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Studies of the orientational order and bilayer thickness in biological and model membranes Monck, Myrna A. 1993

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STUDIES OF THE ORIENTATIONAL ORDER AND BILAYER THICKNESS IN BIOLOGICAL AND MODEL MEMBRANES. Myrna A. Monck B. Sc. (Math/Comp Sci.) St. Francis Xavier University M. Sc. (Biochemistry) University of Ottawa  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 1993  © Myrna A. Monck  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  8/0CHE)-TWY  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Abstract  The hydrocarbon order of a membrane bilayer in the hydrocarbon region is suggested to play a fundamental role in maintaining functional integrity in the biological membrane system, Acholeplasma laidlawii strain B. The hydrocarbon order profile, which can be considered to be linearly related to hydrophobic thickness, has been measured by 2 11  NMR methods for a range of Acholeplasma laidlawii membranes containing exoge-  nously incorporated perdeuteriated palmitic acid and a second fatty acid of increasing unsaturation. The microorganism was grown under conditions where de novo fatty acid biosynthesis was suppressed. At 37°C, the growth temperature, there exists a range of hydrocarbon order compatible with good growth characteristics of the microorganism and outside of which the organism grows poorly or not at all. When grown in the presence of cholesterol which is known to increase the orientational order in the hydrocarbon region, it appears that a small fraction of the cholesterol is solubilized by the membrane. A significant fraction of membrane cholesterol, however, is excluded from the lipid bilayer of the microorganism or is in a membrane domain separate from the rest of the lipid. 2 11 NMR measurements show that this pool of cholesterol is solid -like or crystalline but is available for membrane incorporation using several solubilization methods. It is suggested that the range of hydrocarbon order compatible with good A. laidlawii growth characteristics is maintained even in the presence of cholesterol. Using model membrane systems, T2 relaxation anisotropy measurements have been made for multilamellar vesicles and macroscopically aligned multibilayers by  2 11  NMR  quadrupolar echo techniques. The dominant relaxation mechanism in the multilamellar system is found to be fundamentally different from that in the macroscopically aligned  ii  multibilayers as suggested by the T2 anisotropy found for each. The multilamellar vesicles show an orientation dependence consistent with such mechanisms as collective lipid motions or surface undulations. The macroscopically aligned multi-layers, however, appear to damp out several of these motional modes as suggested by the anisotropy and also an increase in the magnitudes of the relaxation times obtained. A phenomenological theory developed on the basis of similar experimental results suggests that fluctuations in bilayer thickness could be the mechanism responsible for T2 relaxation.  iii  Table of Contents  Abstract^  ii  List of Tables^  v  List of Figures^  vi  List of Abbreviations^  vii  Acknowledgement^ 1  viii  Introduction  1  1.1  1  Membrane Structure ^  1.2  Proteins ^  6  1.3  Cholesterol ^  7  1.4  Lipid Phases  1.5  Lipid Polymorphism ^  11  1.6  The Acholeplasma laidlawii Membrane ^  16  1.7  Membrane Hydrocarbon Order ^  19  1.8  Bilayer Thickness ^  21  1.9  2 11  23  10  ^  NMR Theory ^  1.9.1^Energy Transitions ^  24  1.9.2^Powder Spectra in the Absence of Motion ^  27  1.9.3^The Effect of Lipid Motion  27  1.10 DePakeing ^  ^  29 iv  1.11 Motivation and Thesis Outline ^ 2 Materials and Methods ^ 2.1 Isolation of A. laidlawii Membranes ^  33 35 35  2.2 Preparation of Samples for 2 11 NMR Spectroscopy ^ 35 2.3 Extraction of A. laidlawii Lipids and Preparation of the Polar Lipid Fraction ^  36  2.4 Thin Layer Chromatography ^  37  2.5 Column Chromatographic Separation of A. Laidlawii Membrane Lipids 37 2.6 Determination of Polar Headgroup Composition ^  38  2.7 Determination of Cholesterol ^  38  2.8 Differential Scanning Calorimetry. ^  38  2.9 2 H NMR Measurements ^  39  2.10 T 1 Measurements ^  39  2.11 Derivation of Order Profiles ^  39  3 Influence of Lipid Composition on <S> in A1B Membranes ^41 3.1 Summary ^  41  3.2 Introduction ^  42  3.3 Results ^  43  3.4 Discussion ^  56  4 Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 61 4.1 Summary ^  61  4.2 Introduction ^  62  4.3 Results ^  64  4.4 Discussion ^  77  v  5 Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems ^82 5.1 Summary ^  82  5.2 Introduction ^  84  5.3 Materials and Methods ^  86  5.3.1 Samples ^ 5.3.2 Sample Preparation ^ 5.3.3 Nuclear Magnetic Resonance ^ 5.3.4 Measurement of T2 ^  86 86 87 88  5.4 Results ^  89  5.5 Discussion ^  101  6 Future Directions^  106  7 Bibliography^  108  A^  119  B Phenomenological Theory of Transverse Relaxation in Membranes 120  vi  List of Tables  1.1  Polymorphic Preferences of Membrane Lipids from Eukaryotes . ^  15  3.1  A. laidlawii B Membrane Lipid Composition  48  3.2  Hydrophobic Thickness calculated  4.1  Cholesterol levels in A. laidlawii Membranes of Given Fatty Acid Com-  ^  ^  position ^ 4.2  65  Average Order Parameter vs Temperature for Intact A. laidlawii B Membrane Preparation with Cholesterola ^  5.1  59  Ratios of  T2  layers and  69  (F2)fit coefficients for POPCd 31 and POPC/POPEd 31 bi-  A02  values ^  96  A.1 Values of AV, B(n) and C(n) for POPCd 31 and POPC/POPEd 31 fits . . 119  vii  List of Figures  1.1 Hypothetical Erythrocyte Membrane Bilayer. .  ^2  1.2 Various Lipid classes.  ^4  1.3 Calorimetric tracings and Lipid Phases.  ^9  1.4 Lipid Composition of the Erythrocyte Membrane. ^  12  1.5 Lipid Packing Parameters. ^  14  1.6 A. laidlawii B Membrane Lipids. ^  18  1.7 Acyl chain geometries ^  22  1.8 Deuterium nucleus energy levels and rotation of a single crystal ^ 25 1.9 Typical 2 H NMR Spectrum^  28  1.10 Spectra of P-DPPC d2 membranes ^  30  1.11 Spectra of POPC d31 membranes. ^  32  3.1 Spectra and order profiles of intact and redispersed A. laidlawii Membranes. ^  45  3.2 Order parameter profiles of intact A. laidlawii membranes. ^ 47 3.3 Order Profiles of Intact A1B membranes (at limits of growth) ^ 49 3.4 Order profiles of A. laidlawii membranes and liposomes (grown with Myristic  Acid)  51  3.5 Order Profiles for liposomes derived from A. laidlawii membranes.  53  3.6 Order parameter profiles of A. laidlawii isolated lipid dispersions 55 4.1 2 H NMR spectra for Intact Membranes and Derived Liposomes of A.  laidlawii. ^  66 viii  4.2 Order Profiles for Intact Membranes and Derived Liposomes of A. laidlawii. 68 4.3 2 H NMR spectra for Intact Membranes of A. laidlawii at different temperatures ^  70  4.4 DSC tracings of Intact A. laidlawii membranes with and without Cholesterol. ^  72  4.5 2 11 NMR spectra of 2,2,3,4,6-d5-Cholesterol in A. laidlawii membranes. ^ 74 4.6 2 H NMR spectra and FID of Solid Cholesterol alone and in A. laidlawii membranes ^  76  5.1 Relaxation spectrum of a-DPPC-d2 ^  90  5.2 Relaxation and Pake-doublet spectra of POPCd 31  92  ^  5.3 Anisotropy of relaxation from oriented POPCd 31 and POPC:POPEd 31  ^ 94  5.4 Relaxation spectra of B2, B2 + Leul6 and B2 + Leu24 ^ 99 5.5 Relaxation spectrum of A. laidlawii membranes and powder pattern for reference ^  100  ix  List of Abbreviations  B.1 ALB^Acholeplasma laidlawii strain B ^  127  B.2 DGDG^diglucosyl diacylglycerol ^  127  B.3 DMPC^1-0, 2-0 dimyristoyl phosphatidylcholine ^ 128 B.4 DPPC^1-0, 2-0 dipalmitoyl phosphatidylcholine ^ 128 B.5 DSC^differential scanning calorimetry ^  128  B.6 DSPC^1-0, 2-0 distearoyl phosphatidylcholine ^ 128 B.7 FID^free induction decay ^  128  B.8 FTIR^Fourier transform infrared spectroscopy ^ 128 B.9 GLX^glycolipid-X ^  128  B.10 GPDGDG^glycero-phosphoryl-diglucosyl diacylglycerol ^ 128 B.11 Leul6^Lys2-Gly-Leum-Lys2-Ala- Amide ^  128  B.12 Leu24^Lys2-Gly-Leu24-Lys2-Ala- Amide ^  128  B.13 MGDG^monoglucosyl diacylglycerol ^  128  B.14 MLV^Multilamellar Vesicle ^  128  B.15 0-APG^0-amino acyl phosphatidyl glycerol ^ 128 B.16 NMR^nuclear magnetic resonance ^  128  B.17 PC^phosphatidylcholine ^  128  B.18 PE^phosphatidylethanolamine ^  128  B.19 PEG^Polyethylene glycol 8000 ^  129  B.20 PG^phosphatidyl glycerol ^  129  B.21 POPC^1-0-palmitoyl 2-0-oleoyl phosphatidylcholine ^ 129 B.22 POPE^1-0-palmitoyl 2-O-oleoyl phosphatidylethanolamine^129  B.23 R 1^Longitudinal relaxation rate ^  129  B.24 R2^Transverse relaxation rate ^  129  B.25 T 1^Longitudinal relaxation time ^  129  B.26 T2^Transverse relaxation time ^  129  B.27 TLC^thin layer chromatography ^  129  B.28 TR^repetition time ^  129  xi  Acknowledgement  With great pleasure I Thank: My supervisor, Pieter Cullis for giving me the opportunity to work with such a great group of people and for stimulating discussions during the past four years. Your contribution to Canadian Industry has also been much appreciated. Myer Bloom, for all of the discussions, for access to Room 100 and for enriching my knowledge of NMR. The shady addition for lunch was always a treat. All Room 100 inhabitants: Cyndy, Irene, Frank L., Francois, Alex, Clare, Frank N., Teresa, Michel R. and Jenifer T. for all your help and friendship... Cullis lab inhabitants: Austin, Nancy, Archie, Ajoy, Simon, Dave F., Richard , Troy, Sandy, Paul, Michel L., Nellie, Tom, Barb, Mike, Wendy, Sean, Jeff V., Jeff W., Conrad, Shane, Kim, Steven, Lewis, Laval, Neil and Mick, for hikes, chats, help, advice (I hope I have named all of you). The McElhaney lab: Ron for many contributions to this work, Ruthven for all of the A. laidlawii preparations and Dave Mannock for helpful discussions. The Evan Evans lab: Dave K. for great cappucinos and the occasional Frap experiment, Andrew, Tony and Wieslawa, who are all great mechanics. My family for their love and support over the years. Dedication: Finally, I Dedicate this thesis to two important people in my life: To the memory of Eva Princz, with whom I began graduate work and who was a great friend. To Taun Chapman, my  friend and partner in life, for all his love and encouragement. xii  Chapter 1  Introduction  1.1 Membrane Structure The lipid bilayer of cell membranes is a fundamental molecular assembly in biology due primarily to its role in the maintenance of the functional integrity of the cell or organelle it encloses. While prokaryotic species have a single membrane bilayer which surrounds the entire organism, eukaryotic cells have several membranes. In addition to the plasma membrane surrounding the cell, internal membranes surround the organelles (units of cellular machinery) such as the mitochondrial membrane, nuclear envelope, the endoplasmic reticulum and golgi apparatus. The basic composition of the membrane is particular to the cell or organelle enclosed. The description of the lipid structure in membranes as a bilayer was originally proposed by Gorter and Grendel, 1925 and was further detailed by Danielli and Dayson, 1935. The ability of the lipids to diffuse laterally within the plane of the bilayer was incorporated in the "Fluid Mosaic Model" of Singer and Nicolson, 1972. A depiction of a two dimensional sea of lipids in which proteins are embedded, has been significantly enhanced to depict a many-component system (Figure 1.1). Many different species of lipid, protein, sterol and carbohydrate components comprise the membranes of cells or organelles with various functions and  1  Chapter 1. Introduction  ^  Figure 1.1: A two dimensional model of a membrane bilayer containing proteins, lipids, cholesterol and glycosylated proteins as depicted. Interactions with cytoskeletal proteins are also indicated (Reproduced from Bloom et al., 1991).  2  Chapter 1. Introduction^  3  interact with the internal and external environments of the cell. Membranes with specific functions include, for example, the myelin sheath which encases nervous tissue and which acts as an insulator to allow for the rapid passage of uninterrupted electrical signals down the length of the nerve axon. The selective permeability of the plasma membrane to large and small molecules, which largely maintains their intracellular and extracellular concentrations, is one of the major membrane functions. Part of the diversity in membrane lipid composition may exist to achieve this function of selective permeability while at the same time satisfying packing requirements around integral membrane proteins. The glycerol-based, glycerophospholipids and sphingosine-based, sphingolipids make up the largest structural lipid groups of eukaryotic (animal) membranes with the glyceroglycolipids and sulfolipids, in relatively small amounts, making up the rest. As a percentage of all living cells including the plant kingdom, the glyceroglycolipids are by far the largest membrane structural component (Gurr and Harwood, 1992). It is noteworthy that the glyceroglycolipids contribute the major class of lipids in the mycoplasma species of bacteria. These will be discussed in greater detail in the appropriate sections. The basic structures of some types of these glycerol-based lipids are shown in Figure 1.2. Only glycerolipids have been used in the work presented in this thesis. The amphipathic character, hydrophilic headgroup and hydrophobic hydrocarbon chains, as portrayed in the Figure, promotes lipid assembly into a bilayer structure. The hydrophobic portions are sequestered away from the hydrophilic extracellular and intracellular spaces forming an energetically favorable structure (free energy is minimized) (see Figure 1.2D). The structures of both the chain and headgroup components, along with cholesterol and protein, contribute to membrane function. In the case of glycerolipids, one usually finds an unsaturated fatty acid esterified to the sn2 position of the glycerol backbone and a saturated fatty acid esterified to the snl position (Cullis  ^ ^  4  Chapter 1. Introduction^  A  HO  H — —H  H^ 1  B  cH3 1,  H 3 C — NI — CH I  CH 2  —CH2OH^  HO— C 0^ 0—^ \^ CH 2^  H^ I^I  COO I ±  C  3  HC— NH  CH2^  0^ I^ 0 = P —0—^0  0 I  =  I 0 I^  CH 2^ H^  3  CH  H  P —0— I 0 I CH2  I ^1 H — C — H —^ / C — C — O \ C— 0 0 -0^ /^I^ \^ °^ H^ I \^ /^ 0^ C=0^0^ H / C=0 H^ C=0 H2C \^ \^ / \ / 0=0 H2C /CH2^C=0 H2C^ /^ /^\ H2^ C CH 2^ ,CH 2 \ H2C \^H2C^ / H2C /CH2^ \^H2C CH2 CH2 ^\^H2C / CH 2^\ H2C CH 2^ \ CH2 \ H2C^ / CH 2 H2C^ H2C\^/^ \ H2C\ 2 H2C /\ CH /CH2 / CH 2 H 2 C\ ^ H2C\^ /CH2^ H2C—... s H2O^ H2C H2C / CH 2^x•C''^ \^ H0 2--^ ^ H C \^ /CH2^ .C.,^ \\ H2C ^ / CH Crs>'^ 2^ ) CH2 /CH2^ /^H2C\^ C..>>' H2C\ ^H2C CH 2^\ CH H2C^ /^ / / 2^ H2C^ / CH 2 \^ H2C^H2C \ CH2^ / CH 2 H2C \^\^\^H2C\ / CH /CH2^ / CH2 2^ /^ H2C^ H2C ^ CH2 / \ H 2 C \^ H2C / CH 2^/CH2 H 2C \^ \^H2C\ H 2 C\ CH CH^ / 2^/2 H2C \^H2O^ /CH2 CH 2^\ CH 2 H2C\^ H2C\^H2C \ CH /---"2^ 2^ CH2 H2C H --/ C — C— 0\=-  H  3  C^ H2C\  C H2 ^H C CH3 3^  ^/^\ CH 2 z C^ 3 H C \CH3 H 3  H2O /  D hydrocarbon chains  Figure 1.2: A. 1-0-Palmitoyl, 2-0-01eoy1-3-0-(a-D- glucopyranosyl)- snglycerol B. 1- 0-Palmitoyl, 2- 0- Oleoyl Phosphatidylcholine and C. 1-0-Palmitoyl, 2-0- Stearoyl Phosphatidylserine are used to illustrate phospho- and glycoglycerolipid classes which may be found in various biological membranes in D. is represented a lipid membrane segment where the hydrocarbon chains are sequestered toward the center of the bilayer and the headgroups are in contact with the aqueous phase.  Chapter 1. Introduction^  5  and Hope, 1984). This is depicted in Figure 1.2. In the case of the erythrocyte membrane, the most well studied species, it is usual to find phosphatidylserine and phosphatidylethanolamine containing the most unsaturated fatty acids (Vance and Vance, 1984) where other lipids would be more saturated. The effects of increasing unsaturation in the hydrocarbon region in the absence of other factors is to maintain the bilayer in a fluid state, where more saturated membranes are less flexible or gel-like since they tend to be more tightly packed (see section 1.4 for a description of gel and fluid states). The headgroup imparts similar properties to the bilayer. The relative size of the headgroup appears to play a major role. For instance, under similar conditions of acyl chain composition, ionic strength, pH and temperature phosphatidyl-choline (PC) membranes are more fluid (see section 1.4) than are phosphatidyl-ethanolamine (PE) membranes (Cullis and Hope, 1984). In addition, the PC headgroups are generally larger than are those of PE, under these similar conditions. A smaller size allows for tighter packing of the PE molecules in the membrane. A recent example of the influence of the headgroup on the physical properties of the membrane, measured by 2 H NMR order parameter methods, is found in POPC d31 and POPE d31 model membranes. All other factors of temperature, pH and ionic strength being equal, membranes of POPEd 31 are more ordered than are POPC d31 membranes. This implies a tighter packing of acyl chains in bilayers with the PE headgroup (Lafleur et al. 1990a). The concept of membrane order is discussed in section 1.7 of the introduction. At this point we draw a parallel between the degree of order and the degree of acyl-chain packing in the hydrocarbon region of a membrane. In a new study using skin model membranes (cholesterol:palmitic acid:sphingomyelin 1:1:1 and cholesterol:palmitic acid:ceramide 1:1:1) the influence of the headgroup is suggested to manifest itself in a similar way. The major difference between the outer layer of the skin, the stratum corneum or barrier, and the underlying dermal layers is the  Chapter 1. Introduction^  6  presence of ceramide in the barrier derived, due to the action of sphingomyelinase, from sphingomyelin in the dermis. The effect is to reduce the size of the lipid headgroup. It has been found (Thewalt et al., 1992) that the ceramide is much more solid-like than is sphingomyelin which exhibits liquid-crystalline like properties in 2 H NMR measurements and thus ceramide would be expected to contribute to a more impermeable material. Good reviews of the general physical properties of lipid species can be found (Vance and Vance 1984; Marsh, 1990).  1.2 Proteins Membrane proteins often make up the largest membrane component by weight ( 50% or more in some membranes). The fluid mosaic model of Singer and Nicolson, 1972 described the lipid membrane as a fluid sea in which membrane proteins can carry out their functions. A common theme emphasized throughout this thesis is the functional role of membrane lipids in these interactions. Two main classes of membrane proteins are usually found and are identified by their ease of separation from the lipid bilayer. Peripheral proteins, such as myelin basic protein from the myelin sheath, cyto chrome C from the inner mitochondrial membrane and spectrin of the red blood cell inner monolayer, are to a large extent electrostatically associated with the membrane surface and may be extracted using high salt solutions. The main interactions of peripheral proteins with lipids take place at the interface (in the headgroup region) perhaps mediated by cations such as Ca 2 +. It is surmised that some peripheral proteins (myelin basic protein, for example) may indeed have hydrophobic interactions with the bilayer because of the presence of stretches of hydrophobic amino acids which could penetrate into its hydrophobic core. The second class of membrane proteins, the integral proteins, require detergent  Chapter 1. Introduction^  7  disruption of the hydrophobic interactions with the surrounding membrane lipids to achieve extraction (Vance and Vance, 1984). The tertiary structures of some integral proteins are well known. A group of these including rhodopsin have several transmembrane helices which interact hydrophobically with the lipid matrix, while the cytoplasmic and extracellular domains are more hydrophilic. Many transmembrane transporters, such as the passive sugar transport system in erythrocytes have a well defined transmembrane domain whose function depends on lipid structure. In particular, it has been noted that a characteristic length of hydrocarbon chains is necessary to achieve the correct arrangement of functional domains of the protein across the bilayer for optimal protein function (Gurr and Harwood, 1992). Similar correlations between lipid hydrocarbon length and protein activity have been observed in reconstitution studies of Na+-Mg 2 + ATPase extracted from A. laidlawii B (Silvius and McElhaney, 1980). Since most integral membrane proteins contain transmembrane regions these observations are not surprising. However, most natural membranes contain many different lipid types and cholesterol; thus the important physical properties of lipid molecules interacting with proteins to maintain functional integrity are not immediately obvious. This topic will be addressed further in Chapter 5.  1.3 Cholesterol 0-0H Cholesterol, the most common sterol form found in membrane lamellae, is present in the plasma membranes of eukaryotic cells. Some prokaryotes such as mycoplasma capricolum require sterol for growth and survival but characteristically are incapable of synthesizing it and must obtain it from a host organism. In eukaryotes, cholesterol is primarily found in the plasma membranes of cells and lacking , for the most part, in their organelles. The reason for this remains unknown. However, since cholesterol  Chapter 1. Introduction^  8  imparts mechanical strength to a lipid membrane (Evans and Needham, 1987), increased durability of the plasma membrane compared to the internal membranes may be a factor. The interest here in membrane cholesterol is largely due to the physical properties it imparts to the overall structure. Some most striking physical properties of cholesterol have come from differential scanning calorimetric measurements in which the main lipid melting phase transition, gel-to-liquid crystalline, decreases in membranes containing up to  ti  30% cholesterol and is abolished above  ti  30% (see Fig-  ure 1.3A). Similar studies encouraged the production of a phase diagram based on 2 11 NMR measurements of DPPC/Cholesterol model membranes, (Vist and Davis, 1991). A remarkable feature of these systems is the presence of a "Liquid Ordered" phase characterized by a fluid-like 2 11 NMR spectrum (see section 1.4 and Figure 1.10 for a description of "Liquid Ordered" and an example of a fluid 2 11 NMR spectrum, respectively). Other descriptions of the effects of cholesterol on the lipid bilayer, based on observations from 2 11 NMR studies, suggested that cholesterol disorders the gel phase and orders the fluid phase of a membrane. Thus the sterol functions as an "alloying" agent in the membrane, straightening out (orientationallly ordering) the acyl chains, reducing the average lipid cross-sectional area and thickening the bilayer (Bloom et al., 1991). It has been postulated that the Q hydroxyl group of cholesterol hydrogen bonds with the lipid phosphate group which positiOns the lipid in the bilayer environment (Boggs, 1987) but supporting evidence for this mechanism is yet to be found. The effects on biological function appear as a reduction in the passive bilayer permeability (10 fold for water through membranes containing 30% cholesterol, Koenig et al., 1992) while maintaining the fluid characteristics of the bilayer. In addition, it has been suggested that cholesterol played an important role in the evolution of membranes. This is supported by the observation in precursor sterols of the prokaryotes that the oxygen-requiring enzymatic step to convert the precursor to  9  Chapter 1. Introduction^  A  Endothermic  Pure DPPC  + 5 mol% cholesterol  + 12.5 mol% cholesterol + 20 mol% cholesterol + 32 molo/o cholesterol + 50 mol% cholesterol 290^320^350 Temperature (K)  B  ^Mi0— cellar Or-r,-(r0 0  ,f  4  Bilayer  11 11 Figure 1.3: A) Calorimetric tracings of DPPC dispersions (fully hydrated) with and without cholesterol. B) Lamellar and non-lamellar lipid structures, bilayer, micellar and inverted hexagonal.  Chapter 1. Introduction  ^  10  cholesterol is lacking (Bloch, 1976).  1.4 Lipid Phases Some of the concepts introduced earlier are addressed in this section. As already suggested, membranes can exist in a less-mobile gel-state where the main motions present are a type of axial diffusion of the lipid molecules, or in a fluid liquid-crystalline state where rapid lateral diffusion of the lipid molecules occurs and the lipid acyl chains can undergo fluctuations about individual carbon-carbon segments. These latter motions are often referred to as trans-gauche isomerizations (see Figure 1.7). The particular state depends on temperature, and transitions between these states as a function of temperature can be monitored by various techniques. Those of interest here are 2 H NMR and, probably the most direct method, differential scanning calorimetry (DSC). An example of the calorimetric behaviour of dipalmitoyl -phosphatidylcholine (DPPC) is given in Figure 1.3A. We identify certain useful features of the tracings, namely: the area under the curve (transition enthalpy), the width of the transition at half height (cooperativity) and the transition midpoint (T m ). The transition enthalpy relates to the number of lipid molecules taking part in the transition while the cooperativity reflects the number of molecules undergoing a transition simultaneously. Cholesterol is known to abolish this phase transition, depending on the concentration; however, in the presence of cholesterol, the lipid fatty acyl chains are considered to be highly orientationally "ordered" yet the lipid molecules retain fluid-like motions and thus the "liquid" state of the membrane is maintained. The term "Liquid Ordered" was used to describe these characteristics (Ipsen et al., 1990). Lipid structures (or phases) other than bilayer have been identified for certain lipid  Chapter 1. Introduction^  11  types and are often exhibited at physiological or higher temperatures for dispersions of these single lipid species. The common non-bilayer phases are the inverted hexagonal, micellar and cubic phases. Some of these non-bilayer structures are depicted in Figure 1.3B and their significance is discussed in sections 1.5 and 1.6.  1.5 Lipid Polymorphism The large diversity of lipid types, their structural preferences and their anisotropic distribution across the biological membrane bilayer are subjects of ongoing interest.  The amounts of various lipid types are depicted in Figure 1.4 for the phospholipids phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI); Cholesterol (Chol) and Sphingomyelin(SM) found in the human erythrocyte. Since differences in lipid characteristics are not necessarily observed in an intact biological membrane, it has been general practice to study lipid assemblies of one or two components and derive conclusions based on results obtained from  these simpler systems. An examination of the non-bilayer phase preferences of individual component lipid membranes, commonly referred to as lipid polymorphism, has revealed certain general characteristics of the individual lipid species. Specifically, unsaturated lipids having small, relatively poorly hydrated headgroups such as dioleoyl-phosphatidylethanolamine (DOPE), in isolation, adopt inverted hexagonal structures at temperatures well below physiological (7-12°C for DOPE, Rilfors et  al., 1984). This has been attributed to a cross-sectional area of the lipid headgroup that is smaller than the cross-sectional area of the lipid acyl chains. In general, it is found that lipids which tend to form lamellar structures have well-matched headgroup and acyl chain cross-sectional areas and those which tend to form micelles have a larger headgroup area than acyl chain area. These observations are quantitatively predicted  12  Chapter 1. Introduction^  LIPID TYPE  Percent^of^Total^Lipid  CHOL  23  PE  18  PC  17  SM  18  GL  7  Others  13  Figure 1.4: Phosphatidyl choline (PC), Phosphatidyl ethanolamine (PE) Phosphatidyl serine (PS), Cholesterol (Chol), Glycolipid (GL), Sphingomyelin (SM) and Others are expressed as weight% of total lipid in the erythrocyte membranes (data are from Vance and Vance, 1984).  Chapter 1. Introduction^  13  by a geometrical packing parameter that was first used by Israelachvili and coworkers (1980). The parameter is defined as P=v(a./) -1 where v is the hydrophobic volume, a is the cross-sectional area occupied by the polar headgroup region at the water-membrane interface and l is the hydrophobic length of the acyl chains. Aggregates with values of P < 1 /2 were predicted to form micellar structures. For cylindrical molecules with values of P ',----- 1, bilayers are formed and those molecules with P > 1 give rise to reversed aggregate structures where the polar headgroups are oriented towards the interior aqueous space. A summary of the predicted shapes based on the packing parameter is given in Figure 1.5. These are correlated with typical 2 11 NMR lineshapes. We note that several molecular lipid species possess shapes which promote non -bilayer structures. An extensive treatise of the factors which influence these can be found in Rilfors et al., (1984) and Cullis and deKruijff, (1979). Some of these non-bilayer structures are of immediate interest. To reiterate, lipid species which have a large hydrophobic volume compared to a.1, when dispersed in water, can form inverse hexagonal structures. These formations are highly dependent upon external factors of temperature, pH, ion concentration and the presence of other molecules such as cholesterol and some proteins. Under physiological conditions, these factors become important in determining the phase behaviour of the lipid species. Polymorphism of various synthetic and naturally occuring phospholipids and glycolipids have been investigated using standard methods of X-ray diffraction,  31 P  NMR and 2 11 NMR and freeze-fracture EM (Cullis and deKruijff, 1979, Gruner et al., 1985; Lafleur et al., 1990a;b;c, Sternin et al., 1988; Rilfors et al., 1984). Table 1.1 exhibits polymorphic phase preferences of some lipids from eukaryotes. It is interesting that a significant number of these form thermodynamically stable inverted hexagonal phases. None of these inverted assemblies have been observed in biological  14  Chapter 1. Introduction^  2 H NMR spectra^Phospholipid Phases  Packing Parameter (V/0.1)  Lamellar 2  ...-------' Inverted^Hexagonal  >  1  Isotropic 1.^Vesicles <  1  2.^Inverted  2^  3.^Micelles  micelles  4.^Cubic  Figure 1.5: Typical 2 11 NMR spectra observed for the phases indicated and with the indicated packing parameters. Spectra are from (top) POPE d31 multilamellar vesicles measured at 50°C (middle) POPE d31 dispersions measured at 75°C and (bottom) 100nm vesicles of DOPC initially dispersed with 20% perdeuteriated palmitic acid . The values of the packing parameter are taken from Israelachvili et al., 1980.  Chapter I. Introduction^  15  Table 1.1: Polymorphic Preferences of Membrane Lipids from l ukaryotes. Hexagonal Bilayer^ PC SPM PE PS(pH < 3) PS PG PI PA(d-Ca2+) PA PA(pH < 3) CL(H-Ca2+) CL (data from Vance and Vance, 1984)  systems since the integrity of the bilayer would be compromised. However, in some instances, the appearance of an isotropic line in '1 3 NMR spectra have been attributed to similar formations. It is suggested that these inverted structures play a role in events such as membrane fusion, exocytosis and endocytosis, an idea currently maintained. Indeed Siegel et al., (1989) provided theoretical evidence to suggest that the half-lives of "inverted micellar intermediate" structures are short, in the us to ms range which are too short to detect by NMR methods. Polymorphism is also exhibited by the glyceroglycolipids which is of particular relevance to this thesis. It has been suggested in the A. laidlawii strain A microorganism that the presence of MGDG, an R H forming lipid, balanced in concentration by the bilayer forming, DGDG, PG, cardiolipin and others, provides a regulatory mechanism for maintaining the bilayer formation under growth conditions which would favor 1-I II or micellar formations (Eriksson et al., 1991; Wieslander et al., 1980). For instance, the introduction of cholesterol or unsaturated fatty acids, or growth at an elevated temperature would favor a reduction in the molar ratio MGDG/DGDG. This is because  Chapter I. Introduction^  16  cholesterol is considered to be an 11 .11 phase promoter and unsaturated fatty acids increase the cross-sectional area of the lipid acyl chains preferentially over that of the headgroup as does an increase in temperature. Although this is true to some extent in the evolutionarily related species A. laidlawii strain B, it is emphasized that, for this organism, the maintenance of a fluid lipid bilayer at the growth temperature is the primary reason for altering such ratios (see McElhaney, 1984 for a review). Thus the environmental adaptation is facilitated by the use of H n- or micellar forming lipids. Extensive reviews in the area of lipid polymorphism are presented in Rilfors et al., 1984 and Vance and Vance, 1984.  1.6 The Acholeplasma laidlawii Membrane Although much work has gone into investigating the physical properties of the membrane bilayer, relatively few studies have been carried out on biological membranes mostly due to difficulties in their isolation and/or manipulation. Fortunately, the microorganism Acholeplasma laidlawii is an exception. It provided the biological membrane system for much of the work presented in this thesis for two reasons. First, several members of the mycoplasmas, a cell-wall-less group of prokaryotic microorganisms to which A. laidlawii belongs, have the unique feature of being fatty acid auxotrophic. This means that it is possible to achieve a desired membrane lipid fatty acid composition through addition of the fatty acids to the growth medium while suppressing de novo fatty acid biosynthesis. As is discussed in Chapter 3, this property was exploited in determining the range of membrane order, measured by 2 H NMR order parameter methods, in which the microorganism is seen to maintain good growth characteristics. Second, since the microorganism lacks a cell wall, unlike most prokaryotic cells, the membranes can be isolated by gentle lysis procedures and are easily obtained in large  Chapter 1. Introduction^  17  quantities. This is useful in 2 1-1 NMR studies due to the low sensitivity of the deuterium nucleus. A typical A. laidlawii membrane contains (by weight) 55-65% protein, 30-35% lipid and 10-15% carbohydrate as the major constituents, although the relative levels of these can be varied to some extent (McElhaney, 1984). In addition, low levels (< 5%) of carotenoids, free fatty acids and diacylglycerols are usually found. The predominant lipids found in the membranes of the A. laidlawii B strain are the glycolipids, monoglucosyl diacylglycerol (MGDG) and diglucosyl diacylglycerol (DGDG), and (usually to a lesser extent) phosphatidylglycerol (PG). Membrane lipids usually present in lesser quantities are glycerophosphoryl diglucosyl diacylglycerol (GPDGDG) and an 0amino acid linked PG. It is pertinent to mention another lipid species usually present in trace quantities but which can be found in large amounts under particular conditions of growth (Bhakoo et al., 1987) and which is referred to as G1X in this thesis. The stuctures of these various lipid species are presented in Figure 1.6. Depending on the fatty acid composition, MGDG has been found to form predominantly inverted hexagonal phases at temperatures around 37°C (Wieslander et al., 1978; 1981), whereas DGDG and PG prefer bilayer structures. Recently, it has been found using 2 H NMR methods that GPDGDG dispersions, which are optically clear , form some sort of "isotropic" (micellar) phase in isolation. The significance of the presence of non-bilayer forming lipids in these membranes will be introduced in the next section and discussed further in Chapter 3. Fatty acid compositions which support growth of the microorganism under conditions where de novo fatty acid biosynthesis is suppressed cover a fairly wide range from the relatively short, 14 carbon chains to the relatively long 18 carbon chains. Probably the major restriction on fatty acids which will support growth is the maintenance of  Chapter 1. Introduction^  B A) CH 2 OH /1-0 \ /OH \ N^1  OH 0 H  18  CH2OH )  1-- 0 CH2OH /oH \  L_0^N^/ 0 - CH 2 /oH \ N^A OH 0 CH — R  OH 0H  0-CH 2  CH 2^ R  CH ^ R 1 CH 2^ R^o  E) CH 2 - CH - CH 2 -O -0 ^o (bli OH ^ CH /I- 0 CH  /H o  0  /OH \I  0 - CH 2  OH 0  OH OH C) D) o -0 I^ I o=p o -CH-2^ CH-CH 1^1 2 0 --p-0 -CH- CH CH 2 I^ I ^2^I OH OH CH2^ CH2^OH -  -  CH — R^CH — R I^ I CH-- R^C14- R  CH ^ R CH 2^ R  -  NH 3 +  Figure 1.6: A) Monoglucosyl diacylglycerol (MGDG), B) Diglucosyldiacylglycerol (DGDG), C) Phosphatidyl glycerol (PG), D) Glycerophosphoryl diglucosyl diacylglycerol (GPDGDG), and E) 0-amino acid linked phosphatidyl glycerol (OAPG) (see McElhaney, 1984).  19  Chapter 1. Introduction^  a fluid membrane at the growth temperature. Other aspects pertaining to the physical properties of this biological membrane will be discussed further in Chapters 3 and 4. Excellent reviews concerning biophysical studies of this membrane can be found in McElhaney, 1984; 1989.  1.7 Membrane Hydrocarbon Order A variety of methods have been used to measure orientational order parameters in model and biological membranes, the values of which depend on the technique employed. These include ESR, fluorescence anisotropy, 2 11  19 F-NMR  and 2 H-NMR methods.  NMR methods have been identified as being particularly useful in the measurement  of hydrocarbon order parameters in both model and biological membrane systems (see Seelig, 1977 and Davis, 1983 for reviews). A deuterium NMR spectrum from a deuterium-labelled lipid membrane contains two intense peaks (see Figure 1.10) separated by a value, Avg (kHz) = Scp x 125kHz, called the quadrupolar splitting. A description of how Avg arises is given in the 2 H NMR theory section. The degree of orientational order (or organization) of the environment that a deuterium-labelled carbon atom experiences may be described by an order parameter, SCD = 112(3<COS 2 0> - 1) where we define 0 as the angle between the normal to the bilayer plane and the CD bond axis, the angular brackets denote the time average of cos 2 0. Theoretically Sc ') will vary between -1/2 and 1. However, using .  2  11 NMR (lineshape) methods we have access to the magnitude of the order parameter  and not the sign (see Ipsen et al., 1990); thus the Scp values reported hereafter in this thesis all refer to IS c p I. The higher the value of Scp for a deuteriated carbon atom on a saturated fatty acyl chain, the greater is the degree of order. In the liquid crystalline  Chapter 1. Introduction^  20  phase of lipid bilayers, the greatest degree of orientational order corresponds to the alltrans conformation. In this most ordered fluid phase IScp I = 0.5 since the molecules are rotating rapidly about the bilayer normal and the CD bond axis makes an angle of 90° with the surface normal. Conformational excitations of the type described in the next section result in a decrease of 'Sap I. It is possible to employ 2 11 NMR order parameter methods to measure the order in a membrane system of lipid molecules containing a perdeuteriated fatty acyl chain, such as POPC d31 (see Lafleur et al., 1989). One generally finds an increase in acyl chain flexibility toward the center of the bilayer which may be expressed in an order parameter profile of S c./3 (n) = S(n) measured for each position, n. Characteristically the order profile exhibits a monotonic decrease in order, with a relatively flat region for the first 8 to 10 carbon atoms termed the "plateau region" and a more rapid decrease for the remaining carbon atoms. Such features indicate that little variation in the amplitudes of motion occurs near the headgroup region and that the amplitudes of motion increase toward the center of the bilayer. The methods for deriving an order profile from a superposition of spectra due to a perdeuteriated membrane lipid sample are explicitly described in Chapter 2 and Lafleur et al., 1989. A typical profile for a membrane bilayer system is shown in Chapter 3, Figure 3.1C. It is convenient to compare order profiles using the mean order <S> which, for an acyl chain having N carbon atoms, is simply the average [E„N=2 S(n)]/(N — 1) to be described in Chapter 2. As mentioned earlier (section 1.1), membranes such as POPE d31 which exhibit higher <S> values than POPC d31 at the same temperature are considered to be more ordered (Lafleur et al., 1990b). We note that the value of <S> may be empirically related to the thickness of the bilayer hydrocarbon region as will be shown in the next section.  Chapter 1. Introduction^  21  1.8 Bilayer Thickness An operational indicator of the bilayer hydrophobic thickness is the length of the projection of the acyl chains on the bilayer normal from the carbon 2 position of the snl acyl chain on one leaflet of the membrane to the average carbon 2 position of the acyl chain on the other leaflet. Original work on the relationship between hydrocarbon order and hydrophobic thickness by 2 11 NMR methods was presented by Seelig and Seelig, 1974. A brief outline of the basis for this relationship is given here. Assuming, for a simple model bilayer of DPPC molecules, that an all-trans chain projects into the bilayer at an  A is the length of one (trans) carboncarbon segment, then the largest hydrophobic length possible would be 37.5 A. In the angle normal to the bilayer plane and that 1.25  event of chain disordering by the presence of gauche bonds, this length would decrease by an amount related to the number of chain segments, 1, 9 , whose projection is due on average to gauche conformations, given that l ig = l i cos 60° (60° is the angle of a gauche bond with respect to the bilayer normal, see Figure 1.7). The molecular order parameter S mo i = 1/2(3<cos 2 0'> - 1), where 0' is the angle between carbon-carbon segment 1 and the bilayer normal, is directly related to geometrical disordering due to gauche bonds, see Figure 1.7. If p A and pB denote the probabilities of a segment being in state A (all trans) or B (due to gauche bonds) with P A p B = 1, then the average order over the various orientations is given by S moi = 1/2(p A (3(cos 2 0) - 1) p B 3(cos 2 60 - 1)). The probability of state B is thus given by p B = (1-S mo/ )/1.125. This is related to the length, 1, of a particular chain segment, i, in the following manner: <l i > = p iA 1 p iB 1, cos (60)=1(1 - 0.5p iB ) and the total length <L> of hydrocarbon chain is given by <L>^E,_ 2 < /i >. Thus the thickness is given by 2<L>. Indeed, the thickness 'Here we consider a carbon-carbon segment to be the line joining the midpoint of two consecutive c-c bonds  Chapter 1. Introduction^  22  \ C / C \ C / C  \^\  C^C /^/ C^C  \^\ C^C /^/ C^C \^\  C^C / C  C / C  C  C  \  C- C C  C / C C  all trans tg+t jog t tg Kim<  Figure 1.7: Carbon skeletons of acyls chains depicting (far left) an all-trans conformation, (middle) the introduction of a gauche conformer in between two trans segments and (right) a kink formed by two gauche conformers separated by a trans segment.  Chapter 1. Introduction^  23  measurements obtained by Seelig and Seelig (1974) for DPPC model membranes were in good agreement with thicknesses measured by Xray reflections (Cain et al., 1974). A second theory relating order parameters to chain length (Ipsen et al., 1990) was developed in the same spirit as that of Seelig and Seelig (1974). Such measurements have proven to be useful, for instance, in explaining 2 H NMR spectra of model membranes containing non-bilayer forming lipids. The presence of the non-bilayer, 11  //  ,  forming lipid has been found to increase the membrane hydrocarbon order in binary lipid membranes of POPE:POPC and MGDG:DGDG for example (Lafleur et al., 1991; Monck et al., 1992; Eriksson et al., 1991). Thus, a closer packing of lipid molecules are observed in the presence of FI H forming lipids. Indeed, the permeability coefficient of glucose in similar lipid mixtures decreases as the molar ratio of POPE:POPC is increased (Mui and Cullis, unpublished results). Furthermore, the addition of model peptides, Leul6 and Leu24, whose hydrophobic lengths are not well matched with those of the lipid membranes in which they are dissolved, induce an increase or decrease in the average observed hydrocarbon order (Nezil and Bloom, 1991) depending on whether they have a larger or smaller thickness, respectively. Presumably, this reflects the "stretching" or "shrinking" of the acyl chains to match the hydrophobic length of the peptide. The importance of the hydrophobic thickness in integral membrane protein function has been identified (see Gurr and Harwood, 1992; Mouritsen and Sperotto, 1992 and references therein). As will be discussed in Chapter 5, questions relating to hydrocarbon order/ hydrophobic thicknesses are of current interest.  1.9 2 H NMR Theory At this time, it is useful to introduce some basic NMR theory and outline its application to lipids in membranes. The following discussion develops the theoretical basis for the  Chapter 1. Introduction^  24  NMR measurements obtained from 2 H-labelled membrane lipids. Excellent descriptions of this are given in Seelig, (1977) and Davis, (1983). The approach here follows that of Seelig, (1977). 1.9.1 Energy Transitions  The energy of a deuterium nucleus (spin I = 1) in a magnetic field, H. has two components, Zeeman or magnetic energy, Ern , and quadrupolar energy, EQ . The total energy may be written as  E = En-, + EQ  (1.1)  Em , the magnetic energy term, describes the interaction between the nuclear magnetic moment p and the large magnetic field H.,  Em = —pH. = —gONTriB". (1.2) where ON = --yh is the bohr magneton, g, the so-called nuclear g-factor and m, the spin quantum number defining 2/ + 1 energy levels. A nucleus with spin I = 1 will have m = +1, 0, -1 energy levels. Given a pure Zeeman interaction at the nucleus two transitions are observable as shown in Figure 1.8. Nuclei with spin I > 1/2 have nonspherical charge symmetry at the nucleus and, in the case of the deuterium nucleus, possess an electric quadrupole moment, Q. The interaction of Q with the electric field gradient, VE = V, at the position of the nucleus gives rise to the quadrupolar hamiltonian HQ with energy EQ . The electric field gradient is properly represented as a symmetric, traceless tensor with principal components Vex , Vyy and V. For an spa hybridized carbon nucleus the electric field is approximately axially symmetric with Yz , coincident with the CD bond axis and is defined here as, Vs , = eq. e is the charge on the electron and q is the second derivative of  Chapter I. Introduction^  A  25  m=-1 ^ _ Au m=0  E  Ay + _-  m=+1 Zeeman  Zeeman + Quadrupolar  +Ho  z  B  Figure 1.8: Depicted are A. an energy level diagram for a spin 1 nucleus perturbed by the quadrupolar interaction at the nucleus and B. the results of rotation of a single crystal by angles 0 and 0.  -  Chapter 1. Introduction^  26  the electric potential at the nucleus. Departure from axial symmetry at the nucleus is measured by an asymmetry parameter 77 defined as 77 = Vas — Vyy /Vzz . By definition Vzz > Vex > Vyy,, thus 0 < < 1. In the case of the principal axis coordinate system where V„ is always coincident with the largest field gradient, V /(;' °) = V„, yr ) = 0 and VV' 2) _/6( -G s — Vyy ) = 7-7V i( ' 0) (77 < 0.05 for a CD bond, Seelig, 1977). The Zeeman interaction at high fields (46.175 MHz for our purposes) dominates the quadrupolar interaction (;::-2, 200 kHz). One can treat the quadrupolar interaction as a perturbation, the result of which shifts the energy levels as depicted in Figure 1.8A. For a system in which VV' 2) = 0 and the magnetic field is applied along the z-axis the corresponding energy is written  Em = —gONHorri 41(2 e Q 1) V (2 '°) [3m2 — (-T + 1)] ^(1.3) 1 1) —  In the case of deuterium, I=1, the three shifted energy levels at m=-1, 0, +1 are usually expressed as 1  E+1 = —gONH. + — e QV( 2,0 ) 4 Eo =  1  2  (1.4)  eQV (2 A  (1.5)  E_i = 9/3N-H. + 4 eQV( 2,0 )  (1.6)  The selection rule Am = +1 restricts the allowed transitions to  hv+ = E_ 1 — E0 = gi3 N Ho + 43 eQV (2 ' °) hv_ = E0 — E+1 = g O N Ho — 4 eQV (2 ' °)  ^  ^  (1.7)  (1.8)  giving rise to two transition frequencies (shown in Figure 1.8A) whose separation Av  g  is known as the quadrupolar splitting 3eQ^3 e 2 qQ Avg =^= 2h V(2'°) = 2 h  (1.9)  Chapter 1. Introduction^  27  1.9.2 Powder Spectra in the Absence of Motion e 2 qQ/h is generally referred to as the static quadrupole coupling constant whose value for deuterium labelled lipid molecules is approximately 170 kHz. The Av g varies according to the angle which the CD bond makes with H. This can be best illustrated by looking at the rotation of a single crystal through the Euler angles a, 0 and -y corresponding to a = 0, = B and 'y = 0 (see Figure 1.8b) and is determined by  V( 2 '°)^E  D,20) ( 090) VIC 2 ' )^(1.10)  P=0,±1,±2  1 — V„[(3cos 2 0 — 1)]^ 2  (1.11)  for VV' °) = V„ and VV' 1) = VV' 2) = 0. lie(090) are the appropriate elements of the Wigner Rotation Matrix (Rose, 1957). Substitution of 1.11 into 1.9 shows that the quadrupolar splitting varies with 0 as follows:  Av9 (0) =  3 e 2 qQ 1 2 h 2  (3cos 2 9 — 1)^  (1.12)  The measurement of a polycrystalline sample consists of measuring a powder whose labelled sites are randomly oriented with respect to H. The probability of orientation of a site in the zone B + dB with respect to H o is given by the probability of finding in a zone on a great sphere or, P(8) = (1/2)sinO, where 0 = 90° is the most probable and 0 = 0 is the least probable orientation. The 2 11 NMR spectrum which arises from a superposition of doublets, separated by Av q (0) and weighted by P(9) is referred to as a Pake doublet (G.E. Pake, 1956) as shown in Figure 1.9.  1.9.3 The Effect of Lipid Motion Although the static 2 11 spectrum of a membrane bears similarity to that of a deuterium powder sample, the lipids do not remain motionless. Properties of multilamellar dispersions have been compared with lyotropic liquid crystals (Bloom et al., 1977) where  Chapter 1. Introduction^  Figure 1.9: Simulated deuterium NMR spectrum using line broadening, cs = 0.01. The most intense peaks are separated by 125 kHz.  28  Chapter 1. Introduction^  29  the chains can undergo angular excursions normal to the molecular long axis. This is referred to as disordering and usually occurs by fluctuations such as trans-gauche isomerizations (see Figure 1.7). A convenient measure of the degree of the fluctuations is the so-called order parameter S ti2 defined as S it = 1/2(3< cos 2 Pi > - 1) (i=1,2,3=x,y,z). The term < cos 2 A > denotes the time average of cos 2 0 . We note that since cosO, are direction cosines cos213i = 1 and = 0. In the case of an axially symmetric tensor, the z axis coincident with the symmetry axis, it is evident that  S33 = -2S11 =  -2S 22 . The quadrupolar splitting measurement of a polycrystalline powder was given by equation 1.12 above. In a membrane, if we allow for disordering of the chains, then the electric field gradient tensor is time averaged and the quadrupolar splitting measurement takes the following form v  S33  3 e 2 qQ 1^1 (3 < cos 2 ,3 > —1)- (3cos 2 0 — 1) 2 h 2 2 3 e2q Q S33 1 (3CO3 2 0 = 1) 2 2 h  (1.13) (1.14)  is usually referred to as Sap (the bond order parameter) and is measured at 9 =  90°. It is equation 1.14 which describes the measurements that will be presented in this thesis.  1.10 DePakeing The complexity of overlapping broadline 2 H NMR spectra makes it difficult to extract pertinent data (S c p for example) and derive useful information from the individual powder patterns. Thus the technique of dePakeing was developed (Bloom et al., 1981; Sternin et al., 1983). It can be described as a numerical procedure to deconvolute a Pake-doublet spectrum into its oriented counterpart (depicted in Figure 1.10) where  Chapter 1. Introduction  ^  I^ I^ 1 —20^—10^0^10 Frequency (kHz)  Figure 1.10: 2 H NMR spectra of A. multilamellar vesicles and B. oriented multibilayers of DPPC deuterium labelled in the /3 position of the headgroup. (Spectra are courtesy of C. Morrison).  30  20  Chapter 1. Introduction^  31  the 0° oriented spectrum results. As stated in the last section the orientation refers to the angle between the symmetry axis and H. Since the oriented, F o (x), and Pake doublet, G(w), spectra are related to one another it is possible to derive an oriented spectrum from the corresponding Pake doublet. The dePakeing procedure is described in detail in Sternin et al., (1983). An outline of "how it works" is presented to give the reader an idea of the overall method. The dePakeing procedure of Sternin et al., (1983) is an iterative method involving the calculation of F o (x) from G(w). The spectrum G(w) is stored in a digital form containing N points where G(+ n/N) denotes the spectrum at a given point and F o (x) is replaced by its discrete representation F o (N). For a given spectrum, such as that in Figure 1.10A, one half of the oriented spectrum is first calculated as though each point contributes to an edge (90° orientation). This is an over- estimation of the contribution since intensity is derived from the shoulder (0° orientation), which is of lower intensity, as well as the edge, which contributes greater intensity. The second iteration on the other half of the spectrum under-estimates the contributions since equal edge and shoulder contributions are considered. Since the estimation of F o (N) is improved with each iteration, the spectrum eventually converges to a good estimate of F o (N). The algorithm was derived in this way to enable depakeing of both symmetric (e.g. deuterium) and asymmetric (e.g. phosphorous) spectra. A specific case of the use of dePakeing is shown in Figure 1.11. The spectrum of a sample of POPC d31 multibilayers is presented along with its dePaked counterpart. One can see that the spectrum is much simplified by the dePakeing procedure since it is possible to measure resonance frequencies contributed by a single CD 2 (CD 3 ) group. In general, one can obtain a reasonable dePaked spectrum from a powder spectrum showing a v cx P 2 cos(/3) dependence and where axially symmetric motion is observed. In the case of fluid deuteriated membranes, the procedure works very well in determining  Chapter 1. Introduction  —60  ^  I^I^I^1 —40^—20^0^20 Frequency (kHz)  32  40  Figure 1.11: A. Deuterium NMR spectrum of POPCd31 multilamellar vesicles and B. its dePaked counterpart.  60  Chapter 1. Introduction^  33  the oriented spectrum, as given in figure 1.11 for 0 = 0. As will be shown, the dePakeing procedure has been of great use in the derivation of order parameters in biological membrane systems.  1.11 Motivation and Thesis Outline An understanding of fundamental properties measurable by 2 1--I NMR lineshape and relaxation methods which relate to the hydrocarbon order of the lipid membrane in biological and model membranes are the major goals of this work. The Acholeplasma  laidlawii strain B membrane is an excellent biological membrane system for study due to its fatty acid auxotrophic character. As indicated earlier this means that the desired composition of the A. laidlawii membrane can be controlled to a large extent. Questions which were immediately of interest related to the range of hydrocarbon order/hydrophobic thickness in which the microorganism remains viable. The first  2 1-1  NMR studies of A. laidlawii membranes investigated the fluid character of the membrane (Oldfield et al., 1972, 1976), the similarity of order in A. laidlawii to other biological membrane systems (Kang et al., 1981), similarity to model membrane systems, (Stockton et al., 1977; Rance et al., 1982) and the effects of cholesterol on membrane order (Davis et al., 1980), a comparison of the orientational order in whole membranes and dispersions of A. laidlawii membrane lipids (Jarrell et al., 1982), or the T2 relaxation behaviour of whole membranes (Rance, 1980), and did not focus on the viability of the species in particular. In addition, relatively little information is known about the motions of membranes with thicknesses which vary spatially and/or temporally. Thus it was of interest to study such membranes using 2 11 NMR transverse (T 2 ) relaxation methods which are sensitive to slow motions and would be affected by these variations in thickness. Up to  Chapter 1. Introduction^  34  now few 2 I-I NMR investigations of relatively slow motions occurring in membranes have been carried out. This is probably due to the complexity of motional modes occurring on this timescale, which are poorly understood from a theoretical standpoint. In Chapter 2, common methods used in the work presented in Chapters 3 and 4 are outlined. The methods given in Chapter 5 pertain only to Chapter 5 studies. The influence of fatty acids on the range of order observed in A. laidlawii B membranes is discussed in Chapter 3. This chapter closely follows the paper by Monck et al., 1992 titled "Influence of Lipid Composition on the Orientational Order in Acholeplasma laidlawii Strain B membrane: A Deuterium NMR Study". Presented in Chapter 4 is an investigation of the extent of cholesterol incorporation and the phase state of the bulk of the cholesterol seen by 211 NMR methods. Suggestions for the location of the cholesterol in the A. laidlawii B membrane are discussed. This chapter closely follows the paper by Monck et al., 1993 titled "Evidence for Two Pools of Cholesterol in the Acholeplasma laidlawii Strain B Membrane: A Deuterium NMR and DSC Study". The angular dependence of transverse relaxation times in 2 11 NMR membrane measurements can be shown to be influenced by fluctuations in the thickness of the membrane bilayer in several different membrane systems. This type of motion shows very different transverse relaxation anisotropy from that due to fluctuations in the lipid molecular long axis. Such differences are described in Chapter 5 and suggestions concerning a phenomenological theory of thickness fluctuations and surface undulations are discussed. The "Future Directions" (Chapter 6) section briefly describes further experiments which could be used to answer specific questions on membrane systems such as those presented in the thesis.  Chapter 2  Materials and Methods  The fatty acids used in these studies were obtained from Nuchek Prep Inc. (Elysian, Minnesota). Palmitic acid was perdeuteriated using the methodology of Hsiao et al. (1974), purified by column chromatography and recrystallization from ethanol.  2.1 Isolation of A. laidlawii Membranes The organism A. laidlawii B was cultured in the presence of the desired exogenously supplied fatty acids and cholesterol (where applicable) under conditions where endogenous fatty acid biosynthesis and exogenous fatty acid chain elongation have been inhibited by the inclusion of avidin in the growth medium (see Silvius and McElhaney, 1978). For all cultures in which either fatty acids only or cholesterol and fatty acids were added to the growth medium, they were presented in the form of a mixed micelle in a small amount of ethanol. The membranes were isolated by differential centrifugation after cell lysis by osmotic shock (see Silvius and McElhaney, 1978).  2.2 Preparation of Samples for 2 H NMR Spectroscopy Intact A. laidlawii membranes were prepared for 2 H NMR spectroscopy by a method that is essentially similar to that described by de Kruijff et al. (1976). Briefly, a fresh sample was centrifuged for 20 minutes at 45,000 r.p.m. (Beckman Ti75 rotor) and the pellet resuspended in deuterium-depleted buffered water (20mM Hepes, 100mM  35  Chapter 2. Materials and Methods^  36  NaC1, pH 7.5), recentrifuged as above and resuspended in the same buffer to a final volume of 0.7 ml. Lipid dispersions for 2 H NMR spectroscopy were prepared by the gentle vortexing of the freeze-dried lipid with either the deuterium-depleted buffer or deuterium depleted water at temperatures above that of the gel/liquid-crystalline phase transition temperature of the lipid sample. Under comparable conditions  2 1-1  NMR  spectra of a sample dispersed in deuterium-depleted water were indistinguishable from those of dispersions of the same material in deuterium-depleted buffer.  2.3 Extraction of A. laidlawii Lipids and Preparation of the Polar Lipid Fraction Total lipid extracts were obtained by a modified method of Bligh and Dyer (1959). The aqueous dispersion of intact A. laidlawii membranes was diluted with an equal volume of methanol and the mixture heated at 70°C for 45 minutes. After the mixture had cooled, it was diluted with an equal volume of water and four times its volume of chloroform. After vigorous shaking, the chloroform layer was separated and dried by filtration through chloroform-wetted filter paper and concentrated by rotary evaporation. Next, the lipid concentrate was redissolved in benzene:acetone (1:1 by volume) and the final traces of water removed by azeotropic evaporation of the solvent. The dried lipid extract was redissolved in chloroform and applied to a column of silicic acid (Biosil A, Biorad) in chloroform. The column was washed with at least three volumes of chloroform and the polar lipids were subsequently eluted with methanol. The methanol fraction was subsequently concentrated to dryness by rotary evaporation and the polar lipids were freeze-dried from benzene, flushed with nitrogen and stored at -20°C until required.  Chapter 2. Materials and Methods^  37  2.4 Thin Layer Chromatography  Analytical TLC was used to determine if there was significant lipid degradation during the NMR experiments and preparative TLC was used primarily in the determination of the lipid polar headgroup composition of the A. laidlawii membrane preparations. Samples were analyzed on 0 5mm silica gel G plates which were developed with a solvent system consisting of chloroform:methanol:water (75:25:3, by volume). The lipid components were visualized either by charring (analytical studies) or by staining with iodine (preparative studies). 2.5 Column Chromatographic Separation of A. Laidlawii Membrane Lipids  The chromatographic separation of the MGDG, DGDG and PG present in the total polar lipid extract of A. laidlawii membranes was as follows. The total polar lipid fraction was redissolved in chloroform and applied to a column of silica gel (Davisil grade 634, Aldrich) in chloroform. The column was washed with chloroform and then developed sequentially with 10 column volumes of each chloroform:acetonitrile (75:25 by volume) and chloroform:acetonitrile (1:1 by volume), 4 column volumes of acetonitrile, 10 column volumes of chloroform:methanol (95:5 by volume), 4 column volumes of chloroform:methanol (90:10 by volume), 10 column volumes of each chloroform:methanol (85:15 by volume) and then chloroform:methanol (80:20 by volume) and finally chloroform:methanol (7:3 by volume). Under these conditions, the MGDG elutes in the chloroform:acetonitrile 1:1 fractions, the DGDG in the chloroform:methanol 95:5 fractions and the PG mainly in the chloroform:methanol 90:10 and 80:20 fractions. The fractions containing each of the purified lipid components were separately concentrated by rotary evaporation, lyophylized from benzene and stored at -20°C until required.  Chapter 2. Materials and Methods ^  38  2.6 Determination of Polar Headgroup Composition After separation of the polar lipid components by preparative TLC, the individual components were visualized and the various lipid fractions scraped quantitatively into clean screw-capped tubes. At this stage a known amount of an appropriate phosphatidylcholine was added as an internal standard and the entire mixture was transesterified with 5% H 2 SO 4 in methanol. The lipid components were each quantified by gas chromatographic analysis of the methyl esters formed and from this the polar lipid composition of the given lipid mixture was determined. 2.7 Determination of Cholesterol The cholesterol content of A. laidlawii membranes was determined by the colorimetric method of Watson (1960). Briefly, a known volume of membrane sample was placed in a clean glass test tube and then heated at 110° until all of the water had evaporated. Next, 200 itL of each H 2 O and glacial acetic acid was added followed by 5 mL of a chromogenic solution. The chromogenic solution contained a mixture of 1 volume of 0.25 M 2,5-dimethylbenzenesulfonic acid in glacial acetic acid, 3 volumes of acetic anhydride, and 1 volume of glacial acetic acid. After the samples were allowed to cool (approximately 15-20 min.) 0.6 mL of concentrated H 2 SO 4 was added to each of the above solutions which were then mixed thoroughtly, and allowed to stand for 20 min to enable completion of color development. Afterward, the absorbance was read at 620 nm. (Courtesy of Dr. R.N.A.H. Lewis). 2.8 Differential Scanning Calorimetry. DSC thermograms were recorded with a Microcal MC2 high-sensitivity scanning calorimeter operating at heating rates near 11.5°C/h. (Courtesy of Dr. R.N.A.H. Lewis).  Chapter 2. Materials and Methods^  39  2.9 2 H NMR Measurements 2H  NMR measurements were performed at 46.175 MHz on a home-built spectrometer  (Sternin, 1985). The quadrupolar echo pulse sequence (Davis, 1983) was used with a 300ms recycle delay time, a 50ms interpulse spacing and a 30.5ms ring-down delay. FIDs were acquired with an 8-step phase cycle sequence. Spectral widths for all spectra acquired were either 200kHz or 500kHz. Experiments were repeated for all systems studied with the exception of the myristic acid-containing samples. 2.10 T 1 Measurements A saturation recovery method was used to measure the T 1 relaxation time of the solid cholesterol. The quadrupolar echo pulse sequence (Davis, 1983) was used with a variable TR, a 50-ps interpulse spacing, and a 30.5-,as ring-down delay. All FID's were acquired with an 8-step phase cycle sequence. The spectral width was 500 kHz. The echo height was fit to an exponential function of the form S(T R ) = S (o0) (1-e TR/Ti) -  where S(T R ) is the signal intensity at time TR, T1 is the longitudinal relaxation time, and S( (,) is the signal intensity in the limit that TR >> 5T1. 2.11 Derivation of Order Profiles The spectra of A. laidlawii membranes and derived liposomes recorded at 37°C are typical of lipids bearing perdeuteriated chains in the La phase. Since the lipid system exhibits axial symmetry, it is possible to apply a method previously discussed (Sternin et al., 1988; Lafleur et al., 1989) to derive the general shape of the order profile. Although the order parameter profile derived from using a series of specifically labelled palmitic acid chains provides details of the order at each carbon position (see Seelig and Seelig, 1977), we chose to use the method of Lafleur et al., 1989 for two reasons.  Chapter 2. Materials and Methods ^  40  This latter method can be used to derive the general features of the order profile, and the use of a perdeuteriated palmitic acid minimizes the inherent biological variability inevitable in using membranes derived from different cultures of the microorganism grown using a series of specifically labelled palmitic acid chains. First, dePakeing was performed by the method of Sternin et al. (1983). The dePaked spectrum represents the continuous probability distribution of order for the deuteriated acyl chain.' Assuming that CD2 groups contribute equal intensity to the dePaked spectrum and that there is a monotonic decrease of order from the interface toward the middle of the bilayer, an average value of the quadrupolar splitting Av g was assigned to each methylene group denoted by its acyl chain position n=2,3,...16. The order parameters S(n) were then calculated using equation 2.1 Avg — 43 e2q Q S(n) h  (2.1)  where e 2 qQ/h. 167 kHz is the quadrupolar coupling constant (Davis, 1983). Average order values, mentioned in the text, were determined by calculating an arithmetic mean <S> for the S(n), for 2 < n < 16. Corrections were made for S(16) by a linear extrapolation of S(14) and S(15) (Lafleur et al., 1989).  lAs is often found in studies of this type, a small departure from the Pake doublet shape manifests itself in a small negative-going base line just outside the main dePaked spectrum (see Chapter 3, Figure 3.1). This flat negative spectral region was used to define the base line. Changes in the order profile arising from different plausible choices for the base line are of the order of the random changes resulting from repetition of the experiment. The errors in the derived values of all order parameters are 5-10%  Chapter 3  Influence of Lipid Composition on <S> in A1B Membranes  3.1 Summary 2 I-I  NMR techniques have recently been developed to determine the complete orienta-  tional order profile of lipid bilayers employing lipids containing perdeuteriated palmitic acid (Lafieur,M., Fine,B., Sternin,E., Cullis,P.R., Bloom,M., 1989, Biophys.J. 56:10371041). In this work these techniques have been applied to study order profiles in intact membranes derived from Acholeplasma laidlawii strain B. It is shown that complete orientational order profiles can be readily obtained from the intact membranes of A.  laidlawii B grown on equimolar amounts of perdeuteriated palmitic acid and a nondeuterated fatty acid of varying length and unsaturation. By varying the fatty acid composition employing mixtures of perdeuteriated palmitic acid with myristic, elaidic, oleic or linoleic acid, a range of hydrocarbon order compatible with high rates and extents of cell growth has been obtained where the average order parameter <S> varies over the range 0.140 to 0.176. This same variation in order is seen for liposomes derived from total lipids extracted from these intact membranes. 2 1-I NMR studies on liposomes composed of individual species of the extracted lipids indicate that modulation of the membrane lipid headgroup composition has the potential to play an important role in maintaining the membrane order within this range.  41  Chapter 3. Influence of Lipid Composition on <S> in A1B Membranes^42  3.2 Introduction As indicated in Chapter 1, deuterium nuclear magnetic resonance spectroscopy ( 2 H  NMR) is an important technique for characterizing order and dynamics in the hydrocarbon region of model and biological membranes (Davis et al., 1980; Lafleur et al., 1989; Rance et al., 1982; Jarrell et al., 1982). Orientational order profiles of the hydrocarbon chains have usually been constructed using a series of membranes each containing a specifically CD 2 labelled fatty acyl chain. Recent reports have detailed the development of convenient 2 H NMR procedures to obtain complete hydrocarbon order profiles for lipid systems containing perdeuteriated long chain saturated hydrocarbons. This includes systems containing perdeuteriated tetradecanol (Sternin et al., 1988) as well as systems containing perdeuteriated palmitic acid (Lafleur et al.,1989,1990a). Here we extend this type of measurement to intact biological membranes in which perdeuteriated palmitic acid has been biosynthetically incorporated into the membrane lipids. The membranes of the mycoplasma Acholeplasma laidlawii strain B are ideally suited for such a study since the fatty acid composition of membrane lipids can be widely manipulated by the incorporation of exogenously supplied fatty acids (see, for example, Silvius and McElhaney, 1978), and because these membranes have been extensively studied by many physical techniques (McElhaney 1984, 1989). The use of one preparation containing perdeuteriated palmitic acid, as opposed to a series of preparations each containing a different specifically CD 2 labelled palmitic acid, offers significant advantages with respect to cost and time and minimizes the biological variability inherent in the use of different preparations. Here we show that biosynthetically incorporated perdeuteriated palmitic acid in A. laidlawii allows the straightforward generation of the complete hydrocarbon order profile for a saturated chain. We have employed this  Chapter 3. Influence of Lipid Composition on <S> in A1B Membranes^43  technique to investigate the range of order profiles consistent with normal growth characteristics of this organism. Further, we have examined the hydrocarbon order in liposomal dispersions derived from total membrane lipid extracts and from the major A. laidlawii membrane lipids. Order profiles measured for these systems indicate that the nature of the lipid polar headgroups in the intact A. laidlawii membrane can strongly modulate the order profile.  3.3 Results The first set of experiments was aimed at demonstrating that the 2 11 NMR dePakeing approach for obtaining membrane hydrocarbon order profiles could be applied to an intact biological membrane system. Here, perdeuteriated palmitic acid was biosynthetically incorporated into the membrane lipids of A. laidlawii B. This was achieved by culturing the microorganism in avidin-containing media supplemented with perdeuteriated palmitic acid and another fatty acid. Under these conditions, endogenous fatty acid synthesis is suppressed and the growth of the microorganism is totally dependent on the exogenous supply of fatty acids (Silvius and McElhaney, 1978). It should also be noted that under these conditions the exogenous 16:0d31 is esterified almost exclusively to the snl position of the glycerol backbone (McElhaney and Tourtellotte, 1970; Saito et al., 1977). The 2 11 NMR spectra obtained from the purified A. laidlawii membrane, as well as liposomes prepared from the total extracted lipids, is shown in Figure 3.1A. The "dePaked" spectra derived from these lineshapes are shown in Figure 3.1B. Few of the quadrupolar splittings arising from deuterons at different positions on the chain can be resolved; however, a smoothed orientational order profile can be achieved by assuming a monotonic decrease in order along the acyl chain (Sternin et al., 1988; Lafleur et al., 1989). The resulting order profile derived from the dePaked spectra are shown  Chapter 3. Influence of Lipid Composition on <S> in A1B Membranes ^44  in Figure 3.1C. Two points may be noted: first, as expected, both profiles exhibit the plateau region characteristic of lipid bilayer systems (Seelig and Seelig, 1980); second, the order profile in the intact membrane is essentially the same, within experimental error, as that observed for the liposomes composed of the extracted lipids. A major objective of this study was to determine the range of order profiles compatible with growth of the microorganism A. laidlawii B. Previous studies of fatty acid-enriched and fatty acid-homogeneous cultures of A. laidlawii B have shown that the growth of the microorganism is inhibited when the growth temperature is more than 50° C above the gel/liquid crystalline phase transition temperature of the membrane lipids, or when more than 50% of the membrane lipids are in the gel state at any given growth temperature (McElhaney, 1974; Silvius and McElhaney, 1978; Silvius et al., 1980). Thus the organism will grow poorly or not at all on pure palmitic or pure oleic acid, for example (vide infra). Given these limitations, there may be a range of membrane lipid order within which normal growth and function can occur. It is possible to examine the membrane hydrocarbon order at the limits of growth by varying the effective unsaturation of fatty acids supplied to the A. laidlawii growth medium. We have done this in two ways: 1) by varying the number and type (cis or trans) of double bonds in the fatty acids keeping the saturated/unsaturated ratios constant; and 2) by varying the ratios of saturated and cis-monounsaturated fatty acids. In order to compare the variation in <S> derived from our A. laidlawii membrane preparations with those in fluid model membranes, we have measured <S> for DPP  Cd62  and for DOPC:POPCd 31 (90:10) liposomes. These values correspond to <S> = 0.177 and <S> = 0.133 for the DPPC d62 and DOPC:POPC d31 liposomes, respectively. The values of <S> obtained for most fluid membranes lacking cholesterol normally fall within this range for perdeuteriated palmitic acid chains. Since DPPC  d62  is only fluid  at temperatures above 40°C we have measured the order parameters at 42°C for both  Chapter 3. Influence of Lipid Composition on <S> in A1B Membranes^45  0.30  C 0.24 ---  o 0.18 —  • O O  0.12 —  0.06 _  0.00  1 1 0.^2.^4.^6.^8.^10.^12.  14.  16.  Carbon Number ( n) -  Figure 3.1: Sample 2 H NMR spectra (A) and corresponding dePaked spectra (B) of A. laidlawii B. intact membranes and derived liposomes with a fatty acyl chain composition of 16:0d31/18:1c6.9, 47:53 (mol%). (C) Order profiles derived from B as described in Chapter 2. The ticks beneath the dePaked spectra give the frequency assigned to a carbon position of unit area (Lafleur et al. 1989). Spectra were recorded at 37°C, using the quadrupolar echo pulse technique (Davis 1983). 60000 transients were recorded for each spectrum.  Chapter 3. Influence of Lipid Composition on <S> in AIB Membranes ^46  of the above liposome samples. Based on differences in order profile measurements of the POPC d31 dispersions taken at different temperatures, we estimate that the values of <S> given above for both the DPPCd 62 and DOPC:POPCd 31 (90:10) are approximately 5% lower than expected at 37°C. The order profiles shown in Figure 3.2 are those of A. laidlawii B membranes prepared from fatty acid auxotrophic cultures grown at 37°C in the presence of perdeuteriated palmitic acid and an equimolar quantity of either elaidic (18:1t09), oleic (18:1t09) or linoleic (18:2c09, 012) acids. As may be expected, the order decreases for more unsaturated acyl chain substituents in the sequence 16:0d31/18:1t09 > 16:0d31/18:1c09 > 16:0d31/18:2c09,012 at a given temperature. This trend was not unexpected and is essentially consistent with what one would expect from an examination of the melting points of the three unsaturated fatty acids concerned as well as the results of previous 19 F  NMR studies of MacDonald et al. (1984, 1985a,b). The average order <S> (calcu-  lated as described in Chapter 2) decreased by approximately 20% over the range where <S>=0.173 for the 16:0d31/18:1t09 system and 0.140 for the 16:0d31/18:2c09,012 preparation. It should be noted that, in particular, the linoleate content of the linoleatecontaining membranes (59 mol%, see Table 3.1) is close to the maximum which can be incorporated into A. laidlawii B membranes while supporting normal or near normal growth of the microorganism (Silvius, 1979). Thus, the <S> = 0.140 may well be approaching the lower limits of hydrocarbon order which can support normal growth and membrane function in A. laidlawii B at 37°C. In the next series of experiments, the order profiles were determined for A. laidlawii B membranes in which the ratio of 16:0d31 and 18:1cL9 in the membrane were varied. It has been demonstrated previously that neither highly saturated (e.g. palmitic acid) nor highly unsaturated (e.g. oleic acid) fatty acids alone can support the growth of  A. laidlawii B when made fatty acid auxotrophic by the inclusion of avidin in the  Chapter 3. Influence of Lipid Composition on <S> in A1B Membranes^47  0.40 0.350.300.25_ (JD  0.20_ 0.15- 0^2^4^6^8^10^12  ^  14  Carbon_ Number (ri)  Figure 3.2: Order profiles derived from the dePaked spectra of intact A. laidlawii membranes. All spectra were the sum of 60000 transients, recorded at 37° C. Fatty acid compositions corresponding to individual profiles are as follows: 16:0d31/18:1t09 46:54 mol% (closed rectangles); 16:0d31/18:1c09 47:53 mol% (open rectangles); 16:0d31 /18:2c09, 012 41:59 mol% (open triangles).  ^  16  Chapter 3. Influence of Lipid Composition on <S> in AJB Membranes^48  a  Table 3.1: A. laidlawii B Membrane Lipid Composition fatty acid MGDG DGDG PG OAPG+ GLX compositions GPDGDG 16:0d31/14:0, 4 7 19 9 60 45:54(mol%) 16:0d31/18:1t09, 34 34 26 5 tr 46:54(mol%) 16:0d31/18:1c09, 5 18 1 21 55 78:22(mol%) 16:0d31/18:1c09a, 33 25 15 27 tr 47:53(mol%) 28 16:0d31/18:1c09, 9 tr 34 28 21:79(mol%) 21 24 16:0d31/18:2c09, 012, 32 23 tr 41:59(mol%) Average from analyses of several A. laidlawii (16:0d31/ 18:1c09) preparations.  growth medium. However, the organism will grow normally when the growth medium is supplemented with mixtures of a high-melting and a low-melting fatty acid (Silvius and McElhaney, 1978). These limitations can be rationalized by the suggestion that  A. laidlawii membrane lipids derived from appropriate fatty acid mixtures will have an order (<S>) within the range that can support normal growth and membrane function. Membrane lipids derived from either of the pure fatty acids, however, will result in either membranes that are too disordered, for the 18:1c09 lipid, or membranes containing too much gel-phase lipid for the microorganism to function normally at the growth temperature, for the 16:0 lipid (vide infra). In recent experiments it was found that mixtures of palmitic and oleic acids can support good cell growth provided that the mixture contains no more than 80 mol% of any single conponent (Lewis et al., unpublished results). Assuming that these are close to the limits of saturation consistent with the normal growth and membrane  Chapter 3. Influence of Lipid Composition on <S> in A1B Membranes ^49  0.40_ 0.350.300.25_ CID  0.20_ 0.15_ 0.10_ 0.05_ 0.00 0  2  I^I^■^I^i 4^6^8^10^12  Carbon_ Number (r)  14  Figure 3.3: Order profiles of A. laidlawii intact membranes from spectra recorded at 37 C. The top and bottom profiles correspond to membranes containing the fatty acid compositions: 16:0d31/18:1c09 78:22 mol% and 16:0d31/18:1cA9 21:79 mol% respectively. Methods for deriving the order profiles are described in Chapter 2.  16  Chapter 3. Influence of Lipid Composition on <S> in A1B Membranes ^50  function of the microorganism, we measured the hydrocarbon order in intact A. laidlawii membranes grown in media supplemented with 16:0d31/18:1c09 (80:20) and 16:0d31/18:1c09 (20:80). The observed order profiles are illustrated in Figure 3.3. The systems containing larger amounts of oleic acid are significantly more disordered, as expected. This is expressed by the average order <S> which decreases from 0.176 for the 16:0d31/18:1c09 (80:20) membranes to 0.140 for the 16:0d31/18:1c09 (20:80) containing membranes. Since DSC studies (data not presented) have shown that the upper boundary of the gel/liquid crystalline phase transition of the palmitate (80)/ oleate (20) membranes falls above the growth temperature of 37°C, it seems likely that the order parameters observed with these membranes may be close to the upper limits that are compatible with the normal functioning of A. laidlawii membranes (at 37°C). It should be noted that the 16:0d31/18:1c09 (80:20) membranes gave rise to a spectrum characteristic of a mixture of gel (approximately 50% based on enthalpy estimates from DSC measurements, data not shown) and liquid crystalline phases at 37°C. Thus it was necessary to perform spectral subtraction using a method outlined previously to obtain an order profile for those lipids in the liquid crystalline state (Vist and Davis, 1990). Spectra obtained at 37°C and 42°C were used in this case. We estimate that <S>=0.176 is probably 5% lower than what would be obtained from a pure liquid crystalline membrane preparation based on the difference between order profile determinations of POPC dispersions at different temperatures. In an effort to further probe these limits, we have also examined the order profile of membrane preparations isolated from cultures supplemented with the fatty acids 16:0d31 and 14:0. Studies on A. laidlawii membranes have previously shown that good growth occurs when A. laidlawii is cultured in an avidin-free medium on single fatty acid species with chain lengths in the interval 13 to 19 carbons (Silvius et al., 1980; McElhaney, 1984). The order profiles obtained from intact membranes and liposomes  Chapter 3. Influence of Lipid Composition on <S> in A1B Membranes^51  0.40  1  0.35_ 0.30_  0.25_ -----Z. 0.20 --  0.15_ 0.10  0.05  0 ^ 0  1 2  1^1^1^1^1 4^6^8^10^12 Carbon_ Number (n)  1 ^ 14 16  Figure 3.4: Order profiles of intact A. laidlawii membranes (closed rectangles) and derived liposomes (open rectangles) with the following fatty acid composition: 16:0d31 14:0 46:54 mol%. Spectra were recorded at 37 C. 60000 transients were recorded for each spectrum. Methods for deriving the order profiles are described in Chapter 2.  Chapter 3 Influence of Lipid Composition on <S> in AJB Membranes ^52  composed of the total extracted lipids of A. laidlawii grown on an equimolar mixture of perdeuteriated palmitic acid and myristic acid are shown in Figure 3.4. It may be observed that order in the intact membrane, <S>=0.176, is within the range given above. DSC studies of this system (not shown) indicate that a small proportion (approximately 10-15%) of the membrane lipids are in the gel phase. A gel phase component was not detected in the 2 11 NMR spectrum probably due to a combination of the signal-to-noise ratio obtained and the breadth of frequencies over which such a gel-phase component would normally be observed. The order in the derived liposomes, <S>=0.3, was found to be significantly higher than that in the intact membrane (<S>=0.195), in contrast to the system grown on 16:0d31/18:1cA9 (see Figure 3.1). In an attempt to understand the basis of this difference, the lipid composition of these A. laidlawii membranes was analyzed and compared to the compositions of the other preparations employed in this study. As shown in Table 3.1, the membrane grown on the 16:0d31/14:0 mixture contained large amounts of a glycolipid (referred to as G1X) which was synthesized at the expense of MGDG and DGDG. The structure of this lipid has only recently been characterized in A. laidlawii membranes (Bhakoo et al., 1987) and exhibits a hydrocarbon region comprised of an exogenously supplied fatty acid and a 20-carbon polyprenyl chain. The function of G1X in these membranes is presently unknown but it has been surmised that its flexibility is similar to that of the phytanyl lipids. Further study would be necessary for a complete understanding of the function of G1X in these membranes. It is of interest to compare the range of order observed for the intact A. laidlawii membranes with that observed for liposomes composed of the total extracted lipids as well as individual lipid species. As shown in Figure 3.5, the range of order profiles for liposomes composed of total lipids is very similar to that observed in the intact membranes (Figures 3.2 and 3.3). The <S> values range from 0.176 to 0.140 for  Chapter 3. Influence of Lipid Composition on <S> in AIB Membranes^53  0.15 0.10 0.05 0.00 0^2^4^6^8^10^12 Carbon Number (n)  ^  14  Figure 3.5: Order profiles otained from the dePaked spectra of derived A. laidlawii liposomes. All spectra were the sum of 60000 transients, recorded at 37°C. Symbols used in profiles corresponding to specific fatty acid compositions are as follows: 16:0d31/18:1cL9 78:22 mol% (closed rectangles) 16:0d31/18:1t09 46:54 mol% (open rectangles); 16:0d31/18:1cA9 47:53 mol% (open triangles); 16:0d31/18:2c09,Al2 41:59 mol% (closed triangles); 16:0d31/18:1cA9 21:79 mol% (open circles). Methods for deriving the order profiles are described in Chapter 2.  ^  16  Chapter 3. Influence of Lipid Composition on <S> in ALB Membranes ^54  the 16:0d31/18:1c09 (80:20) and 16:0d31/18:1c09(20:80) systems. Further, it may be noted that, with the exception of the 16:0d31/14:0 preparation noted above, the order profiles of the intact membranes and the derived liposomes are practically identical, indicating that membrane protein does not significantly influence either acyl chain motion or packing in the hydrocarbon region. From the above results and from previous studies (Seelig and Waespe-Sarcevic, 1978), it is evident that the order parameters measured are dependent upon the degree of saturation of the fatty acid composition. However, with A. laidlawii B, it is also known that changes in the fatty acid composition tend to be accompanied by changes in the polar headgroup composition of the membrane lipids (McElhaney, 1984, 1989; Wieslander et al., 1980). The lipids usually found in A. laidlawii B membranes are predominantly the glycolipids MGDG and DGDG, which tend to form inverted hexagonal (HIT) and bilayer phases, respectively (Wieslander et al., 1980) and PG, a bilayer-forming phosphatide present in quantities of about 30 mol% of polar lipid content. Since changes in the polar headgroup composition can also influence the magnitude of the order parameters measured, we have examined the order profiles of these polar lipids. Multilamellar vesicle (MLV) dispersions of MGDG, DGDG and PG extracted from 16:0d31/18:1c09 A. laidlawii membranes were therefore prepared and order profiles determined by 2 11 NMR methods. The results are presented in Figure 3.6. The glycolipids MGDG and DGDG exhibit vastly different 2 H NMR spectra at 37°C. By analogy with 2 11 NMR results obtained for bilayer- and HII-preferring phospholipids (Perly et al., 1985; Lafleur et al., 1990a,b), the DGDG spectrum is characteristic of bilayer structure whereas the MGDG dispersions give rise to a spectrum characteristic of a mixture of (predominantly) HII and bilayer phases at 37°C. Due to the small contribution of the bilayer component (less than 10%), these spectra are not easily  Chapter 3. Influence of Lipid Composition on <S> in AJB Membranes^55  0 30  0.24 —  0.06 —  0.00  2  ^  4^6^9^10^12  ^  114  ^  16  Carbon Number ( n) 0 30  0.24--  0.18 —  0.12 —  0.06 —  0.00  a 2  4  8^10^12  14  16  Car bon Number ( n)  Figure 3.6: Bilayer order profiles of "model membranes". A) PG liposomes (top profile) and DGDG liposomes (bottom profile). B) MGDG:DGDG:PG dispersions in 40:35:25 molar ratios (open rectangles), MGDG:DGDG dispersions in 50:50 molar ratios (closed rectangles). All spectra were recorded at 37°C as for the above figures. Each spectrum giving rise to the order profiles was a sum of 24000 transients. Fatty acyl chain composition was 16:0d31/18:1cA9 47:53 mol%. Methods for deriving the order profiles are described in Chapter 2.  Chapter 3. Influence of Lipid Composition on <S> in AJB Membranes ^56  separable, which would be necessary to apply the dePakeing and integration techniques (Lafleur et al., 1989; Sternin et al., 1988). Thus, an order profile for MGDG liposomes is not presented. As shown in Figure 3.6, both PG and DGDG form bilayers with the characteristic order profile shape although the magnitude of the order in PG bilayers is significantly higher than in DGDG bilayers. MGDG/DGDG/PG (40:35:25) lipid dispersions, mimicking the lipid composition of the parent membrane, were investigated by the above methods. The hydrocarbon order profile obtained (Figure 6B) is essentially the same as that given by the parent, intact membrane (16:0d31/18:1c09 in Figure 3.2), showing that the proportions of the component lipid species are important determinants of the magnitude of the order profile in A. laid lawii. Further, whereas the proportion of PG is relatively unchanged over the range of fatty acids employed, the proportion of DGDG can change considerably along with GPDGDG. As noted previously (Lafleur et al., 1990a and Eriksson et al., 1991) HII-preferring lipids in bilayers can exert an ordering effect. This is also exhibited by liposomes of MGDG/DGDG (1:1). The mixture MGDG/DGDG (1:1) exhibits a 20% increase in <S> over that of the DGDG alone where <S>=0.164 and <S>=0.140 respectively.  3.4 Discussion The major results of this study concern the measurement of hydrocarbon order profiles in intact biological membranes, the implications of the range of order profiles which are compatible with growth, and the roles of individual lipid components in modulating membrane order. With regard to the ability to measure complete order profiles for saturated chains in intact membranes, the 2 11 NMR dePakeing technique clearly offers significant advantages in comparison to previous procedures (Lafleur et al., 1989; Jarrell  Chapter 3 Influence of Lipid Composition on <S> in AJB Membranes^57  et al., 1982) which require growing A. laidlawii on a series of specifically 2 H-labelled fatty acids. As pointed out elsewhere, for model systems (Sternin et al., 1988; Lafleur et al., 1989) the procedure describes the general features of the order gradient without the need for synthesis and, in the present use, biological incorporation of specifically labelled acyl chains. It would clearly be useful to extend these procedures to other biological membranes, such as those of eukaryotes, which are not fatty acid auxotrophs. In this regard, it has been shown that free fatty acids as well as certain long chain alcohols, such as tetradecanol, induce little or no change in membrane order in concentrations up to 20 mol% (Pauls et al., 1983; Lafleur et al. 1990c) and that there is a strong correlation between the magnitude of the order parameters and the shape of the order profile. This suggests that perdeuteriated alcohols could be used as probes of order profiles in other biological membranes, a possibility which is currently under investigation. The range of order profiles compatible with high rates and extents of cell growth were determined to correspond to <S>=0.140 to <S>=0.176. These values appear to fall very close to the maximum and minimum values of <S> = 0.177 ± 5% and <S> = 0.133 ± 5% found for DPPCd 62 and DOPC:POPCd 31 (90:10), respectively, and probably reflect the limits of achievable hydrocarbon order in a fluid membrane lacking cholesterol at physiological temperatures. Since the incorporation of 30 mol% cholesterol in a model membrane would result in <S> > 0.3 (see for example, Lafleur et al., 1990c) one could argue that the maximum possible value of <S> for a "fluid" membrane is actually much higher than that obtained here and therefore the range 0.140 < <S> < 0.176 is narrow in comparison. However, cell growth is greatly inhibited for  A. laidlawii preparations falling outside this range, suggesting a requirement for the hydrocarbon order to fall within this range in order to maintain normal membrane function. The intact membrane preparation of 16:0d31/14:0 is an example of an A.  laidlawii system which meets these conditions. High rates and extents of growth were  Chapter 3. Influence of Lipid Composition on <S> in AlB Membranes ^58  observed for the preparation and <S>=0.176 falls within the above limits. Since the myristate-containing membranes have a component in the gel phase, the order in this system may well be close to the maximum which can be obtained in an A. laidlawii B membrane. The observation of a restricted range in hydrocarbon order may, in turn, reflect a requirement by the organism that other membrane-related parameters are maintained within fixed boundaries. For example, it has been postulated (Ipsen et al., 1990) (see also Seelig and Seelig, 1974 and DeYoung and Dill, 1988) that membrane thickness, 2d, is directly related to the average order parameter by the relation d = dl[a < S >  +1)]  (3.1)  where d is the average projection of the acyl chain along the bilayer normal, d1=19.7 is the length of an all-trans palmitoyl chain (Marcelja, 1974) and a and b are numerical parameters satisfying 0.5a + b=1; values of a=1 and b=0.5 have been used. According to this analysis, the variation in order profiles determined in this study would correspond to a change in hydrophobic thickness of 1.5A (Table 3.2). Other groups have postulated a relationship between parameters such as polymorphism and hydrocarbon order (Epand, 1990). Since the interior of a membrane may be approximated as an incompressible fluid, the choice of lipid cross-sectional area as a membrane coordinate, which is used by others (Thurmond et al., 1991), is equivalent to that of bilayer thickness. However, the details of such quantitative relationships remain, as yet, unestablished. The roles of particular membrane constituents in establishing the order profile observed is of interest. In the exceptional case of the 16:0d31/14:0 preparation, the very different value of <S> obtained for the lipid dispersion relative to the intact membrane suggests that significant protein-lipid interactions may influence the observed order profile. Alternatively, since the DSC studies indicated that more gel phase was  Chapter 3. Influence of Lipid Composition on <S> in AJB Membranes  ^  Table 3.2: Hydrophobic Thickness calculated fatty acids and compositions intact^derived membranes A liposomes 27.4 16:0d31/14:0,45:54(mol%) 26.7 16:0d31/18:1t09,46:54(mol%) 26.7 26.7 16:0d31/18:1c09,78:22(mol%) 26.5 26.6 16:0d31/18:1c09,47:53(mol%) 26.2 26.2 16:0d31/18:1c09,21:79(mo1%) 25.2 25.5 25.2 25.2 16:0d31/18:2c09, Al2,41:59(mol%)  59  A  present in the derived liposomes than in the intact preparation, the proteins may interact with the lipids to reduce the gel-phase component in the membrane to allow continued viability of the A. laidlawii cells during growth. The interpretation of these results is complicated by the presence of the G1X and will require further examination to elucidate. With the exception of the 16:0d31/14:0 preparation, the order profile observed for the intact membrane is effectively identical within experimental error to that for liposomes composed of the total extracted lipids. This indicates that membrane proteins do not significantly perturb acyl chain order and packing, at least for acyl chain lengths of 16 and 18 carbons. This is consistent with previous observations (Seelig and Seelig, 1980; Bloom and Smith, 1985; Bloom, 1979) and, in the spirit of the Mattress Model of Mouritsen and Bloom (1984), suggests that the hydrophobic regions of integral proteins are matched in a manner such that the presence of the protein does not perturb the motional freedom of the lipids on the NMR timescale. The predominant lipid species in A. laidlawii, MGDG, DGDG and PG differ significantly from one another with respect to their polymorphic phase preferences and hydrocarbon order. In addition, from the results obtained, MGDG and DGDG in combination appear to potentially play an important role in establishing the order  Chapter 3. Influence of Lipid Composition on <S> in AlB Membranes ^60  profile in the intact membrane. It is clear that the presence of MGDG, which adopts the HII phase in isolation, increases the order in bilayers formed with MGDG/DGDG mixtures. The increase has been attributed to an increase in lateral pressure induced by the nonbilayer-forming lipid on the acyl chains of the lipid mixture (Lafleur et al., 1990a). This effect has also been observed for POPE in mixtures with POPC (Lafleur et al., 1990a). Wieslander et al. (1980) have shown for the A. laidlawii strain A that the MGDG to DGDG ratio varies for different fatty acid compositions in a manner which can be interpreted as conserving the overall polymorphic preference of the lipid bilayer. Such regulation has not been consistently observed in A. laidlawii strain B (McElhaney et al., 1984 and 1989). As shown in Table 3.1, the only consistent effect observed in A.  laidlawii strain B is a variation in the DGDG to OAPG+GPDGDG ratio as a function of acyl chain unsaturation. Although OAPG and GPDGDG have not received detailed attention in this regard, they also may play a role in maintaining membrane order within defined limits. In summary, this work demonstrates that 2 11 NMR dePaking methods may be used to determine the order gradient in the hydrocarbon region of A. laidlawii membranes grown on perdeuteriated palmitic acid. A relationship apparently exists between hydrocarbon order and high rates and extents of A. laidlawii cell growth. Under natural conditions, the organism may modulate this order via acyl chain and lipid headgroup composition. By restricting the fatty acyl chain composition while allowing for good growth of the organism, the lipid headgroup composition is varied in a manner which can be related to the need to achieve hydrocarbon order profiles lying within a fairly well defined range. The contribution of lipids normally present in lesser amounts in the  A. laidlawii membrane (OAPG and GPDGDG) appear to be important in this regard.  Chapter 4  Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane  4.1 Summary Investigations presented in Chapter 2 have indicated that there exists a well defined range of membrane hydrocarbon order compatible with good growth of the microorganism A. laidlawii B (Monck et al., 1992). Since cholesterol increases hydrocarbon order in membranes, it was of interest to examine the effect of cholesterol on the hydrocarbon order and growth characteristics of A. laidlawii B. Cholesterol is normally absent from A. laidlawii membranes since it is neither biosynthesized, nor required for the growth or survival of the microorganism. However, cholesterol will be incorporated into the membrane if exogenously supplied to the A. laidlawii culture. For membranes prepared from cells grown in the presence of cholesterol, chemical determinations indicated cholesterol represented as much as 40 mol% of the total membrane lipid. However, 2 H-NMR order parameter measurements and DSC studies of the same membrane preparation suggested that cholesterol was present at significantly lower levels (P-., 10-15 mol%) in the membrane lipid bilayer. Further incorporation of cholesterol into the A. laidlawii lipid bilayer was found to occur with an increase in temperature or by lyophilization and rehydration at high temperatures, suggesting that sterol present in a separate pool in the membrane preparation could then gain access to the bilayer. 2 H-NMR  spectra of A. laidlawii membrane preparations containing deuterium labelled  cholesterol indicate that the bulk of the cholesterol present in this separate pool is in  61  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 62  a solid form.  4.2 Introduction Cholesterol and related sterols are major components of at least the plasma membranes of eukaryotic cells. In contrast, prokaryotes rarely synthesize or exhibit a growth requirement for sterols (Nes and McKean, 1977 and Rohmer et al., 1979) a finding of considerable evolutionary significance (Bloch, 1976, 1983; Nes and Nes, 1980 and Bloom et al., 1991). As stated in Chapter 1, many members of the mycoplasmas, require exogenous cholesterol or a closely related sterol for cell growth (Razin, 1982; Rottem, 1979). It has been found that even mycoplasmas which do not require sterol will incorporate variable but significant amounts of exogenous cholesterol into their plasma membranes (McElhaney, 1984, 1989). Since these simple organisms offer many natural advantages in studies of membrane structure and function (Rottem, 1979), both sterol requiring and non-requiring mycoplasma species have contributed greatly to our understanding of the role of cholesterol and related sterols in the membrane (Razin, 1982; Rottem, 1979 and McElhaney, 1984;1989). A number of studies on the effect of cholesterol incorporation on the structure and function of the membranes of the sterol-nonrequiring mycoplasma A. laidlawii B in particular have been carried out using a wide variety of techniques. For example, DSC studies have shown that the incorporation of cholesterol reduces the temperature, enthalpy and cooperativity of the lipid gel to liquid-crystalline phase transition (deKruijff et al., 1972; 1973). 2 11 NMR and ESR spectroscopic studies have demonstrated that cholesterol incorporation substantially increases the degree of hydrocarbon order for membranes in the liquid-crystalline state (Davis et al., 1980; Rance et al., 1982; Jarrell et al., 1983, Butler et al., 1978 and Koblin and Wang, 1981). In addition, the  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 63  incorporation of cholesterol has been shown to reduce the nonelectrolyte permeability (McElhaney et al., 1970, 1973; de Kruijff et al., 1972,;1973), the valinomycin-mediated K+ permeability (van der Neut-Kok et al., 1974), and the rates of glucose uptake (Read and McElhaney, 1975) in A. laidlawii cells and to reduce the ATPase activity in isolated membranes (de Kruijff et al., 1973). In most of the above studies, cholesterol levels of 14-28 mol% were obtained, consistent with the general finding that the sterol-nonrequiring mycoplasmas incorporate substantially less exogenous cholesterol into their membranes than the sterol-requiring mycoplasmas, which typically incorporate cholesterol to levels approaching 50 mol% (Razin, 1982; Rottem, 1979). Moreover, as indicated by the qualitative and quantitative effects of cholesterol on membrane lipid  bilayer organization and on membrane function, it appears from most of the above studies that the majority of the exogenous cholesterol associated with the A. laidlawii B membranes is located in the lipid bilayer. However, in two studies (Davis et al., 1980; Koblin and Wang, 1981) much higher levels of cholesterol incorporation were reported, 39 and 40 mol% respectively. These values seem high for A. laidlawii B, particularly since in both of these studies the membrane lipids were enriched in saturated fatty  acids, which usually results in lower levels of cholesterol incorporation (typically 12-15 mol%) than with lipids enriched in primarily unsaturated fatty acids (deKruijff et al., 1972; 1973; Wieslander and Selstram, 1987; Bhakoo and McElhaney, 1988 and Rilfors et al., 1987). Using 2 H NMR dePakeing methods previously developed (Sternin et al., 1988; Lafleur et al., 1989), we found (see Chapter 3) that membrane hydrocarbon order falls within a defined range if good rates and extents of A. laidlawii growth are to be  observed. Thus changing the membrane composition to achieve a significant increase in order would be expected to influence the growth characteristics of the microorganism. In light of the effects of cholesterol on the plasma membranes of A. laidlawii B listed above, it is of interest to observe the effects of cholesterol on the  2 11  NMR order  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 64  profile derived from A. laidlawii membranes. We note that several different physical techniques have been used to study the membrane of the organism A. laidlawii B, (see McElhaney, 1984; 1989); thus it has been the most well studied of the mycoplasma membranes to date. In the present study we investigate the effect of addition of cholesterol on lipid thermotropic phase behaviour and on the orientational order/hydrophobic thickness in intact A. laidlawii membranes and derived liposomes. The organism was grown in the presence of perdeuteriated palmitic acid and elaidic, oleic or linoleic acid (and cholesterol). 2 H NMR order parameters, DSC studies and chemical methods were used to determine the relative locations and quantities of cholesterol associated with the membrane. Additionally, we have used deuteriated [2,2,3,4,4,6-d6] cholesterol to further examine the phase state of cholesterol associated with the A. laidlawii membrane. The results indicate that two pools of cholesterol exist, both tightly associated with the intact A. laidlawii membrane. Some of the cholesterol is in direct contact with the lipid fatty acyl chains while the bulk of the exogenously supplied sterol is present in a solid form in close association with the membrane.  4.3 Results In Chapter 3 (and in a recent paper, Monck et al., (1992)), we demonstrated that there is a fairly narrow range of hydrocarbon order/ hydrophobic thickness that is compatible with good growth of the microorganism A. laidlawii B. Cholesterol has a (well known) large ordering effect in the hydrocarbon region of model and biological membranes (Huang et al., 1991; Rance et al., 1982; Davis et al., 1980; Gaily et al., 1979; Brown and Seelig, 1978; Stockton and Smith, 1976). Thus, it was of interest to examine the effect of cholesterol on the membrane hydrocarbon order and growth characteristics of  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 65  Table 4.1: Cholesterol levels in A. laidlawii Membranes of Given Fatty Acid Composition fatty acid Cholesterol content composition (mol% total lipid) 16:0d31/18:2c09, 012 29 16:0d31/18:1c09 37 16:0d31/18:1t09 41  A. laidlawii B. For this experiment, equimolar mixtures of cholesterol, perdeuteriated palmitic acid (16:0d31) and elaidic acid (18:1tA9) were presented to the growth medium as a mixed micelle in a small volume (P.,- 500 ml per litre of culture). Progressive growth of the microorganism was observed at a rate (and to an extent) similar to that previously observed (approximately 24 hours at 37°C). The 2 11 NMR spectrum obtained for the intact membranes and extracted lipids are presented in Figure 4.1. Hydrocarbon order profiles and average order parameters, <S>, were derived from 2 1-1 NMR spectra using the dePakeing and integration methods described in Chapter 2. The average order parameter obtained from the intact membrane preparation, <S> = 0.20, is slightly greater than that found for a similar (16:0d31/18:1t09) intact membrane preparation lacking cholesterol; and corresponds to a relatively small 13%) increase in order over <S> = 0.176, the average order parameter for the sample lacking cholesterol. This is surprisingly low given the high levels of cholesterol assayed in the membranes used for this study (41 mol%, see Table 4.1). We note that the <S> value obtained is slightly higher than the upper limit of the range 0.176 > <S> > 0.140 compatible with good growth characteristics for A. laidlawii B. found recently (see Chapter 3 and Monck et al., 1992). The derived liposome dispersion gave rise to the spectrum shown in Figure 4.1B from which an <S> = 0.3 was derived. This contrasts strongly with the value of <S> = 0.2  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 66  I^1^i^i^i^i^I^1^1^1^1 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 Frequency (kHz)  Figure 4.1: Deuterium NMR spectra of A) intact A. laidlawii B membranes and B) derived liposomes containing equimolar mixtures of 16:0d31 and 18:1t09. Both spectra were measured at 37°C and were the result of 60000 scans.  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 67  obtained for the intact membranes. The concentration of cholesterol in this membrane preparation constituted 41 mol% of the total membrane lipid content as determined by chemical methods (see Chapter 2). Because of this large difference between <S> values in the intact membrane and the extracted lipid preparations it appeared that either less of the endogenous cholesterol was interacting with the fatty acyl chains of the intact membrane than with those of the lipid dispersions, or the influence of cholesterol was reduced in the intact membranes, possibly due to the presence of proteins. In order to characterize the generality of this effect, we investigated the amount of cholesterol associated with intact A. laidlawii membranes with different fatty acyl chain compositions. Cholesterol and equimolar mixtures of perdeuteriated palmitic 16:0d31 and either elaidic (18:1t09), oleic (18:1c09) or linoleic (18:2cL9, 012) acids were exogenously supplied to the growth medium. 2 1 I NMR spectra were obtained -  for each of the cholesterol-containing intact membrane and derived liposome samples. Hydrocarbon order profiles were derived using the dePakeing and integration methods (see Chapter 2) and were compared with corresponding samples lacking cholesterol. The results are presented in Figure 4.2. Approximately the same fractional increase in order was observed for each of the above systems. As shown above, a large increase in acyl chain orientational order is obtained upon lipid extraction and redispersion of the lipid mixture at temperatures above the gel/liquid-crystalline main phase transition. It was found that heating the intact membrane also gave this result. Normally, an increase in temperature results in increased motional freedom of the acyl chains in a bilayer which, in a 2 1 1 NMR experiment, is -  measured as a reduction in the quadrupolar splitting or a decrease in the hydrocarbon order. Thus one would expect to see a decrease in hydrocarbon order in A. laidlawii membranes as the temperature is increased. We examined the temperature dependence  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 68  0.40^ 0.35_ 0.30_ 0.250.20 0.15_ 0.10_ 0.05_ 0.000  2  I^I^I^I^I 4^6^8^10^12  16  Carbon. Number (n) 0.40  I^I^I^I^i^I  0.350.30_ 0.25_  0.00  i  0^2^4^6^8^10  16  Carbon Number (n)  Figure 4.2: Order profiles obtained from A) intact A. laidlawii B membranes containing 16:0d31 and 18:2c09, 012 (rectangles), 18:1cA9 (triangles), 18:1t09 (circles) and B) from lipid dispersions of extracted (total) A. laidlawii lipids containing 16:0d31 and 18:1cA9 (rectangles) and 18:1t09 (triangles). The filled symbols represent order parameters from membrane preparations in which cholesterol was added to the growth medium. The open symbols represent order parameters from membrane preparations completely lacking cholesterol.  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 69  Table 4.2: Average Order Parameter vs Temperature for Intact A. laidlawii B Membrane Preparation with Cholesterol' temp (°C) <S> 0.201 37 0.154 47 57 0.178 67 0.185 77 0.195 87 0.177 37 6 0.294 'Fatty acid composition was an equimolar mixture of 16:0d31 and 18:1t09. b Obtained after cooling from 87°C for lh.  of order profiles measured for A. laidlawii membrane preparations containing cholesterol and equimolar mixtures of 16:0d31 and 18:1t09. The results are shown in Figure 4.3 and Table 4.2. As can be seen in the table, from 37°C to 47°C a decrease in <S> is observed as would be expected. However, at higher temperatures, 57°C to 77°C, <S> increases monotonically and then decreases again at 87°C. A second measurement at 37°C after cooling showed <S> to be approximately 50% higher than in the initial measurement. This is the type of result that would be obtained in model membranes if cholesterol was incorporated into the membrane bilayer upon heating. DSC studies of cholesterol-containing and cholesterol-free A. laidlawii membranes were performed to further characterize the effects of heating on the amount of cholesterol interacting with the membrane hydrocarbon chains. DSC thermograms typically found for cholesterol-free and cholesterol-containing A. laidlawii B membranes are presented in Figure 4.4. With both sets of membranes, the membrane lipid chain-melting phase transition occurs as a reversible lower-temperature thermal event, whereas the  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 70  1^■^I^I^I^I^I^I^1^1^i -60 -50-40 -30 -20 -10 0 10 20 30 40 50 60 Frequency (kHz)  Figure 4.3: <S> obtained for an A. laidlawii intact membrane as a function of temperature. For this experiment the microogranism was grown on 16:0d31, 18:1t09 and 20mM cholesterol and spectra were measured at A) 37°C (Initial spectrum) B) 77°C and C) 37°C (after cooling) from 87°C.  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 71  endothermic transitions attributable to the thermal denaturation of the membrane proteins are the broad (and irreversible) higher temperature events. With the cholesterolfree membrane samples, the area under the peak attributable to the gel/liquid-crystalline phase transition of the membrane lipids is usually 10-15% lower in the first heating scan, when compared with subsequent heating scans (see Figure 4.4, left panel). The smaller change in enthalpy in the first scan probably reflects the amount of lipid whose phase behaviour is perturbed prior to the thermal unfolding of the membrane protein. However, in the case of the cholesterol- containing membranes, the thermograms shown in Figure 4.4 (right panel) clearly indicate that the enthalpy of the chain-melting phase transition of the membrane lipids is some 50-55% higher in the first heating scan (A) than in subsequent heating scans (B). A decrease in the enthalpy of the lipid chainmelting phase transition is precisely what would be expected if the high temperature incubation had resulted in an increase in the amount of cholesterol interacting with the acyl chains of the membrane lipids. It is well known that the incorporation of cholesterol into lipid bilayer model membranes reduces both the enthalpy and the cooperativity of the gel/liquid-crystalline phase transition (McElhaney, 1982; Keough, 1984). Previous 2 11 NMR studies of A. laidlawii showed that cholesterol causes a large increase in hydrocarbon order. However, these studies involved sample lyophilization (Rance et al., 1982; Davis et al., 1980) in contrast to the intact wet membrane samples used here. An intact A. laidlawii membrane (16:0d31/18:1t09/cholesterol) preparation was lyophilized and redispersed in deuterium-depleted buffer (see Chapter 2) in order to compare the effect of variation of sample preparation on the values of <S> obtained from 2 11 NMR dePakeing methods. Values of <S> ‘c,-, 0.3 were observed for such samples compared to <S> = 0.20 for non-lyophilized intact membrane samples prepared from  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 72  0  20  40^80^80 100^0^20^40  80  80  100  TEMPERATURE, °C  Figure 4.4: Differential Scanning Calorimetric tracings of an intact A. laidlawii membrane preparation containing 16:0d31, 18:1tA9 without cholesterol (left panel) and with cholesterol (right panel). A. Initial heating scan (scan rate of 10 degrees per hour). B. Final heating scan after cooling for several hours (same rate as in A).  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 73  the same A. laidlawii cell culture. These results indicate that lyophilization and redispersion of intact cells containing large amounts of cholesterol gives rise to the same type of irreversible changes in acyl chain order as the heating and/or solubilization procedures described earlier. Experiments involving 2 11 labelled cholesterol should provide more information on the physical environment of the cholesterol "associated" with the A. laidlawii membranes that exhibit these irreversible effects. Cholesterol, deuterium- labelled in the 2,2,3,4 and 6, ring positions was used in the growth media in place of deuteriated palmitate for the following experiments in which palmitate and elaidate were supplied exogenously. All other conditions concerning the growth of the organism remained the same as in the experiments described earlier. The 2 11 NMR spectra obtained at 37°C for the intact A. laidlawii wet membranes containing deuteriated cholesterol and for their derived liposomes are given in Figure 4.5 (A and C). Following a heating and cooling cycle over the course of 48 hours, during which spectra were taken at 50°C and 70°C (not shown), the intact membrane sample was re-measured at 37°C. This spectrum is shown in Figure 4.5B. The quadrupolar splittings of the individual resonances for the 2,2,3,4 and 6 positions were determined from the dePaked spectrum of the derived liposomes (Figure 4.5C) and were 4kHz, 33kHz, 44kHz for the deuterons on the 6, 2,4 equatorial, 2 axial and the 3 positions on the deuteriated cholesterol respectively. The splittings from the intact wet membrane spectrum (Figure 4.5B) are not as easily obtained by this method due to the poor signalto-noise ratio although some of the splittings are evident and can be estimated from the spectrum. In both cases the splittings correlate well with previously determined splittings of [2,2,3,4,4,6-d5] cholesterol in membranes measured at 35°C and are typical of cholesterol interacting with hydrocarbon chains (see Dufourc, 1983; Kelusky et al., 1983; Dufourc and Smith, 1986; Bonmatin et al., 1990).  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 74  A  B  I^I^I^I^I^I^I^I^I^I^I  —60 —50 —40 —30 —20 —10 0 10 20 30 40 50 60 Frequency (kHz)  I^I^I^I^I^i^I^I^1^1^i —60-50 —40 —30 —20 —10 0 10 20 30 40 50 60 Frequency (kHz)  Figure 4.5: Deuterium NMR spectra of A) and B) intact A. laidlawii membranes and C) derived liposomes containing 16:0/18:1t/.9 50:50 (mol%) and [2,2,3,4,6-d5] cholesterol. Spectra were taken at A) 37°C B) 37°C acquired after cooling for an hour from 70°C and C) 37°C. The intensity of the central peak (due to residual HDO, membrane fragments and small vesicles) was reduced in order to show the details of the broadline spectrum. Quadrupolar splittings in C) were measured from the dePaked spectrum (not shown) and are as indicated in the text .  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 75  The integrated area under a 2 11 NMR spectrum reflects the number of contributing deuterons in the sample of interest. The ratio of the relative integrated areas of the two spectra before and after heating (Figure 4.5A, B), where the spectra were normalized to the number of scans, was 1:2.2 indicating that differences existed in the environments of some of the cholesterol before and after heating. This suggests that a large fraction of cholesterol does not contribute to the type of 2 11 NMR signal arising from a fluid lipid bilayer, consistent with the calorimetric and 2 H NMR results above. A possible organization for the pool of cholesterol not associated with the acyl chains is a solid or crystalline form. In this regard lipids in the solid phase can have long T1 relaxation times which are on the order of seconds, Valic et al. (1979). If the time, TR, between signal acquisition is not long compared with Ti, a loss in signal intensity will occur. A T1 of 4.3 seconds was measured for solid deuteriated cholesterol in a separate experiment (see Valic et al., 1979 for comparison with solid cholesteryl ester). A spectrum of solid [2,2,3,4,6-d5] cholesterol is shown in Figure 4.6A and of [2,2,3,4,6-d5] cholesterol in an intact A. laidlawii membrane preparation in Figure 4.6B. A TR = 20 seconds was used in both cases. Although the signal to noise ratio is poor the broad spectrum (Figure 4.6B) with a Av g = 127 kHz is clearly characteristic of a spectrum of solid [2,2,3,4,4,6-d6] cholesterol. The free induction decay of the spectrum in Figure 4.6B is shown in Figure 4.6C. Two major components can be identified in the figure. The magnitudes of the fast and slower decaying components are denoted by I I and 12 respectively as shown in the Figure. 1 A distortion in the spectrum, derived from 12, has been identified and is due to finite rf pulse width effects as described previously, Bloom et al. (1980). As determined from Bloom et al. (1980) the expected magnitude for the 12 component 1 The measurement of I I does not originate from 0 intensity due to the presence of a slowly decaying component in the FID which contributes to the isotropic peak in the spectrum. This component defines a baseline from which I I is measured.  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 76  A  i^i^I^i^I^I^i^i^1 -200 -160-120 -80 -40 0 40 80 120 160 200 Frequency (kHz)  I^1^i^I^1^1^1 192 224 256 32^64^96^128 160 Point Number  Figure 4.6: Deuterium NMR spectra of A) Solid [2,2,3,4,6-d5] cholesterol B) an intact A. laidlawii preparation containing 16:0/18:1tA9 50:50 mol% and [2,2,3,4,6-d5] cholesterol and C) FID of B with solubilized cholesterol (I i ) and solid cholesterol (I 2 ) components identified. Both spectra in A and B were measured at 37°C with TR=20 seconds. The maximum splitting shown is 127 kHz. We note that in B a line broadening function was applied to the FID prior to Fourier transformation. The applied function involved an exponential decay with T 2 =0.6 seconds.  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 77  given byl2cor, should be, I2corr = 12/0.8 . The ratio of the heights I 1 /I 2c „,., measured at the peak of the echo gives the relative proportions of membrane associated cholesterol to solid cholesterol in the sample. We found a ratio of I1/I2corr = 0.30 in this case which suggests further that the bulk of the cholesterol is in the solid phase.  4.4 Discussion The motivation for this study was largely due to the identification of a range of hydrocarbon order/hydrophobic thickness that is consistent with good growth characteristics of the organism A. laidlawii B. (see Chapter 3 and Monck et al., 1992). It is well known that cholesterol increases orientational order in the hydrocarbon region of model membranes (Brown and Seelig, 1978; Stockton and Smith, 1976; Lafleur et al., 1990b). Cholesterol has also been shown to be effective in this regard in A. laidlawii membranes (Davis et al., 1980; Rance et al., 1982) and this has been thought to be true for A. laidlawii membranes in general (Bloom et al., 1991). However, if the observed range in order 0.176 > <S> > 0.140, (Monck et al., 1992) is necessary for growth of the organism at 37°C, the optimal growth temperature, one would expect that the incorporation of significant amounts of cholesterol, giving rise to <S> >> 0.176, would result in diminished A. laidlawii growth rates. As outlined in the Results section, we found that the presence of cholesterol in the culture medium did not decrease the rate or extent of cell growth compared to cultures lacking cholesterol. In addition, the range of order found in these membranes corresponded to <S> = 0.20 for 16:0d31/18:1t09 down to <S> = 0.149 for 16:0d31/18:2c09,012, an increase of no more than 13% over that of membranes lacking cholesterol in any case. In the previous study (see Chapter 3), it was difficult to determine an upper bound for hydrocarbon order due to poor growth of the organism on long chain saturated fatty acids at 37°C. It was presumed  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 78  that the presence of more than 50% gel state lipid, under these conditions, inhibited  A. laidlawii growth. Given that cholesterol fluidizes a gel state membrane bilayer, it is possible that the value of <S> = 0.20 better approximates the upper bound of the range of order than does <S> = 0.176. A striking feature of these cholesterol-containing membrane systems is the remarkably large <S> values observed for the derived liposomes as compared to those observed for the intact parent membranes. Such a difference is well illustrated by the spectra in Figure 4.1 and the order profiles of Figure 4.2. From these data, it is obvious that cholesterol, in large amounts (see Table 5.1), is not having the same effect on the membrane hydrocarbon order in A. laidlawii B. as was shown previously (Davis et al., 1980; Rance et al., 1982). A logical explanation is that a large fraction of the cholesterol is initially excluded from the intact A. laidlawii membrane lipid bilayer and may become incorporated through perturbation of the membranes, for example, by extraction and resuspension of the total A. laidlawii lipids. Large increases in hydrocarbon order were also observed after incubation at elevated temperatures and/or after lyophilization and resuspension of the intact membranes, provided that resuspension was performed at temperatures well above the gel/liquidcrystalline phase transition temperature of the intact membrane. In addition, the DSC studies of the lipid thermotropic phase behaviour showed a decrease in the enthalpy of transition typical of that due to cholesterol in a membrane, only after the first scan to 70°C (see Figure 4.4). It is possible that protein denaturation influences the increases in hydrocarbon order and decrease in transition enthalpy seen in the  2 1-I  NMR and  DSC measurements, respectively. We cannot rule out this possibility. However, a more reasonable explanation is that a large fraction of the cholesterol, "associated" with the lipid bilayer but not interacting with the lipid acyl chains, is incorporated into the membranes during heating, lyophilization and resuspension of the membranes or during  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 79  lipid extraction and that this is responsible for the large increase in hydrocarbon order or decrease in transition enthalpy. This is discussed more fully below. The use of deuteriated [2,2,3,4,6-d5] cholesterol proved fruitful in determining some physical characteristics of the cholesterol associated with the intact A. laidlawii membrane bilayer. The initial spectrum obtained at 37°C for A. laidlawii membranes grown on 16:0/18:1t09 and [2,2,3,4,6-d5] cholesterol contained a sharp isotropic resonance which, on the basis of further experiment, probably arose from a combination of some small membrane fragments, residual HDO and small vesicles in the sample. The ratio of 1:2.2 observed for the integrated 2 11 NMR signal intensities arising from deuteriated cholesterol before and after heating the A. laidlawii membranes to 37°C is clearly consistent with the presence of two pools of cholesterol. It is estimated that with a T1 = 4.3 seconds, a solid cholesterol signal would contribute approximately 7% of the observable 2 11 NMR signal obtained under the experimental conditions used here (TR = 300ms). The solid cholesterol (see Figure 4.6), although absent from the membrane bilayer, must be closely associated with the A. laidlawii membrane. As discussed in the Introduction, in most of the early studies of the effect of cholesterol on the structure and function of the A. laidlawii B membrane, it appeared that most of the exogenous cholesterol present resided in the lipid bilayer. Thus, although exogenous cholesterol was presented to cultures of this organism in the same manner as in the present study, the existence of two types of cholesterol in these A. laidlawii membranes is not obvious. In the 2 11 NMR studies of Davis et al. (1980) and the ESR studies of Koblin and Wang (1981), where quite high levels of cholesterol were reported, it is possible that these high values arose, at least in part, because of the presence of two pools of cholesterol, one solubilized in the bilayer and one absent from it. Although the values of hydrocarbon order observed by Davis and coworkers for palmitate-enriched A.  laidlawii membranes were compatible with their reported cholesterol levels of 39 mol%,  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 80  we note that the membranes utilized in those 2 11 NMR studies were lyophilized and rehydrated at an elevated temperature. Thus the phenomena described in this study could provide a rationale both for the unusually high levels of cholesterol incorporation observed in the study of Davis et al. (1980) and for their experimental results. Although precise the location of the solid-like pool of cholesterol is not determined by the results presented here, (in principle) the two pools appearing in our spectra may be related to some previously published data on the rates of cholesterol exchange between A. laidlawii cells or isolated membranes and egg phosphatidylcholine/cholesterol vesicles. Davis et al. (1984) reported that in intact cells, about one-half of the cholesterol associated with the cell exhanges relatively rapidly while the other exchanges much more slowly. These workers initially discussed the possibility that the cholesterol present in the outer monolayer of the membrane bilayer could exchange rapidly, while that present in the inner monolayer first had to undergo transverse diffusion (flip-flop) to the outer monolayer before exchange could occur. However, since cholesterol transbilayer movement is believed to be quite rapid in model and in most biological membranes (Philips et al., 1987), this was not a completely convincing explanation for these results. Further experiments by the same workers using unsealed isolated membranes also gave two exchange rates for cholesterol, indicating that the observed differences are not due to a particular transbilayer distribution of cholesterol. A second suggestion was that the slowly exchanging cholesterol is due to some sort of preferential interaction of cholesterol with certain classes of lipids or with certain integral membrane proteins in the A. laidlawii membrane. If the slowly exchanging cholesterol is identified with our solid-like pool, then the interaction presumably gives rise to an immobilization of the cholesterol molecules. It is clear that additional experimental work will be required to firmly establish any of these hypotheses. Perhaps the simplest mechanism for the production of a solid pool of cholesterol is  Chapter 4. Evidence for Two Pools of Cholesterol in the A. laidlawii B membrane 81  crystallization of the cholesterol and its subsequent association with the mycoplasma membrane surfaces. Behaviour analogous to this has been observed for some macrophages (see Johnson et al. (1991) for a review). Such a crystalline state should give rise to Bragg peaks in X-ray diffraction that are characteristic of crystalline cholesterol. In an attempt to observe such peaks we detected none. However, this result does not completely rule out the presence of crystalline cholesterol. In summary, 2 11 NMR and DSC measurements of the effect of cholesterol on A.  laidlawii membranes have given two major results. First, two pools of cholesterol are associated with the membrane bilayer. One is dissolved in the membrane while the other is solid-like, associated with the membrane and can be incorporated in the membrane by various solubilization procedures. Finally, the level of cholesterol residing in the bilayer results in a maximum <S> = 0.20 which is compatible with good growth characteristics of the organism, A. laidlawii B.  Chapter 5  Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems  5.1 Summary 2 11  NMR methods have been used to examine possible mechanisms of T2 relaxation in  multilamellar dispersions and oriented multibilayers of various pure and mixed phospholipid systems. The use of deuterium labelling in lipid membranes provides a convenient probe for obtaining motional and geometrical information on lipids in this environment. Since T2 relaxation times are sensitive to relatively slow motions (7 10 -3 -10 -6 s) on the NMR timescale it is possible to derive information about slow motional processes (surface undulations, diffusion and/or collective motions) which can contribute potential relaxation mechanisms in a lipid environment. 2 11  NMR measurements of the T2 anisotropy of headgroup deuteriated powders (a-  DPPC d2 ) show relaxation behaviour clearly consistent with motional mechanisms due to fluctuations in O. Similar relaxation mechanisms are found for POPC d31 multilamellar dispersions and for POPC d31 bilayers containing cholesterol and both, cholesterol and Leu24, a hydrophobic peptide whose hydrophobic length matches the thickness of the bilayer in which it is solubilized. However, a very different anisotropy of T2 relaxation was found for oriented multibilayers suggesting that a different motional mode provides the dominant relaxation mechanism in these systems. A phenomenological theory describing fluctuations in thickness suggests that random temporal fluctuations in the membrane hydrophobic thickness or those due to lipid lateral diffusion through  82  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^83  neighboring regions of different thickness can provide a potent mechanism for T2 relaxation in membranes. Comparison of the T2 anisotropy measurements (in the oriented systems) with a phenomenological theory (Appendix B) is consistent with such a mechanism. In addition, measurement of the T2 relaxation behaviour in a multilamellar system containing POPC d31 , cholesterol and Leu16, a short peptide which imposes a lateral variation in hydrophobic thickness throughout the membrane plane, is qualitatively consistent with the relaxation theory due to fluctuations in membrane hydrophobic thickness.  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^84  5.2 Introduction  In recent years a certain emphasis has been placed on the dynamical properties of the membrane lipid bilayer. Studies measuring the activity of membrane proteins reconstituted in lipid bilayers have suggested that considerable motional flexibility of the lipid component is necessary for the normal functioning of the proteins and thus the membrane as a whole (see McElhaney , 1984 and Mouritsen and Sperotto, 1992 and the references therein for relevant reviews). Moreover, the extent of hydrophobic matching between proteins and proteins and lipids may have fundamental significance for such processes as protein segregation, or receptor mediated exocytosis/ endocytosis (Mouritsen and Sperotto, 1992). Thus the identification of the motions which either directly or indirectly influence these processes and the conditions of temperature and lipid composition under which they occur are crucial to the understanding of membrane function. Techniques which have been successful in identifying the dynamics of lipids in membranes include, for example, FTIR spectroscopy, various fluorescence methods and NMR spectroscopy.  31 P  and 2 H NMR spectroscopy have proven particularly fruitful  in the identification of various modes of lipid motions including slow motions such as lipid lateral diffusion (Fenske and Jarrell, 1991; Auger et al., 1991; Bloom and Sternin, 1987), surface undulations (Bloom and Evans, 1991; Bloom et al., 1991) or other types of collective motions (Stohrer et al., 1991) and the faster rotations or jump motions  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^85  around the long axis of the lipid molecule ( Jarrell et al. 1987; Auger et al. 1990; Morrison and Bloom, 1993). The presence of membrane surface undulations, in particular, have been identified in model membrane systems as potentially providing potent mechanisms for T2 relaxation (Bloom and Evans, 1991). It has become apparent that mechanical measurements normally associated with hard materials are also important in the study of soft materials such as the model or biological membrane lipid bilayer. The mechanical properties in these systems are characterized by, the elastic bending modulus  k,  elastic area compressibility 1/K a and  shear modulus r, each of which has been measured for certain systems (Helfrich and Servuss, 1984; Evans and Needham, 1987 and Bloom et al., 1991). Progress in this area has facilitated the development of theories describing T2 relaxation processes, particularly with regard to motions which give rise to the various types of collective lipid motions (see Stohrer et al. 1991; Bloom and Evans 1991). In the present study, the 2 11 NMR transverse relaxation time, T2 of various lipid species deuteriated in the headgroup or hydrocarbon regions are determined to try to further characterize slow motional processes occurring in the lipid membrane. Comparisons with effects observed in systems containing cholesterol and either of two integral membrane peptides (Leul6 and Leu24) (Nezil, 1992) are used to gain perspective on the motional processes responsible for T2 relaxation in these systems. It is hoped that the extension of these studies will ultimately provide new insights into membrane lipid-protein and lipid-lipid interactions.  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^86  5.3 Materials and Methods 5.3.1 Samples POPC, POPCd31 and POPEd31 were purchased from Avanti Polar Lipids (Birmingham,Alabama). Cholesterol was purchased from Sigma (St. Louis, MO). The peptides, Leul6 and Leu24 were synthesized using solid phase synthesis methods previously published (Davis et al., 1983). a-DPPC d2 was a generous gift from Dr. Michel Roux, CEN-Saclay, France. 5.3.2 Sample Preparation Multilamellar suspensions containing lipid samples were made by extensive vortexing of lipids or lipid mixtures containing the appropriate dry weight of material in deuterium depleted water for POPC d31 and POPE d31 :POPC and in excess Hepes buffer (50 mM in deuterium depleted water, pH 7.5, 40 mM NaCl) for a-DPPC d2 , at temperatures well above the gel-liquid crystalline phase transition temperature. In addition the DPPC samples were submitted to three freezing (liquid nitrogen) and thawing (50°C) cycles, centrifuged and the pellet was transferred directly to an NMR tube. Oriented multibilayers were prepared using a slight modification of methods previously described (Jarrell et al., 1987). Typically, 30-60mg of lipid, dissolved in chloroform, was smeared on 20 glass slides of average dimensions 10mm x 7mm x 0.15mm. The slides were stacked in a 10mm (O.D.) NMR tube and excess chloroform was pumped off under high vacuum overnight. The sample was then placed in a humid  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^87  atmosphere at room temperature (for POPC d31 ) or 10-15°C above the gel-liquid crystalline phase transition temperature for 72 hours. A solution of 4% polyethylene glycol 8000 by weight in deuterium depleted water was then added to the samples using a 1.0m1 syringe until the glass slides were completely covered (Morrison, 1993). The sample was left to equilibrate for at least 24 hours prior to measurement by NMR. Preparation of the samples containing Leul6 and Leu24 peptides was a slight modification of the procedure by Huschilt et al., (1985). Briefly, the appropriate lipids and peptides were weighed out to achieve the desired component ratios and hydrated using 800/21 of buffer (50mM Hepes, 100mM NaC1 at pH 7.4 in deuterium depleted water). The sample then underwent four cycles of freeze-thaw using liquid nitrogen followed by warming to room temperature. The samples were then pelletted at 25°C using a microBeckman centrifuge and the pellet was transferred to a 10mm (O.D.) NMR tube for measurement by 2 H NMR methods. Intact A. laidlawii membranes were prepared as described in Chapter 2. 5.3.3 Nuclear Magnetic Resonance The 2 11 NMR measurements were performed on a home-built spectrometer operating at 46.175 MHz for deuterium (Sternin, 1985). The quadrupolar echo pulse sequence:90 s y -t-acq,  (Davis et al. 1976) was used for performing T2 relaxation measurements.-790  The value of r was varied to obtain the decay of the echo envelope as a function of pulse spacing. A range of values, typically 50ys to 2ms, was used for r depending on the signal-to-noise ratio. t was typically lOps shorter than T to enable accurate definition of the echo peak. Between 5000 and 12000 transients were recorded for each spectrum. The signals were detected in quadrature using a standard 8 Cyclops phase cycle sequence (Rance and Byrd, 1983). The temperature was controlled using a Bruker model BV-T1000 temperature controller (Bruker Instruments, Inc., Billerica, MA).  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^88  5.3.4 Measurement of T2  The T2 or spin-spin relaxation time may be determined from the decay of the quadrupolar echo or of regions of the frequency spectrum as a function of time. This decay is due to a loss of phase memory of the spins arising from local frequency fluctuations and as such is sensitive to relatively slow motions. In this chapter, we examine the information associated with the dependence of T2 on the angle 0 between the local bilayer surface normal and the external magnetic field. A phenomenological model of the T2 relaxation processes and their dependence on 0 can be found in Appendix B. We refer the reader to Abragam, 1961 for a general theoretical description of T2 relaxation. The angular dependence of T2 relaxation in powder samples can be examined using methods previously developed (Nezil et al., 1991). By fitting the decay of intensities at corresponding points in a series of spectra (F(7)) as a sum of a variable number of exponentials using a non-negative least squares algorithm, a "relaxation spectrum" can be obtained. In a frequency spectrum of a given 2 H site (with a particular value of S(n)) each value of 0 contributes to the magnitude of the intensity. Since a Pake doublet contains two contributions to the intensity in the region between the 90° edges but one elsewhere, one can expect to obtain two T2 relaxation times between the edges and one relaxation time elsewhere for each deuterium site. The resulting relaxation spectrum is parameterized using a weighted average of the relaxation rates determined, 1/T e2ff In the oriented samples, T2 relaxation times were calculated as a function of the pulse spacing for all resolved carbon atoms at certain values of 0. The decay of the spectral peak intensity, I, was adequately fitted by an exponential function of 7 according to 47) = Ioe -2T / T2 where I() is the peak intensity at 7 = 0.  Chapter 5. Anisotropy of  T2  Relaxation in Pure and Mixed Lipid Systems ^89  5.4 Results A system previously selected for the study of T2 relaxation anisotropy (for the purpose of demonstrating relaxation spectra, Nezil et al., 1991) was DPPC bilayers, deuteriumlabelled in the a position of the PC headgroup. The advantage in using this system was that a singly labelled CD 2 species would provide a clean system for study. Thus it is useful to present this data here for comparison with the more complicated spectra discussed below. Spectra of the lipid dispersions were obtained at 50°C (approximately 5 degrees above the main phase transition temperature). The T2 relaxation spectrum was determined for the series of spectra taken as a function of the pulse spacing (90-T-90) using methods described in the materials and methods section. There was essentially one relaxation rate between the shoulders (0°) and edges 90°, and two between the edges, consistent with a single exponential for each angle 0. The Pake doublet, the T2 relaxation spectrum and a fit of the relaxation data to 1  T2  1 =^Bsin20cos20  (5.1)  T20  are shown in Figure 5.1. The reason for the choice of the fit to an equation of this type is outlined in the paper by (Bloom and Evans, 1991) where sin 2 Ocos 2 0 is the angular dependence expected for a lipid membrane undergoing fluctuations about the surface normal. Such fluctuations provide local field fluctuations at the nucleus giving rise to T2  relaxation. A summary of this theory is presented in Appendix B. The coefficients in equation 5.1 were defined using experimental values of 1/7V f  at 0 = 0° and 0 = 54.7°, giving values of 0.30 and 6.7 (ms  -1 ),  respectively. Theis T20  identified with angularly independent relaxation processes. The fit of the orientation  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^90  3 .••••■  w  W  N  CD  2 1 1  H  0  -10^-5^0^5 Frequency (kHz)  ^  10  Figure 5.1: Relaxation spectrum of a-DPPC d2 (solid line) obtained using a NNLS algorithm with a weighted harmonic average of the relaxation rate 1/T 2 to parameterize the relaxation. The relaxation spectrum was fitted by an equation of the form 1/T 2 = 1/T 20 + B sin 2 Ocos 2 0 and plotted as a function of frequency (dashed line). The powder spectrum (top) is presented for reference. All measurements were made at 50°C. (Spectra were courtesy of C. Morrison).  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^91  dependence of T2 suggests that motions such as thermal fluctuations of the orientation of the local surface normal are dominant mechanisms for T2 relaxation in the headgroup deuteriated system. From the coefficient B, one can determine an average value of the correlation time, r0 , for the motion contributing the relaxation process (cf Appendix B). For a-DPPCd 2 with a Avg 7kHz, the value of B is identified with a correlation time of ti 4 x 10's, similar to that predicted by Bloom and Evans, (1991). In order to determine if the same types of motions are dominant in the hydrocarbon region of the bilayer, a similar investigation using chain deuteriated lipids was of interest. POPC d31 multibilayers were measured using the T2 methods described above. POPC d31 was chosen, in part, for reasons of availability. The powder spectrum of POPC d31 perdeuteriated in the palmitoyl chain represents a superposition of powder spectra from each of the deuterium labelled carbons resulting in T2 values which are weighted averages over angular contributions from contributing deuterons. The T2 relaxation spectrum obtained for POPC d31 is presented in Figure 5.2. The prominent feature of this spectrum is that the longest T2 values are found at the 90 and 0 degree orientations suggesting that a T2 relaxation mechanism predominantly due to fluctuations in the local surface normal is the mechanism for all chain labelled positions, as was the case for the headgroup-labelled powder sample of cy DPPCd2. -  We thought that it would be possible to select motions such as surface undulations while diminishing effects due to large membrane curvature using samples where the lipids were macroscopically oriented between glass plates (oriented samples). In the case of POPCd3 1 , one could obtain motional information at the 7 labelled acyl chain segments with resolved splittings and the plateau region (carbons 2-8). Accordingly, the angular variation of T2 has been measured at all (resolved) deuterium-labelled carbon positions of the palmitoyl chain in POPC d31 with the exception of the methyl deuterons. The results are illustrated in Figure 5.3 (left panel) for representative CD2-labelled  Chapter 5. Anisotropy of  7  T2  Relaxation in Pure and Mixed Lipid Systems^92  I^I^I^I^I^I^I^I^I  6— 5— rn 4— 3 2 1—  —50 —40 —30 —20 —10 0 10 20 30 40 50 Frequency (kHz)  Figure 5.2: Relaxation spectrum of POPC d31 (solid line) obtained using a NNLS algorithm with a weighted harmonic average of the relaxation rate 1/T 2 to parameterize the relaxation. The powder spectrum (dotted line) is presented for reference. Note the shoulder (the 0° orientation at r-:.1± 25 kHz) and edge (the 90° orientation at ± 25 kHz) regions which show average relaxation times of 500ms and r-z..-1650ms respectively. The sample was measured at 30°C. (Plot is courtesy of Dr. F. Nezil).  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^93  carbon positions of the palmitoyl chain. All other chain positions showed a similar angular dependence of T2. Two features of the data are particularly notable. First, the anisotropy of T2 relaxation is inconsistent with the theory for surface undulations (T 2 values are shorter for the 0° orientation than for the 90° orientation). Second, compared to the magnitudes of T2 measured for the powder sample 500-700ys), the magnitudes of T2 at particular positions down the chain are significantly longer, ranging from 700ms in the plateau region at at  9 = 0 degrees to 7ms for carbon 15  9 = 70 degrees. It is possible to show that fluctuations affecting the splitting frequency can oc-  cur in both 9 and in <S> (see Appendix B). Fluctuations in <S> could arise, for example, from fluctuations in local membrane thickness, as discussed in Chapter 3. These motions are referred to as fluctuations induced by random diffusion of lipids through regions of different membrane thickness or "thickness fluctuations". According to a phenomenological theory describing such thickness fluctuations (Appendix B), the transverse relaxation rate as a function of  9, 1/T 2 , should show an angular depen-  dence corresponding to [P 2 (cos0)] 2 . As shown in Figure 5.3 (Fl), the T2 data were adequately approximated by a sum of an orientation independent term, A, and a term involving [P 2 (cos0)] 2 in the form 1/T 2 = A + C[P 2 (cos9)] 2 . The data can also be expressed in a more general form that is independent of a specific motional model using a sum of terms P o (cos9), P 2 (cos0) and P 4 (cos0), where P o (cos0) = 1, P 2 (cos0) (3cos 2 0 - 1)/2 and P 4 (cos0) (35cos 4 0 - 30cos 2 0 + 3)/8. We show in Figure 5.3 (F2), that the data were also adequately fitted by an expression of the form  1  Tan)  =  A02  )  ;12  7)  (5.2)  P2(CO30)^P4(COS9)  Reasonable fits, Fl and F2, to the T2 relaxation data were obtained for all carbon positions, n. A comparison of F1 and F2 for each carbon atom using x  2  methods  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems ^94  C13  F2 2  10  10  10  0.8  0.6^0.4 COS 0  0.2  00  10  01.8  0.8  0.8  0.6^0.4 COS 0  0.2  00  0.6^0.4 COS  02  00  0.6^0.4 cos 0  02  00  Figure 5.3: T2 1 values (open rectangles) of representative carbon positions of macroscopically oriented POPC d31 (left panel)and POPC:POPE d31 50:50 (right panel) and the corresponding fits to the data are presented. The data were fitted (solid curves) by F1) 1/T 2 = A + C[P 2 (cosO)] 2 and F2) 1/T 2 = A o A 2 P2 (cos9) A 4 P4 (cos9). All samples were measured at 30°C.  Chapter 5. Anisotropy of  T2 Relaxation in Pure and Mixed Lipid Systems^95  indicated that the fit to equation 5.2 was slightly better in most cases (as can be seen by eye). It is possible to relate the coefficients Atli of equation 5.2, to terms A 0-11n) , B ( n) and C(n) via equations 5.3 - 5.5,  A,(92 ) = AV +  —2  15  B (n) + —1 c(n) 5  A (22 ) = 42 ).f + 2 B(n) + c(n) 1 7 LEL „,„( n ) + 18 c ( n) 420 = A (n)f —^ l-1  42^35 -0,  35  and compare the relative contributions of fluctuations in 0 and thickness to T2 relaxation using the ratio A42 ) /A2 ) . This ratio (calculated for each carbon position) is presented in Table 5.1. 1 (Values of A2(n2 )1 , B (n) , and C(n) are given in Appendix A). Note that this is close to or larger than 1 in most cases. For A 4( n2 ) /A n2 ) r-::% 1, the ratio B'(n)  2  /O n) must be;.:-., 0.175. Thus the contribution from C(n) is larger than from BV(n) for the POPC d31 system studied. We note that if W ( n)(or On)) was zero, the ratio A42 ) /A2 ) would be 1.8(or -2.4). Recall that Bqn) and C(n) refer to the relative magnitudes of contributions from fluctuations in 0 and <S> respectively. Thus thickness fluctuations appear to provide the more dominant contribution to T2 relaxation in the POPCd31 oriented system.  (n f It is of interest to compare the contributions of A02) , the B(n) and C(n) terms to A0 n2 ) in equation 5.3 above and their maximum contributions to the overall T2 relaxation. 'In order to proceed we assume that 42 )1 = A4( 2 )1 = 0 (see Appendix B). This is a reasonable first approximation since A r'2 ) 'f and A '22 ) 'f contribute to both T 1 and T2 relaxation and it is generally found that the orientation dependence of T2 relaxation times is stronger than the orientation dependence of T 1 . Whereas a variation of 20-30% is observed in T1 measurements of the deuterons on the acyl chain (Morrison and Bloom , unpublished results) roughly a factor of 5 is observed for most carbon positions in T2 measurements. 2 The maximum value which sin 2 Ocos 2 0 can attain is one quarter that of [P2(cosO) 2 . Thus we define Bqn)=B(n)/4.  Chapter 5. Anisotropy  of T2  Table 5.1: Ratios of and A02 values  (F2)fit coefficients for POPC d31 and POPC/POPE d31 bilayers  T2  Relaxation in Pure and Mixed Lipid Systems^96  POPCd31 Carbon Number C15 C14 C13 C12 C11 C10 C9 PI  POPC/POPEd31 its -1 A02 x10-4/28-1  A42/A22  A02 X10 -4 /18 -1  A42/A22  0.95 ± 0.20 0.90 + 0.18 1.11 ±0.16 1.08 + 0.11 1.54 + 0.097 0.92 ± 0.41 0.91 ± 0.32 1.05 + 0.095  1.90 + 0.12 2.62 + 0.12 3.03 + 0.12 3.30 + 0.09 3.78 + 0.10 3.97 ± 0.40 4.69 ± 0.29 5.37 + 0.12  0.74 ± 0.30 1.19 ± 0.31 1.14 + 0.28 1.58 + 0.18 1.17 + 0.14 0.92 ± 0.42 0.64 ± 0.45 0.98 ± 0.25  2.22 + 0.167 2.93 + 0.23 3.54 + 0.24 3.81 + 0.17 4.21 + 0.15 5.11 ± 0.47 5.46 ± 0.40 6.09 + 0.34  For most carbon positions we find that A 0( 7.2L)f (2/15)B(') (1/5)C(n ) . This may be expected since A02 )f is due to angular independent relaxation processes and suggests that angular independent relaxation processes make an important contribution to the overall  T2  relaxation in these membrane systems.  In order to get a rough estimate of possible correlation times for any surface undulations present in these systems we have used values of B(n) determined from equations 5.3, 5.4 and 5.5 above. In making these estimates we assume a certain amplitude,  <(89) 2 >, for surface undulations and obtain correlation times from the measurement of  T2.  Thus for B a 71, n) (see Appendix B for details), we estimate that such correlation  times are of the order of 10 -6 - 10 -7 seconds. These are somewhat shorter than in the headgroup-labelled powder samples. Thickness fluctuations should be further pronounced in the fluid phase of a mixed system containing lipids of different intrinsic hydrophobic thicknesses. Since thickness differences had been observed in POPC:POPE d31 dispersions as a function of the ratio  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems ^97  of the two components (Lafleur et al., 1990), a mixture of these lipids was a first choice. Thus T2 measurements were made on oriented samples of POPC: POPE d31 (50:50 mol%). For comparison with pure POPC d31 results, a plot of the T2 data for corresponding CD 2 labelled carbon positions of the palmitoyl chain as a function of is presented in Figure 5.3 (right panel). The values A 4( 2 ) /A (22 ) are presented together with those for the POPC d31 system in Table 5.1. As in the POPC d31 oriented samples, the fits, F2, are slightly better than fits, Fl (determined by x 2 ). Compared with the POPC d31 sample, the ratios  A42)/A2)  are the same within experimental error indicating  that contributions to relaxation from fluctuations in 0 and <S> are similar in both the POPC d31 and POPC:POPE d31 oriented samples. Thus, in the mixed oriented system, the component due to thickness fluctuations appears to make a larger contribution to T2 relaxation than does the component due to fluctuations in 0. However, thickness  fluctuations do not appear to be "enhanced" over those in the  POP Cd31  oriented sample.  Measurements of ternary lipid bilayers are included since they provide a striking demonstration of the influence of hydrophobic mismatch on T2 relaxation. The membranes used were POPC d31 based multilamellar vesicles (powder-like) containing either cholesterol and Leu24 peptide, where the peptide hydrophobic thickness matches that of its membrane environment, or containing cholesterol and Leu16 peptide (Nezil and Bloom, 1991), where the hydrophobic length of the peptide was short causing the average bilayer thickness to decrease by 10%. We can anticipate that POPC d31 molecules undergoing diffusion will encounter regions of very different thicknesses here. Thus lateral variation in thickness obtained in this manner should be reflected in the T2 anisotropy across the spectrum. Accordingly, T2 was measured for three powder systems: 1) POPC d31 multilayers containing 30mol% cholesterol; 2) System 1 containing 30% Leu24 peptide by weight; and 3) System 1 containing 30% Leul6 peptide by weight. The relaxation spectra of these are presented in Figure 5.4. The shapes of  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^98  the relaxation spectra are very distinctive. It is evident that those for systems 1 and 2 have the same general features in that corresponding edges and the shoulders show similar relaxation times. We interpret these results to mean that the anisotropy of T2  relaxation for all labelled CD 2 positions could be fit by a function that includes  sin 2 Ocos 2 0 terms. However, the relaxation spectrum for system 3 is clearly different. It is suggested, from the shape of this relaxation spectrum, that the mechanism of relaxation could be described by a function involving [P 2 (cost9)] 2 terms, as indicated by the theory for random thickness fluctuations. Finally, some preliminary T2 relaxation studies were performed using A. laidlawii membranes containing the fatty acid composition, 16:0d31/14:0 to determine if such relaxation techniques can be applied to biological membranes. This system was chosen because of some results of Chapter 3, where intact membranes containing these fatty acids appeared to be thinner than their derived liposomes. Thus certain features of the frequency spectra of B2 and B2 + Leul6 membranes are similar to the derived liposomes and intact membranes, respectively, of A. laidlawii containing 16:0d31/14:0 fatty acids. The relaxation spectrum and Pake-doublets spectrum taken at r = 50fts (for reference) are presented in Figure 5.5. Although noisy, we find that similar features of the T2 relaxation spectra of B2 and B2 + Leul6 membranes also appear in the  T2 relaxation spectrum of this A. laidlawii membrane system. This suggests that surface undulations and/or thickness fluctuations may also be important in this biological membrane system.  ^  Chapter 5. Anisotropy of  7  T2  Relaxation in Pure and Mixed Lipid Systems^99  1  6  1 0 i^I^I^I^1^I —40 —30 —20 —10 0^10 20 Frequency (kHz)  30  Figure 5.4: Relaxation spectra are shown for the POPC d3i :Cholesterol bilayer containing 30mol% cholesterol B2 (solid line), with 30% (by weight) Leul6 peptide (double line) or with 30% (by weight) Leu24 peptide added. All samples were measured at 30°C. (Plot is courtesy of Dr. F. Nezil.)  40  Chapter 5. Anisotropy of  —30  ■  —20  T2  Relaxation in Pure and Mixed Lipid Systems^100  1^1^i —10^0^10 Frequency (kHz)  20  30  Figure 5.5: A) Relaxation spectrum and B) Superposition of powder patterns for reference are shown for the A. laidlawii intact membranes grown using the fatty acids, 16:0d31 and 14:0. The sample was measured at 37°C.  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^101  5.5 Discussion Some of the earliest observations of surface undulations were obtained from light microscopic studies of erythrocytes (Brochard and Lennon, 1975). Scattering of the incident light was due to random changes in the surface normal of the erythrocyte membrane producing an effect which is commonly known as the "flicker effect". More recent observations, also at long wavelength scales, were made using giant vesicles (Evans and Rawicz , 1990) where it was possible to observe undulations under the light microscope. From the long wavelength measurements, Evans and Rawicz (1990) found that it was possible to predict undulations occurring on the mesoscopic (— 200 A) length scale. Only a few investigators have directly studied these and similar types of motions on the mesoscopic length scale, which is within the realm of 2 H-NMR transverse relaxation measurements (Bloom and Evans, 1991; Stohrer et al., 1991, Watnick et al., 1991). We have studied several different membrane systems both macroscopically oriented multibilayers and multilamellar vesicles using 2 H-NMR T2 relaxation methods to derive information, on the types and modes of motions dominant in lipid membrane systems. It appears that the headgroup deuteriated a-DPPC d2 and chain deuteriated POPCd31 samples in the fluid phase show T2 relaxation processes clearly consistent with motions resulting from fluctuations of the orientation of the local surface normal. Results using chain deuteriated oriented samples, surprisingly, give a different dominant relaxation mechanism. The main mechanism suggested for the oriented samples is supported by a phenomenological theory of thickness fluctuations which describes T2 relaxation due to random diffusion of lipid molecules through regions of different thickness. These results are discussed within the framework of the theory for surface undulations and thickness fluctuations. It is assumed throughout that the influence of dipolar interactions to relaxation are negligible.  Chapter 5. Anisotropy of  T2  Relaxation in Pure and Mixed Lipid Systems ^102  It is of interest to compare the modes of surface undulations in the a-DPP Cd2 membranes and the macroscopically oriented POPCd 31 multibilayers using the estimated  re(n)values and the angular dependence of the relaxation times obtained. The  values of Tt(i n) can be interpreted as averages over all undulational modes and longer values correspond to longer wavelengths. Then, for TP ) --:-., 10 -5 s, longer wavelength modes must be present, on average, than for 7-P ) ,c..--, 10 -6 -10 -7 s. This suggests that longer wavelength modes present in the powder a-DPPC d2 samples are damped out in the oriented multibilayers. Thus the relative contribution of surface undulations to relaxation in the oriented samples could be expected to be small. Furthermore, a small contribution to relaxation from thermal fluctuations in the orientation of the surface normal in the oriented samples is qualitatively indicated by the lack of similarity of T2  relaxation times at the edge and shoulder orientations. Quantitively the ratios of  A42 ) /A22) ;,-, 1 suggest that the contribution from undulational type motions is .c.. --, 1/5 that of fluctuations in thickness in both oriented multibilayer systems presented. The above observations are also supported by a comparison of T2 values in oriented and powder POPC d31 samples. The average value of T2 measured for such a powdersystem was 1515 ,us. The average value of T2 in the corresponding oriented sample, calculated from values measured at all carbon positions for all angles and weighted by siner, is1513 ,us, significantly longer than that in the powder. Thus some motions contibuting to transverse relaxation in the powders must be absent in the oriented samples. We suggest that the On) term contributing to the relaxation in the oriented samples can also be present in the powder samples. To demonstrate this we examine the POPC d31 relaxation spectrum (cf. Figure 5.2) and the A02 )-f and On) (see Appendix A) values of the plateau region at 0 = 0. At this angle the intensity of the plateau region 'Sin 0 represents the probability density of nuclei with a given value of 0 in a powder sample  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^103  is relatively clean (i.e. contains essentially no intensity from other nuclei or angles) and surface undulations do not contribute to T2 relaxation. The 1/T 2 value of the powder spectrum in this region, Re, 2000 s -1 , is larger than the sum Arniateauf cptateau, ti 1200 5 -1 suggesting that the CP l a t eau term may contribute at least as much to T2 relaxation in the powder as in the oriented sample. At the present time one can derive only relative contributions to T2 relaxation from thickness fluctuations. It is suggested here that these are dominant in the macroscopically oriented multibilayers investigated. It is possible to estimate some plausible upper and lower bounds for correlation times due to random thickness fluctuations assuming that these arise from random diffusion through regions of different thickness. Using an approximation for these correlation times (see Appendix B), Te = < A >/D, D = 4 x 10' cm 2 /s (a typical diffusion constant for some fluid membranes (Bloom et al, 1978; Cullis, 1976)), and an appropriate range of areas, we can estimate the correlation times over which the phenomenological theory describing thickness fluctuations is valid. Since the average cross-sectional area for a lipid molecule such as POPC is 40-60 A 2 the motions must be slower than 10 -8 s. At the other extreme, the correlation time  must be faster than the magnitude of T2 (< 7 ms for the longest T2 values measured here) for the motion to be a mechanism for relaxation. Thus 10's < r < 10's gives a range of allowable correlation times. As an example of a plausible correlation time, a value of 7, of the order of 10,as would allow a lipid molecule to diffuse over an area of 4000 A 2 , Pi 70-100 lipid molecules. The relative contributions to T2 relaxation from fluctuations in <S> and 0 in oriented POPC d31 and POPC:POPE d31 are of interest. One would anticipate that the mixed system containing lipids of different intrinsic thickness should show a larger contribution from fluctuations in <S> than from fluctuations in 0. The similarity in the ratios A 4( '2'. ) /A  )  1) for all carbon positions of both POPCd 31 and POPC:POPEd31  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^104  oriented samples suggests that there is no relative increase in fluctuations in <S> for the mixed system. Thus if such differences occur, they are not detectable by these methods perhaps because, by <S>, the hydrophobic lengths (measured by <S>) of POPC d31 and POPE d31 membranes differ by only a few percent. It could be useful, with similar methods, to study DMPC:DSPC oriented samples for example, where hydrophobic lengths of the individual components are different by more than 10%. As indicated in the Results section the POPC d31 :Chol system containing either the Leul6 or Leu24 peptide provides an elegant system for the study of the influence of thickness differences in a membrane. Significant qualitative information can be derived from the data. For the POPC:Chol and POPC:Chol: Leu24 systems, a distinctive shape of the relaxation spectrum was obtained. The angular dependence of relaxation is clearly consistent with an undulational type of lipid motion mentioned earlier. However, in membranes containing the short peptide, Leu16, the dominant relaxation mechanism, suggested by the shape of relaxation spectrum, appears to be provided by random thickness fluctuations. These membrane systems containing a specifically labelled lipid could provide excellent model systems for further testing of such theories. Using the biological membrane A. laidlawii B, as much as could be discerned, features of the relaxation spectrum resembled both, spectra exhibiting sin 2 0cos 2 9 relaxation behaviour and [P 2 (cose9)] 2 relaxation behaviour. If membrane topology was such that some regions of the membrane were protein deficient and others were protein enriched (assuming that lipid-protein hydrophobic mismatch was occurring) then the predominant lipid fluctuations in each of these regions could be from fluctuations in 0 and <S> respectively. It is evident that one must initially clarify the relative contributions of these types of fluctuations in model and then biological membranes. In general the hydrophobic regions of proteins and lipids in natural membranes are probably relatively well matched under normal conditions. One would expect that  Chapter 5. Anisotropy of T2 Relaxation in Pure and Mixed Lipid Systems^105  in lipid-protein or lipid-lipid interactions there is a threshold level of mismatch above which segregation of individual species occurs (Mouritsen and Sperotto, 1991). Indeed it has been demonstrated that the presence of > 50% gel state lipid (thick membranes), substantially reduces the activity of certain integral membrane proteins ( see McElhaney, 1984; Mouritsen and Sperotto, 1991 for reviews); presumably since high protein activity cannot exist with this level of gel-state lipid in the membrane. In summary, the results presented here indicate that both surface undulations and random thickness fluctuations can provide dominant mechanisms for T2 relaxation in membrane systems. Surface undulations or other types of collective motions have been suggested for some systems (Bloom and Evans, 1991; Stohrer et al. 1991). Thickness fluctuations are motions which we suggest to be present in membrane lipid bilayers, particularly pronounced where significant hydrophobic mismatch of membrane components occurs. The establishment of the presence of such motions in membranes with further study may suggest certain consequences for membrane function and dynamics.  Chapter 6  Future Directions  It is conceivable that the range of order consistent with the limits of growth of A. laidlawii as identified in chapters 3 and 4 would be similar in a wide range of fluid plasma membranes. However, for membranes containing high levels of cholesterol, one would expect a significant increase in the order observed. For instance, the membrane lipids of model stratum corneum, (ceramide, cholesterol and free fatty acids 1:1:1) appear to be highly ordered and potentially inhomogeneous mixed in the membrane. An examination of the average order/thickness in other biological membranes would be of interest to determine the distribution of thicknesses present in active membranes. Diffusion rates and mechanisms can provide significant information about the lateral distribution of membrane components and their miscibility. Such techniques would be useful in clarifying some questions which have arisen during the course of this thesis. It is noted that several techniques have been developed to measure diffusion in membranes. In particular the pulsed field gradient NMR methods of Lindblom et al. (1976, 1977) enable one to study not only diffusion rates, but to identify non-micellar isotropic phase  structure and the presence of regions of bounded diffusion in membrane bilayers. Such methods could be useful in the study of, for instance, A. laidlawii membranes such as those containing the fatty acid combination 16:0/14:0 discussed in chapters 3 and 5. Some questions which arise from this work are whether an inhomogeneous distribution of membrane constituents exists in this system and if so, to what extent. Fluorescence recovery after photobleaching (see Knowles, 1992) and 2D  106  31 P-NMR  Chapter 6. Future Directions ^  107  (Fenske and Jarrell, 1991) methods have been identified as useful techniques in measuring diffusion constants. We have attempted to directly compare the diffusion coefficients, D, in POPC and POPE membranes with these two techniques. The POPC diffusion coefficients D 3x10 -8 cm 2 /s and 5x10 -7 cm 2 /s from FRAP and  31 P  NMR  respectively are comparable. However, the POPE diffusion coefficients are different by more than an order of magnitude D 2.5x10 - 8 cm 2 /s and 5x10 -7 cm 2 /s from FRAP and  31 P-NMR  measurements respectively. Difficulties with both techniques are identi-  fied. For instance, using 2D  31 P  NMR techniques an accurate vesicle size is necessary  for the correct determination of D. This is not always easily achieved with standard techniques particularly if vesicle aggregation and/or a large distribution in size or shape are common (as may be the case with POPE vesicles). The requirement of a fluorescently labelled lipid, accurate definition of the bleach spot size, temperature regulation and vesicle manipulation are often drawbacks using FRAP techniques particularly if homogeneity of vesicle constituents is compromised. We note that in particular, improvements in the NMR methods to better define the distribution of vesicle shapes and sizes is necessary to define an accurate value of D for these measurements. The slow motions present in membranes are of interest. The results identifying the various motions, fluctuations in 9 and thickness, in the POPC d31 and POPC:POPE d31 systems and the Leu16/Leu24 containing membranes provide qualitative information on the motions predominant in these systems. Further development of the theory describing the T2 relaxation mechanisms will provide more quantitative methods of identifying the motions responsible for the relaxation. We hope that the eventual extension of these methods to biological membranes, such as A. laidlawii strain B of various fatty acid compositions, will achieve further understanding of the dynamics of biological lipid membranes.  Chapter 7  Bibliography  Abragam A. (1961) Principles of Nuclear Magnetism London:Oxford University Press. Auger, M., Carrier, D., Smith I.C.P. and Jarrell, H.C. (1990) J. Am. Chem. Soc. 112, 1373-1381. Auger, M.A., Smith, I.C.P and Jarrell, H.C. (1991) Biophys. J. 59, 31-38. Bhakoo, M., Lewis, R.N.A.H and McElhaney, R.N. (1987) Biochim. Biophys.  Acta 922, 344-345. Bhakoo, M., and McElhaney, R.N. (1988) Biochim. Biophys. Acta 945, 307-314. Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917. Bloch, K. 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(1985a)  Biochemistry 24, 177-184. MacDonald, P.M., Sykes, B.D. and McElhaney, R.N. (1985b) Biochemistry 24, 2237-2245. Marcelja, S. (1974) Biochim. Biophys. Acta. 367, 165-176. Marsh, Derek (ed.) (1990) Handbook of Lipid Bilayers, CRC Press Inc., Boca Raton, Florida. McElhaney, R.N. and Tourtellotte, M.E. (1970) Biochim. Biophys. Acta. 202, 120-178. McElhaney, R.N. (1974) PAABS REVISTA 3, 753-767.  Bibliography^  114  McElhaney, R.N. (1982) Chem. Phys. Lipids 30, 229-259. McElhaney, R.N. (1984) Biochim. Biophys. Acta. 779, 1-42. McElhaney, R.N. (1986) Biochem. Cell. Biol. 64, 58-65. McElhaney, R.N. (1989) CRC Crit. Rev. Microbiol. 17 1-32. McElhaney, R.N., DeGier, J. and van Deenen, L.L.M. (1970) Biochim. Biophys.  Acta. 219, 245-247. McElhaney, R.N., DeGier, J. and Van Der Neut-Kok, E.C.M. (1973) Biochim.  Biophys. Acta. 298, 500-512. Monck, M.A., Bloom, M., Lafleur, M., Lewis, R.N.A.H., McElhaney, R. N. and Cullis, P.R. (1992) Biochemistry 31, 10037-10043. 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Lipids 9, 69-81. Oldfield, E., Meadows, M. and Glaser, M. (1976) J. Biol. Chem. 251, 6147-6149. Pauls, K.P., MacKay, A.L. and Bloom, M. (1983) Biochemistry 22, 6101-6109. Perly, B., Smith, I.C.P. and Jarrell, H.C. (1978) Biochemistry 17, 2727-2740. Philips, M.C., Johnson, W.J. and Rothblat, G.H. (1987) Biochim. Biophys.  Acta., 906, 223-276. Rance, M. (1980) Ph.D. Thesis, University of Guelph, Guelph, Ontario, Canada. Rance, M., Jeffrey, K.R., Tulloch, A.P., Butler, K.W. and Smith, I. C.P. (1982)  Biochim. Biophys. Acta. 688, 191-200. Rance, M. and Byrd, R.A., (1983) Obtaining high-fidelity spin-1/2 powder spectra in anisotropic media: phase-cycled Hahn echo spectroscopy. J. Magn. Reson., 52 221-240. Razin, S. (1982) in Organization of Prokaryotic Cell Membranes (Ghosh, B. K., Ed.) CRC Press, Boca Raton, Fl. Read, B.D. and McElhaney, R.N. (1975) J. Bacteriol. 123, 47-55. Reif, F. (1965) Fundamentals of Statistical and Thermal Physics, McGraw-Hill, New York.  Bibliography^  116  Rilfors, L., Lindblom, G., Wieslander, A. and Christiansson, A. (1984) Lipid Bilayer Stability in Biological Membranes In Membrane Fluidity, eds. Morris Kates and Lionel Manson, 205-245, Plenum Publishing Corporation. Rilfors, L., Wikander, G. and Wieslander, A. (1987) J. Bacteriol. 169, 830-838. Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 847-851. Rommel, E., Noack, F., Meier, P. and Kothe, G. (1988) J. Phys. Chem., 92, 2981-2987. Rose, M.E. (1957), Elementary Theory of Angular Momentum Wiley and Sons Inc., New York. Rottem, S. (1979) in The Mycoplasmas (Barile, M.F. and Razin, S., Eds.) Vol. 1, Academic Press, New York. Saito, Y., Silvius, J.R. and McElhaney, R.N. (1977) Arch. Biochem. Biophys. 182, 443-454. Seelig, J. and Seelig, A. (1974) Biochemistry 13, 4839-4845. Seelig, J. and Seelig, A. (1977) Biochemistry 16, 45-50. Seelig, J. (1977) Q. Rev. Biophys., 10, 353-418. Seelig, J. and Seelig, A. (1980) Q. Rev. Biophys. 13, 19-61. Seelig, J. and Waespe-Sarcevic, N. (1978) Biochemistry 17, 3310-3315. Siegel, D.P., Burns, J.L., Chestnut, M.H. and Talmon, Y. (1989) Biophys. J. 56, 161-169.  Bibliography^  117  Silvius, J.R. and McElhaney, R.N. (1978) Can. J. Biochem. 56, 462-469. Silvius, J.R. (1979) Ph.D. Thesis, University of Alberta, Edmonton Alberta, Canada. Silvius, J.R., Mak, Nanette and McElhaney, R.N. (1980) Biochim. Biophys. Acta. 597, 199-215. Singer, S.J. and Nicolson, G.L. (1972) The fluid mosaic model of cell membranes.  Science, Wash., 175, 720-731. Sternin, E. (1985) Data acquisition and processing: a systems approach Rev. Sci.  Instrum., 56, 274-282. Sternin, E., MacKay, A.L. and Bloom, M. (1983) J. Magn. Reson. 55, 274-282. Sternin, E., Fine, B., Bloom, M., Tilcock, C.P.S., Wong, K.F. and Cullis, P.R. (1988) Biophys. J. 54, 689-694. Stockton, G.W., Johnson, K.G., Butler, K.W., Polnaszek, D.F., Cyr, R. and Smith, I.C.P. (1975) Biochim et Biophys. Acta. 401, 535-539. Stockton, G.W., Johnson, K.G., Butler, K.W., Tulloch, A.P., Boulanger, Y., Smith, I.C.P., Davis, J.H. and Bloom, M. (1977) Nature 269, 267-268. Stockton, G.W. and Smith, I.C.P. (1976) Chem. Phys. Lipids 12, 251-263. Stohrer, J., Graner, G., Reimer, D., Weisz, K., Mayer, C., and Kothe, G. (1991) Collective lipid motions in bilayer membranes studied by transverse deuteron spin relaxation. J. Chem. Phys. 95, 672-678.  Thewalt, J., Kitson, N., Araujo, C., MacKay, A.L. and Bloom, M. (1992) Biochem.  Biophys. Res. Commun. 188, 1247-1252.  Bibliography^  118  Thurmond, R.L., Dodd, S.W. and Brown, M.F. (1991) Biophys. J. 59, 108-113. Valic, M.I., Gorrisen, H., Cushley, R.J. and Bloom, M. (1979) Biochemistry 18, 854-859. Vance, D.E. and Vance, J.E. (eds.) (1984) Biochemistry of Lipids and Membranes, The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Cali-  fornia. 25-72. van der Neut-kok, E.C.M., De Gier, J., Middelbeek, E.J. and van Deenen, L.L.M. (1974) Biochim. Biophys. Acta. 332, 97-103. Vist, M.R. and Davis, J.H. (1990) Biochemistry 29, 451- 464. Watnick, P.A., Dea, P. and Chan, S.J. (1990) P.N.A.S., 87, 2082-2086. Watson, D. (1960) Clin. Chim. Acta. 5, 637-643. Wieslander, A. and Selstam, E. (1987) Biochim. Biophys. Acta. 901, 250-254. Wieslander, A., Christiansonn, A., Rilfors, L. and Lindblom, G. (1980) Biochemistry 19, 3650-3655.  Appendix A  Table A.1: Values of A, (L )f , 13 ( n ) and C(n ) for POPC d31 and POPC/POPE d31 fits POPC/POPEd31 s -1 POPCd31 s -1 A fl2 )f Carbon B(n) B n) C(n) A(j2)f B (n) Number (n) C15 61.6 324.3 427.3 71.4 455.1 450.4 ± 25.6 + 122.6 + 31.6 + 36.5 + 174.5 + 44.6 C14 120.1 376.3 461.3 155.5 262.8 514.5 ± 25.0 + 119.7 + 30.8 + 51.2 ± 24.4 + 62.4 C13 160.5 301.6 513.7 195.6 323.9 575.6 ±25.2 ± 120.7 ± 31.1 ± 51.9 ± 247.8 ± 63.3 C12 164.1 364.4 588.0 206.5 304.2 670.0 ± 19.8 + 95.0 ± 24.4 ± 38.1 +18.2 ± 46.5 C11 230.6 127.5 651.1 224.8 383.6 723.3 + 20.7 + 99.0 ± 25.5 + 32.4 ± 154.6 ± 39.5 C10 184.3 550.8 698.5 259.3 653.0 824.5 + 85.4 + 409.2 ± 105.3 ± 102.6 + 489.7 ± 125.2 269.1 522.5 652.7 C9 272.2 885.1 778.2 ± 60.6 ± 290.7 + 74.7 ± 87.7 ± 418.8 + 107.0 P1 289.4 565.9 863.2 318.2 710.3 978.6 ± 25.5 ± 122.0 + 31.4 ± 73.7 ± 351.6 ± 89.9 (  119  Appendix B  Phenomenological Theory of Transverse Relaxation in Membranes  Effect of molecular motion on 2 H NMR properties  1  The NMR spectra of fluid bilayer membranes exhibit axial symmetry with respect to the bilayer normal 71. We mean by this that molecular motions are axially symmetric with respect to ii so that, for example, in the presence of an external magnetic field H., the averaging of the quadrupolar interaction of a spin-1 nucleus by the molecular motions that are fast on the "NMR time scale" gives rise to a quadrupolar splitting 2w which depends only on the angle 0 between H. and 77. More precisely, for a deuteron ( 2 H) replacing a H on a C-H bond and approximating the electric field gradient (efg) as an axially symmetric tensor about the C- 2 H bond characterized by an angular frequency  w Q P-- 27r x 1.25 x 10 5 s -1 (Davis, 1983; Seelig, 1977) , one can express the splitting parameter in the form  co = wQP2(cos0)Scp (B.1) where P2 (,u) = (3u 2 — 1)/2 is a Legendre polynomial, and the orientational order parameter  Sap =  S20 = < P2(cos/3) -5fastmotions is a measure of the averaging of the  quadrupolar interaction due to modulation of the angle 0 between the C- 2 H bond direction and it' by fast motions. The motions that give rise to this motional averaging are also responsible for longitudinal (spin-lattice) and transverse relaxation, commonly characterized by the time 1  This appendix was a contribution from Dr. Myer Bloom 120  Appendix B. Phenomenological Theory of Transverse Relaxation in Membranes 121  constants T 1 and  T2  respectively. The relaxation rates may be conveniently expressed  in terms of the spectral densities of the correlations functions of the fluctuating spindependent interactions. The correlation functions are characterized in turn by a spectrum of correlation times 7 -. (Davis, 1983; Abragam, 1961). The relevant spectral densities contributing to T i-1 are evaluated at w o and 2w o , where w o is the angular Larmor frequency in the field H., while T2 1 has contributions from spectral densities evaluated at zero frequency, in addition to those evaluated at w o and 2w o . From a definitive study of the dependence of T i-1 on w o in DMPC extending to very low magnetic fields (Rommel et al., 1988), it appears that the correlation times fall into two classes, those associated with relatively fast motions having values Tc < TIPs.'-, 10 -8 s that contribute to the field dependence of T1 1 only at high fields, i.e . corresponding to w o > 27r x 10 7 s -1 , and those associated with relatively slow motions having values of Tc > Tcs ''.'"', 10 -6 s that contribute appreciably to T i-1 only at very low magnetic fields. The  slow motions would be expected to contribute to T2 1 , but not to T i-1 under the conditions of the experiments reported in this paper . We shall make use of this empirical demonstration of the clumping of the correlation times into two categories, long and short, to develop a tentative model for transverse relaxation in terms of two distinct contributions as follows:  1  — = Rs + R2  (B.2)  T2 2  where 11,12 may be inferred from T 1 measurements, and especially from the dependence of T 1 on w o . It is assumed to have its origin in motions for which Tc < 7 1. In the -  same spirit a theory for 14 may be constructed using approximations appropriate for relatively slow motions. Such a theory is presented in the following paragraphs where  we characterize .IT; as the adiabatic contribution to the transverse relaxation rate.  Appendix B. Phenomenological Theory of Transverse Relaxation in Membranes 122  One of the features of transverse relaxation of special interest is its orientation dependence. A general form for the orientation dependence of spin-lattice and transverse relaxation rates for spin-1 systems, valid within the Redfield approximation, has been derived recently (Morrison and Bloom, 1993). For axial symmetry, this takes the form  1^A^A Di^n\^A ni^n\ y.,. =^11-2i/-2 COSu) ii4iFACOSu) -  (B.3)  for Ti = T1 z , T i , or T2, where the coefficients A 0 1, A2, and A4, are dependent on co., correlation times, the orientational order parameters S20 and S40 and the thermodynamic variables required to characterize the system. Adiabatic Contributions to Transverse Relaxation We suppose that the fast molecular motions discussed above establish a quadrupolar splitting 2w given by equation B.1 in terms of Sc.!) and 0. The slower motions modulate w adiabatically, where the word adiabatic is used in the same sense as in pages 427ff of the "bible" of Abragam(1961). We assume that the adiabatic motions may be divided into two classes, those that modulate 0 and SCD independently, with effective correlation  times Teo and  TcA,  respectively. We associate these motions with:  (i) fluctuations in local curvature that give rise to changes in 9 without any appreciable changes in bilayer thickness, and, consequently, without changes in SCD. (ii) fluctuations in bilayer thickness x or equivalently the membrane area A, that give rise to fluctuations in SCD without any appreciable changes in 0. It is emphasized that the assumption of independence of these types of motions cannot be justified on theoretical grounds at the present time. It is likely that the actual modes responsible for the adiabatic motions involve a coupling between membrane curvature and thickness fluctuations. The assumption of codependence should be considered as a first approximation in the establishment of a rigorous theory of adiabatic  Appendix B. Phenomenological Theory of Transverse Relaxation in Membranes 123  motion. Explicit connections between x, A and  Sap for acyl chains have been discussed by  several authors (see, e.g. Ipsen et al., 1990). Thermodynamic considerations allow one to relate the mean squared fluctuations < (60) 2 > and < (SA) 2 > to measurable properties of membranes. These two types of fluctuations are governed, under some conditions, by the so-called curvature energy K, (Bloom and Evans, 1991) and the isothermal area compressibility (1/K a ) (Bloom et al., 1991; Lipowsky, 1991), respec-  tively, as follows:  < (80)2 >,_< 92 > < >2_ kBT  Am  4rn,  A < (86) 2 >,-‹ A 2 > <  > 2 = BT Ka  (B.4)  (B.5)  where A m and A, in B.4 are the maximum and minimum wavelengths for curvature fluctuation modes, respectively, and K a is defined in terms of the fractional variation of area with surface tension u as 1/K a = (1/A)(OA/Oa)T. Expressions analogous to equation B.5 can be found in many texts (see, eg. Reif, 1965; page 300) for volume fluctuations in three dimensional systems. It is easy to show that the thermal fluctuations in 0 and A, given by equations B.4 and B.5 respectively, give rise to renormalizations of the quadrupole splitting of only a few percent, which may be ignored for present purposes. For example curvature fluctuations lead to a reduction in Scp by < P2 (cas60) > 1-3/2< (60) 2 >. From the values used in the paper by Bloom and Evans (1991) of n a P.,- 5 x 10'ergs and A m /A m 20, equation B.4 gives < (60) 2 > 1.8 x 10 -2 at room temperature. Similarly, the typical value of K a 200 dynes/cm for fluid membranes (Needham and Evans, 1988), gives k B T/K a 2A 2 << < A > 2 , also leading to small changes in the quadrupole splittings for practical cases.  Appendix B. Phenomenological Theory of Transverse Relaxation in Membranes 124  If these motions give rise to uncorrelated fluctuations in the quadrupolar splitting they result in adiabatic relaxation given by the classic formula (Abragam, 1961) for the transverse relaxation rate arising from a fluctuating interaction characterized by a second moment AM 2i and correlation time  Tc i  satisfying the condition AM 2i 7- << 1,  R;^E R2 i = E Am20-ci^  (B.6)  (8466) A 2 < (89)2 > Tc9 + (sco/aA),2 < (SA)2 > TcA^  (B.7)  = c,), 2 [-i9^• SLj szn 2 Ocos 2 < (69) 2 > 7,9 + [P2(cos9)] 2 [(8ScD/SA)B] 2 < (811) 2 > TcAl Bsin 2 8cos 2 0 C[P2 (cos0)] 2^(B.8) where the coefficients B and C are defined by Eqs. B.5 and B.8. Returning to Eq. B.2, identifying R2 with i =2 and the coefficients A02 , A22 A4 2 in Eq. B.3, and ,  expressing sin 2 0cos 2 0 and [P 2 (cos9)] 2 in terms of the Legendre polynomials, we can write  1^A — /102 A22P2(cosO) A42P4(cosO) T2  (B.9)  where A02 = + 1E 2 B C  +  A22 =^,7 + B 2C  A42 = 4,2 —  LB+ T5c 18  Theoretical Predictions for the B and C coefficients in adiabatic Transverse Relaxation:  Appendix B. Phenomenological Theory of Transverse Relaxation in Membranes 125  A theory for contributions of thermally induced curvature fluctuations to the transverse relaxation rate (i.e. the B coefficient) has been published (Bloom and Evans, 1991). In this theory, the curvature fluctuations are expressed in terms of a spectrum of curvature modes of wavelengths A q having correlation times as a product of terms of the form < (8190 2 > terms of < (890 2 >  Tcq  Tcq .  Furthermore  Tq  Tcq  and B is expressed  may be expressed in  with the help of Eqs. B.4 and B.8. The major uncertainty is  in the damping mechanism. While < (8 9 q ) 2 > can be obtained reliably from thermodynamic considerations and the equipartition theorem,  Tcq  should depend sensitively  on factors such as the degree of hydration, tension and the multi-layer character of the membranes, that, in turn, depend on the method of preparation and thermal history of the membranes. Within the range of uncertainty of these factors, it was concluded that the predicted values of T2 due to this mechanism could easily fall in the observed range of approximately 100 to 1000 ,us typically found for 2 11 NMR in membranes (Bloom and Evans, 1991). We should further emphasize that a second mechanism, associated with order-  director fluctuations (Stohrer et al., 1991), has been proposed for a sin 2 9cos 2 0 contribution to R. Experiment must ultimately distinguish between these different possibilities. It may well be that both contribute to R,,s2 , with curvature fluctuations possibly being of greater importance for spins located in the head-group and order-director fluctuations for spins on the chains. We are unaware of any published theory for the contributions of area (or equivalently  thickness) fluctuations to 112. Again one can construct a theory based on sinusoidal modes of area fluctuations which take into account an empirical or theoretical connection between Sap and membrane area A. In order to get a feeling for the order of magnitude anticipated for the C term in N, we look at the predicted value of C for deuterons on acyl chains for which we have found ( to our surprise) that an area mode  Appendix B. Phenomenological Theory of Transverse Relaxation in Membranes 126  characterized by < A > gives a contribution to R; that is independent of < A >. In order to calculate C in Eq B.8, we first model the hydrophobic region of a fluid membrane as an incompressible fluid and make use of the empirical relationship between bilayer thickness x, its maximum value in the fluid phase x m (or, alternatively, the corresponding membrane area A =< A > with its minimum value A m ) and the average chain order parameter S (Ipsen et al., 1990)  x = ^aS -I- 13, with 0.5a + /3 =1 A xm  (B.13)  We then note that the order parameter profile corresponding to the variation of  Scp with chain position has been found to have a universal form for a given value of S (Lafleur et al., 1990) so that one can express aS cp/aA in Eq. B.8, for a given S, in terms of the experimentally measurable coefficients ECD = aSaDiaS as follows:  aScD Ecn as^r,  aA =  Am  ECD ^  (B.14)  '-  From the definition of C in Eq. B.8, making use of Eq. B.5 and assuming that fluctuations in area are relaxed by spatial diffusion so that  TcA  pA/D, where D is  the translational diffusion coefficient for diffusive motions parallel to the plane of the membrane and p is a dimensionless coefficient of order unity, we write  C = pELL0,Q2 (S  a  )2  kB T  (B.15)  DICa  For the typical values of W Q = 2ir x 1.25 x 10 5 s -1 , T = 300K ,Dcse, 4 x 10 -8 cm 2 s -1 -  and K a 200 dynes cm -1 , we obtain C ti 3000pE6(S+ /3/a) 2 indicating that C is of the same order of magnitude as B; thus both the area and curvature fluctuations are capable of producing values of T2 as short as about 100 its.  


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