanning the bilayer will have reduced

diffusion by a factor of two relative to another molecule of similar radii residing only in

one monolayer.26 The presence of WALP19 in both high viscosity regions of the

membrane is also a contributing factor to why the transmembrane peptide needs greater

energy to diffuse along the lipid bilayer.

WALP19 also has tryptophan residues that anchor on both membrane surfaces, and thus

would also need more energy to overcome interactions with the lipid head group

compared to interactions between the similar headgroups of DMPE and DMPC. Further

experiments can be done to test the energy requirement of the interfacial interactions of

the peptide with the lipid headgroups by changing the flanking residues of the peptide 6,8

and observing the diffusion behavior with respect to temperature.

3.3. Relaxation issues

The longitudinal relaxation time (T1) and transverse relaxation time (T2) of the ethylene

oxide protons for both DMPE-PEG2000 and WALP-PEG2000 are graphed in Figure 3.5,

as measured using the PFG STE pulse sequence. Keeping all the variables of the pulse

sequence constant, the delay time between the first and second or the second and third

pulse is varied and the corresponding intensity decay is fitted according to the Stejskal-

Tanner equation.27

As seen in Figure 3.5, the PEG tag always has a longer T1 than T2 for the temperature

range of where the DMPC/DMPG/DHPC bicelles remains aligned, so the investigated

diffusion times can be lengthened with much signal attenuation due to relaxation.

However, when diffusion is very slow, as per the PEGylated peptide, diffusion times

longer than T1 have to be set in order to have a well-defined decay where the diffusion

34

coefficient is quantifiable. Diffusion times as long as 1000 ms considerably reduced the

signal, so the number of scans have to be increased significantly, making the experiment

a lengthy one. A gradient driver that can deliver stronger gradients could circumvent this

problem, since the signal intensity is proportional to the square of the gradient pulse

amplitude.

Figure 3.5. Longitudinal and transverse relaxation times T1 and T2 of the DMPE-PEG2000 and WALP19-PEG2000 1H resonance at 3.55 ppm versus temperature in negatively magnetically-aligned DMPC/DMPG/DHPC, R=0.10, = 20 wt% mixtures.

Homogenous broadening of resonance linewidths is related to the transverse relaxation

time T2.28 Protons undergoing fast isotropic motion exhibit sharp narrow resonances and

have long T2 relaxation times. The PEG group retains some rapid internal motion even

while anchored to the surface of a magnetically aligned bicelle. However, more

relaxation mechanisms are apparently at play, giving rise to some line broadening. This

effect is more pronounced when the PEGylated molecule incorporated in the membrane

undergoes slower diffusion.

35

Figure 3.5B shows that the T2 of the PEG protons attached to the anchored WALP19 has

decreased to less than a third of those from DMPE-PEG2000. Ideally, the delay between

the first and second 90° pulses should be much less than the T2 so that signal will not be

significantly attenuated due to transverse relaxation effects. However, instrumental and

hardware considerations, which include being limited by the power of the gradient driver,

dictate that the delay should be a minimum of 6 ms. For the WALP-PEG2000, this

translates to significant broadening of resonances and loss of signal compared to the

DMPE-PEG2000, as can be seen in the spectra at Figure 3.6.

Figure 3.6. 1H PFG STE NMR spectra at 30°C of (A) DMPE-PEG2000 and (B) WALP19-PEG2000 incorporated in magnetically-aligned DMPC/DMPG/DHPC, R=0.10, = 20 wt% mixtures at 1 mol% with respect to DMPC. Resonances shown are those associated with the PEG tag. Spectra are acquired with a gradient strength of 0.94 T/m and a diffusion time of 1000 ms and are processed with 5 Hz line broadening.

Figure 3.6 shows diffusion filtered 1H spectra of DMPE-PEG2000 and WALP-PEG2000

in negatively magnetically-aligned bicelles above the transition temperature of DMPC.

The diffusion filter is necessary to eliminate the lipid 1H resonances that overlap and

obscure the PEG signal. Figure 3.6A shows DMPE-PEG2000 having a sharp peak at 3.55

ppm with 40 Hz linewidth, with a shoulder at 3.45 ppm and a triplet at 3.2 ppm. At the

1H NMR chemical shift (ppm) 1H NMR chemical shift (ppm)

A B

36

same conditions, WALP-PEG2000 at Figure 3.6B shows a broader 3.55 ppm peak with a

125 Hz linewidth, while the shoulder at 3.45 ppm became a more distinct peak. The

triplet at 3.22 ppm, however, is now the prominent feature.

___

Figure 3.7. 1H NMR spectra at 25°C of a 750 Da methoxy PEG (mPEG) sample in D2O. The bulk of the ethylene oxide groups contribute to the resonance at 3.60 ppm while the multiplet at 3.53 ppm are from those groups near the ends of the polymer chain. The methoxy caps give the resonance at 3.28 ppm.

The PEG tag used for both DMPE and WALP19 are mono-functionalized with a

methoxy group at the end of the polymer chain. As seen in Figure 3.7, the protons of the

terminal methyl give a sharp singlet at 3.28 ppm. This resonance correlates with the

triplet at 3.22 ppm exhibited by the anchored PEG in the aligned bicelles. The splitting of

the signal into a triplet is possibly due to residual dipolar coupling that emerges when the

PEG group has limited mobility, being anchored to the bilayer surface.

The distinctive resonance for free PEG in aqueous solution is a sharp singlet at 3.60 ppm

from the ethylene oxide protons, with a diminutive multiplet at 3.54 ppm attributed to the

37

ethylene oxide groups at the PEG termini. These peaks correlate to the 3.55 ppm peak

and 3.45 ppm shoulder, respectively, of the PEGylated DMPE and WALP19

incorporated in negatively magnetically-aligned DMPC/DMPG/DHPC bicelles. The

broadening and loss of signal from the ethylene oxide protons of the anchored PEG,

especially from the resonance at 3.60 ppm, is a concerning observation, since it limits the

efficacy of the diffusion measurement using PFG STE.

The changing profile of the spectra could not be attributed to the equilibrium of PEG

“mushroom” and “brush” conformations, since the PEG used in the sample is only at 1

mole% with respect to DMPC, which is much less than the critical “overlap”

concentration where the transformation takes place.2,17 However, one can speculate that

the ethylene oxide groups in the anchored PEG in the bicelle are experiencing a chemical

shift anisotropy which contributes to the T2 relaxation rate.28 The effect seems to become

more pronounced when the PEG anchor is slowly diffusing, as with the case for

WALP19-PEG2000. The slow lateral movement in addition to the proximity to the high-

viscosity region in of the triple-layer membrane could also make the PEG group more

susceptible to dipolar interactions with the surface of the bilayer, which is another

relaxation mechanism.28 For future experiments, the magic angle spinning (MAS)

technique can be employed to eliminate the unwanted broadening mechanisms for more

effective monitoring of the diffusing species through pulsed field gradients.29–31

3.4. Conclusions

This study has demonstrated the use of PFG NMR in measuring the lateral diffusion of a

peptide in negatively aligned bicellar model membranes. DMPC/DHPC bicelles retain

negative alignment between 28°C to 40°C with a small concentration of the alpha-helical

transmembrane peptide WALP19-PEG2000 incorporated with the membrane. Within this

temperature range, the PEG tag can be monitored using the 1H STE PFG NMR technique

to report diffusion coefficients of the model transmembrane peptide WALP19, and

compared with that of a PEGylated DMPE lipid.

38

Increasing temperature results in faster diffusion for both the WALP19-PEG2000 and

DMPE-PEG2000 lipid in the bicelle and both fit the free-area model for lateral diffusion

in this size range. The observation that the transmembrane peptide has slower diffusion

and requires larger activation energy to diffuse than the DMPE lipid is consistent with the

triple layer model with high viscosity surface-interface regions of the bilayer which limit

the diffusion of the membrane components. However, to make the PFG NMR technique

more amenable to examine slow-diffusing systems such as peptides in membranes,

further studies should be done to overcome relaxation effects and increase the signal

from the sample.

3.5. References

(1) Axelrod, D. Lateral Motion of Membrane Proteins and Biological Function. J.

Membr. Biol. 1983, 75, 1–10.

(2) Soong, R.; Macdonald, P. M. Lateral Diffusion of PEG-Lipid in Magnetically

Aligned Bicelles Measured Using Stimulated Echo Pulsed Field Gradient 1H

NMR. Biophys. J. 2005, 88, 255–268.

(3) Macdonald, P. M.; Saleem, Q.; Lai, A.; Morales, H. H. NMR Methods for

Measuring Lateral Diffusion in Membranes. Chem. Phys. Lipids 2013, 166, 31–44.

(4) Killian, J. A.; Salemink, I.; de Planque, M. R. R.; Lindblom, G.; Koeppe, R. E.;

Greathouse, D. V. Induction of Nonbilayer Structures in

Diacylphosphatidylcholine Model Membranes by Transmembrane -Helical

Peptides: Importance of Hydrophobic Mismatch and Proposed Role of

Tryptophans. Biochemistry 1996, 35, 1037–1045.

(5) Killian, J. A. Hydrophobic Mismatch Between Proteins and Lipids in Membranes.

Biochim. Biophys. Acta BBA - Rev. Biomembr. 1998, 1376, 401–415.

(6) Killian, J. A. Synthetic Peptides as Models for Intrinsic Membrane Proteins. FEBS

Lett. 2003, 555, 134–138.

39

(7) Holt, A.; Killian, J. A. Orientation and Dynamics of Transmembrane Peptides: The

Power of Simple Models. Eur Biophys J 2010, 39, 609–621.

(8) De Planque, M. R. R.; Kruijtzer, J. A. W.; Liskamp, R. M. J.; Marsh, D.;

Greathouse, D. V.; Koeppe, R. E.; de Kruijff, B.; Killian, J. A. Different

Membrane Anchoring Positions of Tryptophan and Lysine in Synthetic

Transmembrane -Helical Peptides. J. Biol. Chem. 1999, 274, 20839 –20846.

(9) Liu, L.; Li, S.; Goto, N. K.; Deber, C. M. Threshold Hydrophobicity Dictates

Helical Conformations of Peptides in Membrane Environments. Biopolymers

1996, 39, 465–470.

(10) Pomroy, N. C.; Deber, C. M. Solubilization of Hydrophobic Peptides by

Reversible Cysteine PEGylation, ,. Biochem. Biophys. Res. Commun. 1998, 245,

618–621.

(11) Pomroy, N. C.; Deber, C. M. Conjugation of Polyethylene Glycol via a Disulfide

Bond Confers Water Solubility Upon a Peptide Model of a Protein

Transmembrane Segment. Anal. Biochem. 1999, 275, 224–230.

(12) Macdonald, P. M.; Soong, R. Diffusion NMR and Bicelle Morphology. Can. J.

Chem. 2011, 89, 1021–1035.

(13) Picard, F.; Paquet, M.-J.; Levesque, J.; Bélanger, A.; Auger, M. 31P NMR First

Spectral Moment Study of the Partial Magnetic Orientation of Phospholipid

Membranes. Biophys. J. 1999, 77, 888–902.

(14) Worcester, D. L. Structural Origins of Diamagnetic Anisotropy in Proteins. Proc.

Natl. Acad. Sci. U. S. A. 1978, 75, 5475–5477.

(15) Cullis, P. R.; de Kruijff, B. Lipid Polymorphism and the Functional Roles of

Lipids in Biological Membranes. Biochim. Biophys. Acta 1979, 559, 399–420.

(16) Triba, M. N.; Warschawski,, D. E.; Devaux, P. F. Reinvestigation by Phosphorus

NMR of Lipid Distribution in Bicelles. Biophys. J. 2005, 88, 1887–1901.

40

(17) Soong, R.; Macdonald, P. M. PEG Molecular Weight and Lateral Diffusion of

PEG-ylated Lipids in Magnetically Aligned Bicelles. Biochim. Biophys. Acta BBA

- Biomembr. 2007, 1768, 1805–1814.

(18) De Gennes, P. G. Conformations of Polymers Attached to an Interface.

Macromolecules 1980, 13, 1069–1075.

(19) Flory, P. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY,

1971.

(20) Hristova, K.; Needham, D. Phase Behavior of a Lipid/Polymer-Lipid Mixture in

Aqueous Medium. Macromolecules 1995, 28, 991–1002.

(21) Nieh, M.-P.; Raghunathan, V. A.; Pabst, G.; Harroun, T.; Nagashima, K.; Morales,

H.; Katsaras, J.; Macdonald, P. Temperature Driven Annealing of Perforations in

Bicellar Model Membranes. Langmuir 2011, 27, 4838–4847.

(22) Du, H.; Chandaroy, P.; Hui, S. W. Grafted Poly-(ethylene Glycol) on Lipid

Surfaces Inhibits Protein Adsorption and Cell Adhesion. Biochim. Biophys. Acta

BBA - Biomembr. 1997, 1326, 236–248.

(23) Soong, R.; Macdonald, P. M. Influence of the Long-Chain/Short-Chain

Amphiphile Ratio on Lateral Diffusion of PEG-Lipid in Magnetically Aligned

Lipid Bilayers as Measured via Pulsed-Field-Gradient NMR. Biophys. J. 2005, 89,

1850–1860.

(24) Ramadurai, S.; Holt, A.; Krasnikov, V.; van den Bogaart, G.; Killian, J. A.;

Poolman, B. Lateral Diffusion of Membrane Proteins. J. Am. Chem. Soc. 2009,

131, 12650–12656.

(25) Almeida, P. F. F.; Vaz, W. L. C. Chapter 6 Lateral Diffusion in Membranes. In

Structure and Dynamics of Membranes; Lipowsky, R.; Sackmann, E., Eds.;

Handbook of Biological Physics; North-Holland, 1995; Vol. Volume 1, pp. 305–

357.

41

(26) Liu, C.; Paprica, A.; Petersen, N. O. Effects of Size of Macrocyclic Polyamides on

Their Rate of Diffusion in Model Membranes. Biophys. J. 1997, 73, 2580–2587.

(27) Tanner, J. E. Use of the Stimulated Echo in NMR Diffusion Studies. J. Chem.

Phys. 1970, 52, 2523.

(28) Keeler, J. Understanding NMR Spectroscopy; 2nd ed.; John Wiley & Sons, Ltd:

United Kingdom, 2010.

(29) Gaede, H. C.; Gawrisch, K. Lateral Diffusion Rates of Lipid, Water, and a

Hydrophobic Drug in a Multilamellar Liposome. Biophys. J. 2003, 85, 1734–1740.

(30) Pampel, A.; Kärger, J.; Michel, D. Lateral Diffusion of a Transmembrane Peptide

in Lipid Bilayers Studied by Pulsed Field Gradient NMR in Combination with

Magic Angle Sample Spinning. Chem. Phys. Lett. 2003, 379, 555–561.

(31) Pampel, A.; Zick, K.; Glauner, H.; Engelke, F. Studying Lateral Diffusion in Lipid

Bilayers by Combining a Magic Angle Spinning NMR Probe with a Microimaging

Gradient System. J. Am. Chem. Soc. 2004, 126, 9534–9535.

42

Chapter 4: Morphological Characterization of

DMPC/CHAPSO Bicellar Mixtures via NMR

Portions of this chapter have been published by Li, Ming; Morales, Hannah Hazel;

Katsaras, John; Ku erka, Norbert; Yang, Yongkun; Macdonald, Peter; Nieh, Mu-Ping, in

the paper "Morphological Characterization of DMPC/CHAPSO Bicellar Mixtures: A

Combined SANS and NMR Study". Langmuir 2013 (Accepted) DOI: 10.1021/la402799b

Much of what has been established concerning the morphological transformations

undergone by bicelles has been contributed by small angle neutron scattering (SANS)

studies,1–5 aided and abetted by NMR,6–11 electron microscopy,12 and other techniques.

Most such studies have been performed on the canonical bicellar mixture of the

detergent-like, short-chain phospholipid dihexanoyl phosphatidylcholine (DHPC) with

dimyristoyl phosphatidylcholine (DMPC) introduced by Sanders and Schwonek.6

Although early reports on bicelles used the bile salt 3-(cholamidopropyl)dimethyl-

ammonio-2-hydroxyl-1-propanesulfonate (CHAPSO) as the detergent component,13,14

CHAPSO has been largely displaced by DHPC since the resulting mixture then consists

of phospholipids only. Nevertheless, CHAPSO is known to confer certain advantages

such as better functionality of particular bicelle-reconstituted membrane proteins15,16 and

enhanced pH17 and thermal stability17,18 relative to DHPC-containing bicelles.

Comparisons of DHPC- versus CHAPSO- containing bicelles reveal much in common,

such as the presence of bicelle disks at high detergent ratios and the ability to orient in a

magnetic field at higher lipid concentrations and temperatures above the gel-to-liquid-

crystalline phase transition of DMPC.13 However, to the best of our knowledge, the

structural phase diagram of DMPC/CHAPSO mixtures has not yet been resolved. Thus, it

is not clear whether, and to what extent, CHAPSO-containing bicelles undergo

morphological transformations analogous to those observed with DHPC-containing

bicelles.

43

Performing studies on the morphological changes undergone by DMPC/CHAPSO

bicelles would help researchers better understand the system and effectively utilize it as a

biomembrane mimic for membrane proteins and membrane-associated molecules. In this

report, 31P NMR was used to characterize the quality of magnetic alignment while 1H

pulsed field gradient (PFG) NMR diffusion studies were conducted to understand the

structural and dynamic behavior of DMPC/CHAPSO bicelles as a function of

temperature and surface charge density (molar ratio of DMPG).

4.1. Magnetic alignment of DMPC/DMPG/CHAPSO mixtures from 31P NMR

31P NMR spectroscopy is an excellent tool for examining the quality of bicellar magnetic

alignment9 and has been used here to compare the thermal stability of magnetic

alignment in DMPC/CHAPSO, DMPC/DMPG/CHAPSO and DMPC/DHPC mixtures,

all having q = 3. All were examined at = 25 wt%, since at low the magnetic

alignment can become problematic regardless of the composition.

Figure 4.1 compares the 31P NMR spectra of DMPC/DHPC, DMPC/CHAPSO and

DMPC/DMPG/CHAPSO mixtures, all having q = 3 and = 25 wt%, as a function of

temperature. For the DMPC/DHPC mixture, at 25°C, a temperature close to the Tm of

DMPC, the spectrum consists of a broad powder pattern line shape with a width on the

order of 45 ppm, indicating a powder distribution of DMPC orientations,19 and

demonstrating that magnetic alignment has not yet occurred. Whatever overall

reorientational motions the bicellar assemblies experience, they are slow on the timescale

defined by the inverse of the powder pattern width of 45 ppm. For this composition, the

narrow resonance at the isotropic frequency (0 ppm) is due to DHPC, which occupies

highly-curved edge regions and, hence, experiences near isotropic motional averaging,

thus producing a narrow resonance. At a temperature just above 25°C the broad DMPC

powder pattern disappears and is replaced by a narrow resonance at a frequency of

roughly -12 ppm as shown in Figure 4.1 for the specific instance of 35°C. This is

indicative of a uniform alignment of the bicellar aggregates oriented such that the normal

to the plane of the bilayer lies perpendicular to the magnetic field direction. This so-

44

called negative magnetic alignment arises spontaneously due to the negative magnetic

susceptibility anisotropy of the bicellar assembly.20 The second, and smaller, resonance

at roughly -5 ppm is assigned to DHPC. The fact that DHPC’s 31P NMR resonance no

longer appears at the isotropic frequency has been taken to indicate that DHPC is now in

fast exchange between edge and planar regions of the bicelles, as per the “mixed bicelle

model”.10 At 45°C, as shown in Figure 4.1, a third resonance appears at -13.8 ppm,

similar to that observed by Triba et al,10 and which was attributed to deformed vesicles,

and undoubtedly corresponds to a separate and more highly-ordered population of lipids.

Figure 4.1. 31P NMR spectra of DMPC/DHPC and DMPC/CHAPSO mixtures, q = 3 and = 25 wt%, with differing DMPG content, at the indicated temperatures.

For the DMPC/CHAPSO mixture, at 25°C a powder pattern spectrum was not observed:

rather, a broad featureless spectrum was obtained, centered at 0 ppm. This is indicative

of assemblies undergoing overall reorientational motions at a rate intermediate to the

45

inverse of the powder spectrum width of 45 ppm. This difference versus the

DMPC/DHPC mixture may be due to the same structural effects which produce the

different viscosity temperature minimum of 4°C for DMPC/DHPC versus 15°C for the

DMPC/CHAPSO mixture.

At a temperature above 298 K the DMPC/CHAPSO mixture yields a single narrow

resonance at a frequency indicative of a highly negatively aligned DMPC population.

Note that CHAPSO contains no phosphorus atoms and so yields no 31P NMR resonance.

The example in Figure 4.1 was obtained at 45°C and was chosen to contrast with the

corresponding DMPC/DHPC mixture which indicates multiple DMPC populations in

different environments at that same temperature. Eventually, at temperatures exceeding

60°C the spectrum splits into two resonances, the dominant one at the isotropic frequency

and the other at -13.3 ppm. At such elevated temperatures there are, therefore, two

populations of DMPC: one undergoing isotropic motions and one corresponding to

deformed vesicles. Note that undeformed MLVs would yield a powder pattern line shape

similar to that seen at 25°C for DMPC/DHPC, rather than the narrow resonance at -13.3

ppm which is indicative of continued magnetic alignment. This indicates the presence of

a highly-ordered, closely-stacked lamellar domain, which may be from deformed vesicles

which contain defects stabilized by DHPC or CHAPSO and are thus presumably

magnetically alignable. At such elevated temperatures, CHAPSO may also phase

separate from DMPC, forming non-alignable CHAPSO-rich / DMPC-depleted micelles

co-existing with CHAPSO-depleted / DMPC-rich lamellae.

For DMPC/DMPG/CHAPSO mixtures, whether having R=0.01 or 0.10, the 31P NMR

spectra indicated basically comparable behavior to that obtained with DMPC/CHAPSO.

Note that for the R=0.10 case, the 31P NMR resonance of DMPG becomes visible as a

shoulder on the DMPC resonance. At approximately 65°C, separation into two

populations is again apparent, one exhibiting an isotropic frequency and the other

apparently more ordered. With increasing DMPG content from R=0 to 0.01 to 0.10, the

fractional population exhibiting an isotropic resonance declines precipitously. This

indicates greater thermal stability of the alignable phase in the presence of surface charge

due to the inclusion of DMPG.

46

Overall, these 31P NMR spectra demonstrate that the DMPC/CHAPSO and

DMPC/DMPG/CHAPSO mixtures have a wider temperature range over which a

homogeneous magnetically alignable phase is observed than found for DMPC/DHPC

mixtures. Thus, CHAPSO-containing systems exhibited a significantly greater thermal

stability than the DHPC-containing ones.

Figure 4.2. 31P NMR residual chemical shift anisotropy of DMPC as a function of temperature in various negatively magnetically-aligned, q = 3, = 25 wt% mixtures: DMPC/DHPC ( ), DMPC/CHAPSO ( ), DMPC/DMPG/CHAPSO, R=0.01 ( ), DMPC/DMPG/CHAPSO, R=0.10 ( ).

4.5

5.5

6.5

7.5

8.5

9.5

10.5

11.5

25 35 45 55 65

DM

PC 31

P N

MR

resid

ual C

SA (p

pm)

=bi

c is

o

Temperature (°C)

47

The quality of the magnetic alignment in these bicelle mixtures can be examined in

greater detail via a comparison of the residual 31P NMR chemical shift anisotropy

| | = | | exhibited under particular circumstances, where is the observed

DMPC chemical shift for a particular bicellar composition at a particular temperature,

while is the isotropic chemical shift. Figure 4.2 illustrates such a comparison as a

function of temperature for the different mixtures of interest here. In each instance

increased with increasing temperature. While this observation seems contradictory to the

notion that strong thermal fluctuations should decrease the ordering of the lamellae, it can

be understood to arise from the formation of larger discs, ribbons or lamellae upon the

coalescence of smaller aggregates. In the case of DMPC/DHPC mixtures, this

coalescence is driven by the increased miscibility of DHPC with DMPC with increasing

temperature, as suggested elsewhere,10,11,21 which decreases the amount of DHPC

available to populate edge regions. 31P NMR cannot directly monitor CHAPSO in

bicelles, but the fact that similar behavior is observed with both DHPC-containing and

CHAPSO-containing mixtures suggests that a similar increase in CHAPSO miscibility

with DMPC upon increasing temperature might be occurring.

The higher ordering for DHPC- containing versus CHAPSO-containing DMPC mixtures

evident in Figure 4.2 has been observed previously by Sanders and co-workers,13,22 and is

believed to arise from the greater efficiency with which CHAPSO masks edge regions,

thus allowing more “edge” per mole and, hence, smaller aggregates (or lamellae

containing more defects) having lower orientational order.

At any one temperature, increased for the DMPC/DMPG/CHAPSO mixture with

increasing DPMG content from R=0 to 0.01 to 0.10. This may be understood to arise

from a progressive “stiffening” of the bilayers with increasing DMPG as the associated

surface charge dampens thermal undulations within and between the membranes, thereby

enhancing their ordering.23–26 Nevertheless, even at 10 mol% DMPG, i.e., R=0.10, the

DMPC/DMPG/CHAPSO mixture exhibits a smaller than that of DMPC/DHPC

mixtures, i.e., lacking DMPG entirely, at the same temperature and lipid concentration.

48

31P NMR provides a useful overview of the phase homogeneity, magnetic alignment and

orientational order present in such mixtures. However, certain morphologies, such as the

ribbons and perforated lamellae, provide essentially identical 31P NMR spectra.

Diffusion NMR, on the other hand, may differentiate such details,27 and these studies will

be presented next.

4.2. Water and PEG diffusion in interbicellar spaces

1H PFG STE NMR diffusion studies were undertaken to compare CHAPSO-containing

and DHPC-containing systems, where PEG-1000 was incorporated as a diffusion probe

of the bicellar interstitial space. In particular, it was of interest to investigate whether it

was possible to observe the so-called ribbon morphology, where non-exponential and

diffusion-time-dependent diffusive decays may be observed.28

Figure 4.3. 1H PFG STE NMR spectra of negatively magnetically-aligned DMPC/DMPG/CHAPSO, R=0.10, = 25 wt% mixtures at 60°C as a function of increasing gradient pulse amplitude.

Increasing gradient strength

49

Figure 4.3 shows a series of 1H PFG STE NMR spectra for DMPC/DMPG/CHAPSO

mixtures, q = 3, R=0.10 and = 25 wt%, acquired at 60°C, at which temperature

negative magnetic alignment is still present. The spectra contain only two resonances,

one at 4.3 ppm corresponding to water, and the other at 3.4 ppm arising from the ethylene

oxide protons of PEG-1000. Other possible resonances are not visible due to residual

dipolar interactions which shorten their transverse relaxation times relative to the delays

in the PFG STE pulse sequence. The spectral series in Figure 4.3 was acquired with

increasing gradient amplitude, , so that diffusion leads to a progressive decrease in

signal intensity across the series. Obviously, water diffuses more quickly than does PEG-

1000. The diffusion coefficient may be extracted from the intensity decay as per the

Stejskal-Tanner equation.29

For the case of a molecule diffusing within the interstitial spaces between magnetically-

aligned lamellae, the diffusion coefficient will be anisotropic since diffusion along the

direction of the channels separating adjacent planar bilayer surfaces, i.e., perpendicular to

the bilayer normal and characterized by a diffusion coefficient , will be relatively

facile, while diffusion across such channels, i.e., in a direction parallel to the bilayer

normal and characterized by a diffusion coefficient , will be obstructed by the

intervening surface of the adjacent lamellae. The experimentally measured diffusion

coefficient depends then on the orientation of the applied field gradients. For the case of

field gradients applied along the z-direction, parallel to the direction of the main

magnetic field, as in the present case, the measured diffusion coefficient, Dzz, is related to

the diffusion tensor components in the magnetically-aligned bicelle reference frame via

Equation 4.1:

= + (4.1)

where is the angle between the bilayer normal and the direction of the applied field

gradients.30,31 For these negatively magnetically-aligned bilayers = 90° so the

measured diffusion coefficient is D .

50

Figure 4.4. Semi-logarithmic plots of the normalized intensity of the water resonance and PEG-1000 resonance from 1H PFG STE NMR spectra of negatively magnetically-aligned DMPC/DMPG/CHAPSO, R=0.10, = 25 wt% mixtures at 60°C as a function of the experimental factor, k = ( g )2·( – /3)/10-10 for different experimental diffusion times: 50 ms ( ), 100 ms ( ), 200 ms ( ).

Figure 4.4 shows representative diffusion decays obtained via 1H PFG STE NMR

measurements on negatively magnetically-aligned DMPC/DMPG/CHAPSO mixtures

(q=3.0, R=0.10) at 60°C. Intensity decays for water and for PEG-1000 are plotted for

different diffusion times . All exhibit monoexponential behavior and diffusion time

independence, indicative of normal Gaussian, non-restricted diffusion. Similar behavior

was obtained for all bicellar compositions investigated here within the range of

temperatures over which stable magnetic alignment was observed. From the slope one

1.0E-01

1.0E+00

0 0.2 0.4 0.6 0.8

I/I 0

( g )2 ( – /3) 10-10

PEG Water

51

extracts the particular diffusion coefficient. For water, the diffusion coefficient was on

the order of 3.0 x 10-9 m2s-1 which is roughly 30% of the bulk water diffusion coefficient

at the same temperature. This reduction is due in large measure to the population of lipid

bilayer-bound water present in such situations. The PEG-1000 diffusion coefficient was

found to equal 3.2 x 10-10 m2s-1, representing an approximately 60% reduction in the

diffusion coefficient relative to PEG-1000 free in solution at the same temperature. This

reduction can be attributed to confinement of the PEG-1000 between the walls of the

channels formed by the magnetically-aligned membranes, further verifying the lamellar

structure.32

Given the diffusion coefficient D and the experimental diffusion time t= , one may

calculate the root-mean-square (rms) displacement undergone by the diffusant during the

course of the measurement as per Equation 4.2:

= (4 ) (4.2)

For water, the rms displacements fall in the range between 17 and 50 m for lower

temperature-shorter diffusion time measurements (T=303 K, =50 ms) versus higher

temperature-longer diffusion time measurements (T=333 K, =200 ms). For PEG-1000

the same range of experimental conditions yield rms displacements between 5 and 15

m. The PFG NMR diffusion decays demonstrate, therefore, that these diffusants

experience unrestricted normal diffusion over these length scales.

52

Figure 4.5. Arrhenius plot of the natural logarithm of the diffusion coefficients of water and PEG-1000 versus reciprocal temperature for various negatively magnetically-aligned q = 3, = 25 wt% mixtures: DMPC/DHPC ( ), DMPC/CHAPSO ( ), DMPC/DHPC/CHAPSO, R=0.01 ( ), DMPC/DMPG/CHAPSO, R=0.10 ( ).

Figure 4.5 is an Arrhenius plot of the temperature dependence of both water and PEG-

1000 diffusion coefficients in the different bicelle systems. The comparison demonstrates

little systematic difference in the diffusion behavior across these various bicelle

compositions for either diffusant. The activation energies for diffusion are listed in Table

4.1 for individual bicelle compositions. For water, the activation energies are similar to

the literature value for pure water (18.8 kJ/mol between 15°C and 45°C).33 For PEG-

1000, the activation energies were significantly higher than that for the diffusion of free

-23.0

-22.5

-22.0

-21.5

-21.0

-20.5

-20.0

-19.5

-19.0

2.90 3.00 3.10 3.20 3.30 3.40

ln (D

)

1/Temperature (K-1) 10-3

53

PEG-1000 in aqueous solution, as might be expected given its size relative to the

confinement dimensions of the inter-lamellar spacings.32

Table 4.1. Activation energies for water and PEG-1000 diffusion in various negatively magnetically aligned bicelles.

Short Chain Lipid CHAPSO CHAPSO CHAPSO DHPC Control

(Aqueous solution) R=DMPG/DMPC 0 0.01 0.10 0

(kJ / mol)

Water 18.8±1.7 19.1±1.6 18.6±1.5 15.9±5.5 19.7±1.2

PEG-1000 21.6±1.2 22.2±1.5 22.3±1.4 25.0±4.5 20.0±2.4

4.3. Thermal stability of CHAPSO bicelles

The many useful properties of bicellar mixtures depend, in fact, on a degree of non-ideal

mixing of the detergent with the long chain lipid. This limited miscibility means that the

“curvaphilic” detergents, having a high positive spontaneous curvature, tend to occupy

edge regions, while the “curvaphobic” long chain lipids, with their slightly negative

spontaneous curvature, prefer planar regions.34,35 In the gel phase this miscibility is at its

lowest, so the line integral of edge regions is at its greatest, producing small discoidal

assemblies. Above the gel-to-liquid-crystalline phase transition temperature, detergent

miscibility increases. For DHPC, increasing miscibility with DMPC with increasing

temperature, as observed here and elsewhere,10 progressively diminishes the DHPC

available to cover edge regions, thus decreasing the line integral of edge regions and

forcing morphology adaptions including fusion into larger assemblies (ribbons and/or

perforated lamellae). Larger assemblies exhibit greater orientational order, as manifest in

the increasing 31P NMR chemical shift anisotropy with increasing temperature in Figure

4.2. The fact that CHAPSO-containing mixtures exhibit the same trend suggests a

similarly increased miscibility with increasing temperature. Because CHAPSO lacks any

phosphorus atom, the current 31P NMR measurements cannot address this question

directly, nor that of the degree to which DHPC and CHAPSO might differ in that regard.

54

Some other approach, such as 2H NMR of deuterium-labeled CHAPSO, might well be

able to do so.

The general correlation between the size of bicellar assemblies and their orientational

order as manifest in the 31P NMR residual chemical shift anisotropy22 suggests that

CHAPSO-containing assemblies are significantly smaller or contain more defects than

DHPC-containing assemblies, i.e., the line integral of edge regions per mole of detergent

is greater for CHAPSO than for DHPC. An inspection of their different chemical

structures, as shown in Figure 4.5, shows that CHAPSO’s amphiphilicity arises from the

presence of apposing hydrophobic and polar faces, rather than from the hydrophobic tail

and polar head group arrangement in DHPC. As proposed even in the earliest studies of

CHAPSO-containing bicelles,13 CHAPSO may lie flat on a hydrophobic surface, thereby

masking a greater hydrophobic surface area per molecule, than DHPC which is conical

and assembles into a hemi-spherical coating. Note that it is not necessary, therefore, to

postulate differential bilayer miscibility for DHPC versus CHAPSO in order to explain,

qualitatively, the different behaviors of the assemblies formed upon mixing with DMPC,

although such a differential miscibility may well exist.

Figure 4.6. Chemical structures of CHAPSO and DHPC.

Hydrophobic face most likely associating with edges of bicelle assemblies

CHAPSO

Polar face

DHPC PO-

OO

N+O

H

O

O

O

O Polar headgroup

Hydrophobic tail

55

As to the high temperature instability observed with DHPC, Triba et al.10 observed a

similar evolution in their 31P NMR spectra. The upfield resonance was ascribed to

vesicles co-existing with DHPC-enriched perforated lamellae. These authors argue that

the progressive movement of DHPC into the bilayer with increasing temperature

eventually forces vesiculation as the line tension energy cost exceeds the bending energy

cost. With CHAPSO the picture is somewhat different at high temperature, aside from

the fact that the instabilities occur at temperatures above that at which DHPC-containing

mixtures exhibit instability. As has been reported previously,13 CHAPSO-containing

mixtures at such temperatures clearly divide into two DMPC-containing populations: one

experiencing isotropic motion and therefore being small in size, and a second

experiencing anisotropic motion and therefore being large in size. The latter population

apparently remains magnetically aligned.

It is tempting to speculate that the isotropically averaging DMPC population is enriched

with CHAPSO, but the data reported here do not directly address that question. If,

however, this is the case, the 31P NMR spectra in Figure 4.1 would indicate that DMPG

encourages retention of CHAPSO in planar regions, versus formation of the isotropic

phase, possibly due to favourable hydrogen-bonding interactions between the two. Even

for DHPC-containing bicellar mixtures, however, DMPG has been shown to stabilize

against the thermally driven transition to multilamellar vesicles.

Another factor influencing the temperature at which vesicles form, apart from migration

of detergent from edges into bilayer regions, is the rigidity of the bilayer. It is possible

that CHAPSO’s cholesterol-like planar structure might induce a certain rigidity of the

membrane, inhibiting the self-folding. Small-angle nuclear scattering (SANS)

measurements on the same systems were performed by our collaborators Prof. Mu-Ping

Nieh, Ming Li, and Dr. Yongkun Yang in the University of Connecticut and Dr. John

Katsaras and Dr. Norbert Ku erka at the Canadian Neutron Beam Centre, National

Research Council in Chalk River, ON. The results indicated a smaller d-spacing of the

lamellar structures in the DMPC/CHAPSO mixture (< 63 Å) relative to the

DMPC/DHPC mixture ( 64.5 Å), which suggest a less repulsive undulation force in the

former, consistent with the notion of a more rigid membrane.36,37

56

4.4. Conclusions

This study of CHAPSO-containing mixtures has focused on differences relative to

DHPC-containing mixtures, since both have been widely used for solubilizing and

reconstituting membrane proteins, among other uses. 31P NMR spectroscopy of

negatively magnetically-aligned bicelles show that bicelles formulated with DHPC as the

short chain lipid component exhibit greater order that those formulated with CHAPSO.

Nevertheless, the upper temperature range limit at which magnetic alignment could be

maintained was significantly higher for CHAPSO-containing relative to DHPC-

containing bicelles. Both these effects reflect a more efficient stabilization of bicelle edge

regions by CHAPSO versus DHPC. We postulate that this is due to the different

amphiphilic molecular architectures of CHAPSO and DHPC, but this hypothesis requires

further study. Nevertheless, the addition of surface charge stabilizes smaller aggregates

and tends to dampen differences between CHAPSO- and DHPC-containing mixtures.

Diffusion of both water and PEG-1000 along the channels between the various negatively

magnetically aligned bicelles was normal Gaussian and followed Arrhenius-type

temperature dependence with similar activation energies for all the bicelle systems

studied examined. Thus, on the rms displacement scale of tens of microns, the diffusion

measurements gave no indication of the presence of a ribbon morphology in any of these

bicelle systems. However, it cannot be excluded that such morphology exists with

characteristic “cross-over” dimensions on sub-micron distance scales, since the PFG

NMR diffusion measurements would not distinguish these from normal diffusion.

Knowledge from these data must be then combined with other techniques such as 2H

NMR, SANS, and TEM, to better provide a physical foundation for utilizing these

zwitterionic mixtures (both DMPC/DHPC and DMPC/CHAPSO) in structural studies of

membrane-associated proteins and elsewhere.

57

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62

Chapter 5: Summary

This report has shown that lateral diffusion of a PEGylated WALP19 peptide in

magnetically aligned DMPC/DHPC bicelles can be measured using 1H STE PFG NMR.

The results are consistent with the free-area model for diffusion, indicating that the cross-

sectional area of the alpha-helical peptide is within the size range of the bilayer lipids.

The triple layer model of the lipid membrane can also account for the differences in the

diffusion coefficients and activation energies of the transmembrane peptide and the

bicellar DMPE lipid. The experiment has to be further optimized, however, to counter

relaxation effects and low signal to noise ratio.

Differences between DMPC/CHAPSO and the DMPC/DHPC bicellar mixtures have also

been discussed in this report. Although bicelles with DHPC exhibit greater order than

those with CHAPSO, as revealed by 31P NMR spectroscopy, CHAPSO-containing

bicelles maintain magnetic alignment at higher temperatures. This characteristic can

prove useful in employing magnetically-aligned DMPC/CHAPSO bicelles for studies of

membrane-associated biomolecules at a wider temperature range than what the canonical

DMPC/DHPC composition allows. For instance, bicelles formulated with CHAPSO as

the short chain lipid component can be utilized for diffusion studies of WALP19-

PEG2000, as the effect of T1 relaxation in of the PEG group is lessened at higher

temperatures.

For the CHAPSO-containing bicelles, further studies need to be done regarding the

morphological changes that occur with increasing temperature and surface charge.

Combined data from NMR, SANS, and TEM would give a better physical picture of the

bicellar mixtures in order for them to be used more effectively in studies of membrane-

associated biomolecules.