Lateral diffusion and fluorescence microscope studies on a monoclonal antibody specifically bound to supported phospholipid bilayers

Supported phospholipid bilayers prepared by Langmuir-Blodgett techniques were introduced recently as a new model membrane system [Tamm, L.K., & McConnell, H.M. (1985) Biophys. J. 47, 105-113]. Here, supported bilayers are applied to study the lateral diffusion and lateral distribution of membrane-bound monoclonal antibodies. A monoclonal anti-trinitrophenol antibody was found to bind strongly and with high specificity to supported phospholipid bilayers containing the lipid hapten (trinitrophenyl)phosphatidylethanolamine at various mole fractions. The lateral distribution of the membrane-bound antibodies was studied by epifluorescence microscopy. The bound antibodies aggregated into patches on a host lipid bilayer of dimyristoylphosphatidylcholine below the lipid chain melting phase transition and redistributed uniformly on fluid-phase supported bilayers. Lateral diffusion coefficients and mobile fractions of fluorescent phospholipid analogues and fluorescein-labeled antibodies were measured by fluorescence recovery after pattern photobleaching. The lateral diffusion coefficients of the membrane-bound antibodies resembled those of the phospholipids but were reduced by a factor of 2 in the fluid phase. The lipid chain melting phase transition was also reflected in the lateral diffusion coefficient of the bound antibody but occurred at a temperature about 3 deg higher than the phase transition in supported bilayers of pure phospholipids. The antibody lateral diffusion coefficients decreased in titration experiments monotonically with increasing antibody surface concentrations by a factor of 2-3. Correspondingly, a relatively small decrease of the antibody lateral diffusion coefficient was observed with increasing mole fractions of lipid haptens in the supported bilayer.     

Lateral diffusion of small compounds in human stratum corneum and model lipid bilayer systems.

An image-based technique of fluorescence recovery after photobleaching (video-FRAP) was used to measure the lateral diffusion coefficients of a series of nine fluorescent probes in two model lipid bilayer systems, dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (40 mol%), as well as in human stratum corneum-extracted lipids. The probes were all lipophilic, varied in molecular weight from 223 to 854 Da, and were chosen to characterize the lateral diffusion of small compounds in these bilayer systems. A clear molecular weight dependence of the lateral diffusion coefficients in DMPC bilayers was observed. Values ranged from 6.72 x 10(-8) to 16.2 x 10(-8) cm2/s, with the smaller probes diffusing faster than the larger ones. Measurements in DMPC/cholesterol bilayers, which represent the most thorough characterization of small-solute diffusion in this system, exhibited a similar molecular weight dependence, although the diffusion coefficients were lower, ranging from 1.62 x 10(-8) to 5.60 x 10(-8) cm2/s. Lateral diffusion measurements in stratum corneum-extracted lipids, which represent a novel examination of diffusion in this unique lipid system, also exhibited a molecular weight dependence, with values ranging from 0.306 x 10(-8) to 2.34 x 10(-8) cm2/s. Literature data showed that these strong molecular weight dependencies extend to even smaller compounds than those examined in this study. A two-parameter empirical expression is presented that describes the lateral diffusion coefficient in terms of the solute's molecular weight and captures the size dependence over the range examined. This study illustrates the degree to which small-molecule lateral diffusion in stratum corneum-extracted lipids can be represented by diffusion in DMPC and DMPC/cholesterol bilayer systems, and may lead to a better understanding of small-solute transport across human stratum corneum.     

Domain formation in model membranes studied by pulsed-field gradient-NMR: the role of lipid polyunsaturation

The effects of increased unsaturation in the sn-2 fatty acyl chain of phosphatidylcholines (PCs) on the lipid lateral diffusion have been investigated by pulsed-field gradient NMR. Macroscopically oriented bilayers containing a monosaturated PC, egg sphingomyelin, and cholesterol (CHOL) have been studied at temperatures between 0 degrees C and 60 degrees C, and the number of double bonds in the PC was one, two, four, or six. For PC bilayers, with and without the incorporation of egg sphingomyelin and CHOL, the lateral diffusion increased with increasing number of double bonds, as a consequence of the increased headgroup area caused by the unsaturation. Addition of CHOL caused a decrease in lipid diffusion due to the condensing effect of CHOL on the headgroup area. Phase separation into large domains of liquid-disordered and liquid-ordered phases were observed in the ternary systems with PCs containing four and six double bonds, as evidenced by the occurrence of two lipid diffusion coefficients. PC bilayers with one or two double bonds appear homogeneous on the length scales probed by the experiment, but the temperature dependence of the diffusion suggests that small domains may be present also in these ternary systems.     

Obstructed diffusion in phase-separated supported lipid bilayers: a combined atomic force microscopy and fluorescence recovery after photobleaching approach

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.     

Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker

There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphology of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coefficients (0.8-1.2 x 10(-8) cm(2)/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b(5) and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were observed in the polymer-supported bilayers. Approximately 25% of cytochrome b(5) diffused with a diffusion coefficient of approximately 1 x 10(-8) cm(2)/s, and 50-60% diffused with a diffusion coefficient of approximately 2 x 10(-10) cm(2)/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of approximately 3 x 10(-9) cm(2)/s, and two-thirds diffused with a diffusion coefficient of approximately 4 x 10(-10) cm(2)/s. A model for the interaction of these proteins with the underlying polymer is discussed.     

An NMR study of the temperature dependence of the coefficient of water self-diffusion through lipid bilayer membranes

The temperature dependence of the coefficient of water self-diffusion through plane-parallel lipid multilayers of the phospholipid dioleoylphosphatidylcholine oriented on a glass support has been studied in the temperature range of 20-60 degrees C by the method of NMR with magnetic field pulse gradient. The values of the coefficients of transbilayer water diffusion are by four orders of magnitude less than for bulky water and ten times less than the coefficients of lateral diffusion of the lipid under the same conditions. The temperature dependence of the coefficient of water diffusion is described by the Arrhenius law with an apparent activation energy of about 41 kJ/mol, which far exceeds the activation energy for the diffusion of bulky water (18 kJ/mol). The experimental data were analyzed using a "dissolving-diffusion" model, by simulating the passage of water through membrane channels, and by analyzing the exchange of water molecules in states with different modes of translation mobility, including pore channels and bilayer "defects". Each of the approaches used made it possible to take the significance of bilayer permeability for the apparent energy of activation of water diffusion into account and estimate the energies of activation of water diffusion in the hydrophobic moiety of the bilayer, which were found to be close to the values for bulky water. The coefficients of water diffusion in the system under examination and the coefficients of permeation of water through the bilayer were estimated, and the effect of bilayer "defects" on the coefficients of water diffusion along and across bilayers was studied.     

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.     

Lateral diffusion coefficients of separate lipid species in a ternary raft-forming bilayer: a Pfg-NMR multinuclear study

By isotopical labeling lipid lateral diffusion coefficients for each of the membrane constituents, including cholesterol, have been measured by 1H, 2H, and 19F pulsed field gradient NMR spectroscopy in macroscopically oriented lipid bilayers. This provides a means of obtaining detailed dynamic and compositional information in raft-forming lipid bilayers without introducing foreign molecules into the systems. The raft systems studied contained dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DPPC)/cholesterol at the molar ratios of 42.5:42.5:15 and 35:35:30 in excess water. At temperatures below 30 degrees C the raft system forms large (>1 microm) domains of a liquid ordered (l(o)) phase, in which the lipid lateral diffusion was approximately 5 times slower than for the lipids in the surrounding liquid disordered (l(d)) phase. Within each domain all lipid species showed the same diffusion coefficient, despite the very different structures of cholesterol and phospholipids. DPPC partitions exclusively into the l(o) domains, whereas cholesterol and dioleoylphosphatidylcholine were distributed in both l(o) and l(d) phases. The cholesterol concentration was found to be 10-20 mol % in the l(d) domain and 30-40 mol % in the l(o) domain. Comparison of these results with data from sphingomyelin-containing systems suggests that DPPC interacts more weakly with cholesterol than does sphingomyelin.     

Lateral diffusion of gramicidin S, M-13 coat protein and glycophorin in bilayers of saturated phospholipids. Mean field and Monte Carlo studies

We have developed a general model that relates the lateral diffusion coefficient of one isolated large intrinsic molecule (mol. wt. greater than or approximately 1000) in a phosphatidylcholine bilayer to the static lipid hydrocarbon chain order. We have studied how protein lateral diffusion can depend upon protein-lipid interactions but have not investigated possible non-specific contributions from gel-state lattice defects. The model has been used in Monte Carlo simulations or in mean-field approximations to study the lateral diffusion coefficients of Gramicidin S, the M-13 coat protein and glycophorin in dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC) bilayers as functions of temperature. Our calculated lateral diffusion coefficients for Gramicidin S and the M-13 coat protein are in good agreement with what has been observed and suggest that Gramicidin S is in a dimeric form in DMPC bilayers. In the case of glycophorin we find that the 'ice breaker' effect can be understood as a consequence of perturbation of the lipid polar region around the protein. In order to understand this effect is necessary that the protein hydrophilic section perturb the polar regions of at least approx. 24 lipid molecules, in good agreement with the numbers of 29-30 measured using 31P-NMR. Because of lipid-lipid interactions this effect extends itself out to four or five lipid layers away from the protein so that the hydrocarbon chains of between approx. 74 and approx. 108 lipid molecules are more disordered in the gel phase, so contributing less to the transition enthalpy, in agreement with the numbers of 80-100 deduced from differential scanning calorimetry (DSC). An understanding of the abrupt change in the diffusion coefficient at a temperature below the main bilayer transition temperature requires an additional mechanism. We propose that this change may be a consequence of a 'coupling-uncoupling' transition involving the protein hydrophilic section and the lipid polar regions, which may be triggered by the lipid bilayer pretransition. Our calculation of the average number of gauche bonds per lipid chain as a function of temperature and distance away from an isolated polypeptide or integral protein shows the extent of statically disordered lipid around such molecules. The range of this disorder depends upon temperature, particularly near the main transition.     

Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature'). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.     

Effect of cholesterol on the structure and dynamic properties of unsaturated phospholipid bilayers

Molecular dynamics (MD) computer simulations of five different hydrated unsaturated phosphatidylcholine lipid bilayers built up by 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules with 40 mol% cholesterol, and the same five pure phosphatidylcholine bilayers have been performed at 303 K. The simulation box of a lipid bilayer contained 96 phosphatidylcholines, 64 cholesterols, and 3840 water molecules (48 phosphatidylcholine molecules and 32 cholesterols per layer and 24 water molecules per phospholipid or cholesterol in each case). The lateral self-diffusion coefficients of the lipids in these systems and mass density profiles with respect to the bilayer normal have been analyzed. It has been found that the lateral diffusion coefficients of phosphatidylcholine molecules increase with increasing number of double bonds in one of the lipid chains, both in pure bilayers and in bilayers with cholesterol. It has been found as well that the lateral diffusion coefficient of phosphatidylcholine molecules of a lipid bilayer with 40 mol% cholesterol is smaller than that for the corresponding pure phosphatidylcholine bilayer.     

Solubility of carbon dioxide in lipid bilayer membranes and organic solvents

Partition coefficients of carbon dioxide into lipid bilayers (liposomes) and organic solvents were measured as a function of temperature. The molar partition coefficient of CO2 into liposomes of egg lecithin at 25 degrees C was 0.95 (ml CO2/ml lipid)/(ml CO2/ml saline). The addition of an equimolar amount of cholesterol to the egg lecithin decreased the partition coefficient by about 25%. The partition coefficients for CO2 into liposomes at 25 degrees C were lower than the partition coefficients into octanol (1.3), hexadecane (1.5) and olive oil (1.7). The results are discussed in terms of the solubility-diffusion model of non-electrolyte transport through lipid bilayer membranes.     

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

The translational diffusion of bovine rhodopsin, the Ca2+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein greater than or equal to 3000/1) in liquid-crystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100 000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X 10(-8) cm2/s. In consideration of the sizes of the membrane-bound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbrück, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113]. Lipid diffusion was also examined in th same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X 10(-8) cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Tr?uble, H., & Sackmann. E. (1972) J. Am. Chem. Soc. 94, 4499-4510] may have to be invoked to explain the lipid diffusion behavior.     

Comparative study of molecular dynamics,diffusion, and permeability for ligands in biomembranes of different lipid composition

A comparative study of several model lipid bilayers of different composition, which included analysis of kinetic parameters of model lipid bilayers and permeability of bilayer membranes for small molecules, has been carried out. The conformity of results of numeric experiments to experimental data (structure of membrane lipid bilayers, lateral diffusion coefficients, and relative permeability of biomembranes for ligands) is discussed in the framework of a standard molecular dynamics protocol.     

Temperature dependence of water self-diffusion through lipid bilayers assessed by NMR

The temperature dependence of the coefficient of water self-diffusion across plane-parallel multib-ilayers of dioleoylphosphatidylcholine oriented on a glass support was studied in the 20–60°C range by pulsed field gradient NMR. The coefficient for transbilayer diffusion of water proved almost four orders of magnitude smaller than for bulk water, and 10 times smaller than that for lateral diffusion of lipid under the same conditions. The temperature dependence obeyed the Arrhenius law with apparent activation energy of 41 kJ/mol, much higher than that for bulk water (18 kJ/mol). The experimental data were analyzed using the “dissolution-diffusion” model, by simulating water passage through membrane channels, and by examining water exchange in states with different modes of translational mobility, including pore channels and bilayer defects. Each approach could take into account the role of bilayer permeability and assess the apparent activation energy for water diffusion in the hydrophobic part of the bilayer, which proved close to the value for bulk water. Estimates were obtained for water diffusion coefficients in the system, coefficients of bilayer permeability for water, and the influence of bilayer defects on the lateral and transverse diffusion coefficients.     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

A pulsed field gradient NMR was used to study lateral diffusion in the cholesterol-containing oriented bilayers of saturated (dipalmitoyl- and dimyristoyl-) phosphatidylcholines, upon their limiting hydration. Similar dependences of lateral diffusion coefficients on temperature and cholesterol concentration were observed, which agree with phase diagram showing the presence of the regions of disordered and ordered liquid-crystalline phases and a two-phase region. Under the same conditions, the lateral diffusion coefficient of dipalmitoylphosphatidylcholine is lower, which agrees qualitatively with its larger molecular weight. The comparison of data for dipalmitoylphosphatidylcholine with previous results for dipalmitoylsphingomyelin-cholesterol bilayers under the same conditions, in spite of similarity of phase diagrams, shows large (two–three times) differences in the lateral diffusion coefficient and a different profile of its dependence on cholesterol concentration. The comparison of data for dimyristoylphosphatidylcholine with previous results shows that the values of lateral diffusion coefficient and the shape of its dependence on cholesterol concentration coincide at high concentrations (>15 mol%) but differ at lower concentrations The revealed disagreement may be caused by the fact that the measurements were carried out at different water content in the system. At limiting hydration (more than 35% of water), the lateral diffusion coefficient for lipids decreases when cholesterol concentration rises, while at water content about 25% (as a result of equilibrium hydration from vapors) the lateral diffusion coefficient of phosphatidylcholine may be independent of cholesterol concentration. This is the consequence of the denser packing of molecules in the bilayer at reduced water content, an effect that competes with the ordering effect of cholesterol.     

Lateral diffusion of gramicidin S,M-13 coat protein and glycophorin in bilayers of saturated phospholipids: Mean field and Monte Carlo studies

We have developed a general model that relates the lateral diffusion coefficient of one isolated large intrinsic molecule (mol. wt. ?1000) in a phosphatidylcholine bilayer to the static lipid hydrocarbon chain order. We have studied how protein lateral diffusion can depend upon protein-lipid interactions but have not investigated possible non-specific contributions from gel-state lattice defects. The model has been used in Monte Carlo simulations or in mean-field approximations to study the lateral diffusion coefficients of Gramicidin S, the M-13 coat protein and glycophorin in dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC) bilayers as functions of temperature. Our calculated lateral diffusion coefficients for Gramicidin S and the M-13 coat protein are in good agreement with what has been observed and suggest that Gramicidin S is in a dimeric form in DMPC bilayers. In the case of glycophorin we find that the ‘ice breaker’ effect can be understood as a consequence of perturbation of the lipid polar region around the protein. In order to understand this effect is is necessary that the protein hydrophilic section perturb the polar regions of at least approx. 24 lipid molecules, in good agreement with the numbers of 29–30 measured using ³¹P-NMR. Because of lipid-lipid interactions this effect extends itself out to four or five lipid layers away from the protein so that the hydrocarbon chains of between approx. 74 and approx. 108 lipid molecules are more disordered in the gel phase, so contributing less to the transition enthalpy, in agreement with the numbers of 80–100 deduced from differential scanning calorimetry (DSC). An understanding of the abrupt change in the diffusion coefficient at a temperature below the main bilayer transition temperature requires an additional mechanism. We propose that this change may be a consequence of a ‘coupling-uncoupling’ transition involving the protein hydrophilic section and the lipid polar regions, which may be triggered by the lipid bilayer pretransition. Our calculation of the average number of gauche bonds per lipid chain as a function of temperature and distance away from an isolated polypeptide or integral protein shows the extent of statically disordered lipid around such molecules. The range of this disorder depends upon temperature, particularly near the main transition.     

The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers

Pulsed field gradient NMR was utilized to directly determine the lipid lateral diffusion coefficient for the following macroscopically aligned bilayers: dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), palmitoyloleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC) with addition of cholesterol (CHOL) up to approximately 40 mol %. The observed effect of cholesterol on the lipid lateral diffusion is interpreted in terms of the different diffusion coefficients obtained in the liquid ordered (l(o)) and the liquid disordered (l(d)) phases occurring in the phase diagrams. Generally, the lipid lateral diffusion coefficient decreases linearly with increasing CHOL concentration in the l(d) phase for the PC-systems, while it is almost independent of CHOL for the SM-system. In this region the temperature dependence of the diffusion was always of the Arrhenius type with apparent activation energies (E(A)) in the range of 28-40 kJ/mol. The l(o) phase was characterized by smaller diffusion coefficients and weak or no dependence on the CHOL content. The E(A) for this phase was significantly larger (55-65 kJ/mol) than for the l(d) phase. The diffusion coefficients in the two-phase regions were compatible with a fast exchange between the l(d) and l(o) regions in the bilayer on the timescale of the NMR experiment (100 ms). Thus, strong evidence has been obtained that fluid domains (with size of micro m or less) with high molecular ordering are formed within a single lipid bilayer. These domains may play an important role for proteins involved in membrane functioning frequently discussed in the recent literature. The phase diagrams obtained from the analysis of the diffusion data are in qualitative agreement with earlier published ones for the SM/CHOL and DMPC/CHOL systems. For the DOPC/CHOL and the POPC/CHOL systems no two-phase behavior were observed, and the obtained E(A):s indicate that these systems are in the l(d) phase at all CHOL contents for temperatures above 25 degrees C.     

Free volume model for lipid lateral diffusion coefficients. Assessment of the temperature dependence in phosphatidylcholine and phosphatidylethanolamine bilayers

The lipid lateral diffusion coefficients, DT, in fluid-phase phosphatidylcholine and phosphatidylethanolamine bilayers have been analysed in terms of the free-volume diffusion model by fitting the expression: DT = AT exp[- B/(T - T0)] to the observed temperature dependence, where A, B and T0 are the parameters to be optimized. Application of an unconstrained optimization procedure to data obtained from excimer formation (Galla et al. (1979) J. Membrane Biol. 48, 215-236) and from fluorescence photobleaching (Vaz et al. (1985) Biochemistry 24, 781-786) provides statistical evidence for a free-volume model as opposed to a simple Stokes-Einstein model (T0 = 0), only in certain cases. In the instances for which the parameter T0 can be determined with a reasonable degree of accuracy, it is found that this characteristic temperature at which the free volume extrapolates to zero lies below the bilayer gel-to-fluid phase transition temperature and does not coincide with the pre-transition temperature for phosphatidylcholines.