Cholesterol effects on a mixed-chain phosphatidylcholine bilayer: a molecular dynamics simulation study.

A molecular dynamics simulation of a mono-cis-unsaturated 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer containing approximately 22 mol% of cholesterol (POPC-Chol) was carried out for 15 ns. An 8-ns trajectory was analysed to determine the effects of Chol on the membrane properties and compare it with that on the fully saturated 1,2-dimyristoyl-phosphatidylcholine bilayer containing approximately 22 mol% of Chol (DMPC-Chol). The study suggests that the experimentally observed weaker effect of Chol on the POPC than DMPC bilayer might result from a different vertical localisation of the Chol hydroxyl group (OH-Chol) in both bilayers: in the POPC-Chol bilayer, OH-Chol is placed approximately 3 A higher in the bilayer interface than in the DMPC-Chol bilayer. Because of the rigid cis double bond in the beta-chain of POPC, Chol fits worse to the POPC-Chol membrane environment and is pushed up, in effect all Chol ring atoms are, on average, located above the double bond. Both in mono-cis-unsaturated and fully saturated PC bilayers, Chol induces stronger van der Waals interactions among the chains, whereas its interactions with the chains are weak. In contrast to DMPC, the smooth alpha-face of the Chol ring lowers the order of POPC chains, whereas the rough beta-face increases the order.

Effects of a carane derivative local anesthetic on a phospholipid bilayer studied by molecular dynamics simulation.

Molecular dynamics (MD) simulations of two hydrated palmitoyloleoylphosphatidylcholine (POPC) bilayers each containing eight carane derivative (KP-23) local anesthetic (LA) molecules in neutral (POPC-LA) or protonated (POPC-LAH) forms were carried out to investigate the effect of KP-23 and its protonation on the bilayer. 3-ns trajectories were used for analyses. A pure POPC bilayer was employed as a reference system. In both POPC-LA and POPC-LAH systems a few KP-23 molecules intercalated into the bilayer and moved near the bilayer/water interface. They were located on the hydrophobic core side of the interface in the POPC-LA bilayer, but on the water phase side in the POPC-LAH bilayer. The order of the POPC chains was higher in the POPC-LA bilayer than in the pure POPC bilayer and was lower in the POPC-LAH bilayer. Interactions between polar groups of KP-23 and POPC or water were responsible for a lower hydration of POPC headgroups in POPC bilayers containing KP-23 than in the pure POPC bilayer. KP-23 molecules were found to form aggregates both in POPC-LA and POPC-LAH bilayers. Due to higher amphiphilicity of LAH, the LAH aggregate was more micelle-like and larger than the LA one. The results demonstrate the rapid timescales of the initial processes that take place at and near the bilayer interface as well as details of the atomic level interactions between local anesthetic and the lipid matrix of a cell membrane.

Cholesterol effects on the phosphatidylcholine bilayer nonpolar region: a molecular simulation study.

A 15-ns molecular dynamics (MD) simulation of the fully hydrated dimyristoylphosphatidylcholine-cholesterol (DMPC-Chol) bilayer in the liquid-crystalline state was carried out to investigate the effect of Chol on the hydrocarbon chain region of the bilayer. The last 8-ns fragment of the generated trajectory was used for analyses. As a reference system, a pure DMPC bilayer (M. Pasenkiewicz-Gierula, Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi, 1999, Biophys. J. 76:1228-1240) simulated for 14 ns was used. The study shows that a Chol-induced increase of the bulk molecular order parameter along both beta- and gamma-chain is mainly caused by a decrease of the average tilt of the chains, because the bulk average number of gauche rotamers/myristoyl chain is not significantly changed by Chol. Nevertheless, for DMPCs located near Chol molecules both the number of gauche rotamers/chain and the chain tilt are decreased. The magnitude of the Chol effect on the PC alkyl chains depends, in addition to the PC-Chol distance, on the side of the Chol molecule (alpha- or beta-face) that the chains are in contact with. This study provides some new insight into the properties of the coexistence region of the partial phase diagram for DMPC-Chol bilayers.

Cholesterol effects on the phospholipid condensation and packing in the bilayer: a molecular simulation study.

A 15-ns molecular dynamics simulation of the fully hydrated liquid-crystalline dimyristoylphosphatidylcholine-cholesterol (DMPC-Chol) bilayer containing approximately 22 mol% Chol was carried out. The generated trajectory was analysed to investigate the mechanism of the Chol condensing effect on DMPC hydrocarbon chains and the influence of Chol on the chain packing in the membrane. Chol was found to induce stronger van der Waals interactions among the chains, whereas its interactions with the chains were weak. In the DMPC-Chol bilayer, as in the DMPC bilayer, DMPC chains were regularly packed around a chosen chain but around a Chol molecule they were not. DMPC gamma chains made closer contacts with Chol than the beta chains.

The effect of the Glu342Lys mutation in alpha1-antitrypsin on its structure, studied by molecular modelling methods.

The structure of native alpha1-antitrypsin, the most abundant protease inhibitor in human plasma, is characterised primarily by a reactive loop containing the centre of proteinase inhibition, and a beta-sheet composed of five strands. Mobility of the reactive loop is confined as a result of electrostatic interactions between side chains of Glu342 and Lys290, both located at the junction of the reactive loop and the beta structure. The most common mutation in the protein, resulting in its inactivation, is Glu342-->Lys, named the Z mutation. The main goal of this work was to investigate the influence of the Z mutation on the structure of alpha1-antitrypsin. Commonly used molecular modelling methods have been applied in a comparative study of two protein models: the wild type and the Z mutant. The results indicate that the Z mutation introduces local instabilities in the region of the reactive loop. Moreover, even parts of the protein located far apart from the mutation region are affected. The Z mutation causes a relative change in the total energy of about 3%. Relatively small root mean square differences between the optimised structures of the wild type and the Z mutant, together with detailed analysis of 'conformational searching' process, lead to the hypothesis that the Z mutation principally induces a change in the dynamics of alpha1-antitrypsin.

Effects of phospholipid unsaturation on the membrane/water interface: a molecular simulation study.

Molecular dynamics (MD) simulations of fully hydrated bilayers in the liquid-crystalline state made of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) or 1-palmitoyl-2-elaidoyl-phosphatidylcholine (PEPC) were carried out to investigate the effect of the incorporation of a double bond in the phosphatidylcholine (PC) beta-chain (cis or trans) on the membrane/water interface. The bilayers reached thermal equilibrium after 3 and 1 ns of MD simulations, respectively, and productive runs were carried out for 3 ns for each bilayer. As reference systems, the 1,2-dimyristoyl-phosphatidylcholine (DMPC) bilayer (M. Pasenkiewicz-Gierula, Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi, 1999, Biophys. J. 76:1228-1240) and DMPC-cholesterol (Chol) bilayer containing 22 mol % Chol (M. Pasenkiewicz-Gierula, T. Róg, K. Kitamura, A. and Kusumi, 2000, Biophys. J. 78:1376-1389) were used. The study shows that at the interface of POPC, PEPC, and DMPC-Chol bilayers, average numbers of PC-water and PC-PC interactions are similar and, respectively, greater and smaller than in the DMPC bilayer. The average area/PC in mono-unsaturated bilayers is approximately 4 A(2) larger than in the DMPC bilayer; nevertheless, a strong correlation was found between a single molecular area (SMA) of a PC and the number of interactions this PC makes; i.e., PCs (either saturated or unsaturated) with the same SMA form similar numbers of intermolecular links. The numbers and corresponding SMAs are distributed about averages pertinent to each bilayer. No significant difference between cis and trans bonds was found.

Fast lipid disorientation at the onset of membrane fusion revealed by molecular dynamics simulations.

Membrane fusion is a key event in vesicular trafficking in every cell, and many fusion-related proteins have been identified. However, how the actual fusion event occurs has not been elucidated. By using molecular dynamics simulations we found that when even a small region of two membranes is closely apposed such that only a limited number of water molecules remain in the apposed area (e.g., by a fusogenic protein and thermal membrane fluctuations), dramatic lipid disorientation results within 100 ps-2 ns, which might initiate membrane fusion. Up to 12% of phospholipid molecules in the apposing layers had their alkyl chains outside the hydrophobic region, lying almost parallel to the membrane surface or protruding out of the bilayer by 2 ns after two membranes were closely apposed.

Comparative investigations of hydroxyamine carane derivative and its R,S-diastereoisomers with strong local anesthetic activity.

Our previously conducted pharmacological screening led as to the discovery of the strong local anesthetic activity of the compound designated as KP-23. Earlier crystallographic studies revealed that the compound KP-23 crystallized in diastereoisomeric form in lowest symmetry. The aim of these comparative investigations was to evaluate anesthetic activity of KP-23 and its R,S-diastereoisomers, which were synthesized at the Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology.

Molecular dynamics generation of nonarbitrary membrane models reveals lipid orientational correlations.

This report addresses the following problems associated with the generation of computer models of phospholipid bilayer membranes using molecular dynamics simulations: arbitrary initial structures and short equilibration periods, an Ewald-induced strong coupling of phospholipids, uncertainty regarding which value should be used for surface tension to alleviate the problem of the small size of the membrane, and simultaneous realization of both order parameters and the surface area. We generated a computer model of the liquid-crystalline L-alpha-dimyristoylphosphatidylcholine (DMPC) bilayer, starting from a configuration based on a crystal structure (rather than from an arbitrary structure). To break the crystalline structure, a 20-ps high-temperature pulse of 510 K (but not 450 or 480 K) was effective. The system finally obtained is an all-atom model, with Ewald summation to evaluate Coulombic interactions and a constant surface tension of 35 dynes/cm/water-membrane interface, equilibrated for 12 ns (over 50 ns total calculation time), which reproduces all of the experimentally observed parameters examined in this work. Furthermore, this model shows the presence of significant orientational correlations between neighboring alkyl chains and between shoulder vectors (which show the orientations of the lipids about their long axes) of neighboring DMPCs.

Cholesterol effects on the phosphatidylcholine bilayer polar region: a molecular simulation study.

A molecular dynamics (MD) simulation of a fully hydrated, liquid-crystalline dimyristoylphosphatidylcholine (DMPC)-Chol bilayer membrane containing approximately 22 mol% Chol was carried out for 4.3 ns. The bilayer reached thermal equilibrium after 2.3 ns of MD simulation. A 2.0-ns trajectory generated during 2.3-4.3 ns of MD simulation was used for analyses to determine the effects of Chol on the membrane/water interfacial region. In this region, 70% of Chol molecules are linked to DMPC molecules via short-distance interactions, where the Chol hydroxyl group (OH-Chol) is 1) charge paired to methyl groups of the DMPC choline moiety ( approximately 34%), via the hydroxyl oxygen atom (Och); 2) water bridged to carbonyl ( approximately 19%) and nonester phosphate ( approximately 14%) oxygen atoms, via both Och and the hydroxyl hydrogen atom (Hch); and 3) directly hydrogen (H) bonded to carbonyl ( approximately 11%) and nonester phosphate ( approximately 5%) oxygen atoms, via Hch ( approximately 17% of DMPC-Chol links are multiple). DMPC's gamma-chain carbonyl oxygen atom is involved in 44% of water bridges and 51% of direct H bonds formed between DMPC and Chol. On average, a Chol molecule forms 0.9 links with DMPC molecules, while a DMPC molecule forms 2.2 and 0.3 links with DMPC and Chol molecules, respectively. OH-Chol makes hydrogen bonds with 1.1 water molecules, preferentially via Hch. The average number of water molecules H bonded to the DMPC headgroup is increased by 7% in the presence of Chol. These results indicate that inclusion of Chol decreases interlipid links and increases hydration in the polar region of the membrane.

Molecular dynamics simulation studies of lipid bilayer systems.

The main structural element of biological membranes is a liquid-crystalline lipid bilayer. Other constituents, i.e. proteins, sterols and peptides, either intercalate into or loosely attach to the bilayer. We applied a molecular dynamics simulation method to study membrane systems at various levels of compositional complexity. The studies were started from simple lipid bilayers containing a single type phosphatidylcholine (PC) and water molecules (PC bilayers). As a next step, cholesterol (Chol) molecules were introduced to the PC bilayers (PC-Chol bilayers). These studies provided detailed information about the structure and dynamics of the membrane/water interface and the hydrocarbon chain region in bilayers built of various types of PCs and Chol. This enabled studies of membrane systems of higher complexity. They included the investigation of an integral membrane protein in its natural environment of a PC bilayer, and the antibacterial activity of magainin-2. The latter study required the construction of a model bacterial membrane which consisted of two types of phospholipids and counter ions. Whenever published experimental data were available, the results of the simulations were compared with them.

Molecular dynamics of a vasopressin V2 receptor in a phospholipid bilayer membrane.

Molecular dynamics simulations were carried out for a V2 receptor (V2R) model embedded in a dimyristoylphosphatidylcholine (DMPC) bilayer. Both free and ligand-bound states of V2R were modeled. Our initial V2R model was obtained using a rule-based automated method for GPCR modeling and refined using constrained simulated annealing in vacuo. The docking site of the native vasopressin ligand was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. The primary purpose of this work was to investigate the usefulness of MD simulation of an integral membrane protein like a GPCR receptor, upon inclusion of a carefully parameterized surrounding lipid membrane and water. Physical properties of the system were evaluated and compared with the fully hydrated pure DMPC bilayer membrane. The solvation interactions, individual lipid-protein interaction and fluctuations of the protein, the lipid, and water were analyzed in detail. As expected, the membrane-spanning helices of the protein fluctuate less than the peripheral loops do. The protein appears to disturb the local lipid structure. Simulations were carried out using AMBER 4.1 package upon constant number-pressure-temperature (NPT) conditions on massively parallel computers Cray T3E and IBM SP2.

Charge pairing of headgroups in phosphatidylcholine membranes: A molecular dynamics simulation study.

Molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine (DMPC) bilayer membrane in the liquid-crystalline phase was carried out for 5 ns to study the interaction among DMPC headgroups in the membrane/water interface region. The phosphatidylcholine headgroup contains a positively charged choline group and negatively charged phosphate and carbonyl groups, although it is a neutral molecule as a whole. Our previous study (Pasenkiewicz-Gierula, M., Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi. 1997. J. Phys. Chem. 101:3677-3691) showed the formation of water cross-bridges between negatively charged groups in which a water molecule is simultaneously hydrogen bonded to two DMPC molecules. Water bridges link 76% of DMPC molecules in the membrane. In the present study we show that relatively stable charge associations (charge pairs) are formed between the positively and negatively charged groups of two DMPC molecules. Charge pairs link 93% of DMPC molecules in the membrane. Water bridges and charge pairs together form an extended network of interactions among DMPC headgroups linking 98% of all membrane phospholipids. The average lifetimes of DMPC-DMPC associations via charge pairs, water bridges and both, are at least 730, 1400, and over 1500 ps, respectively. However, these associations are dynamic states and they break and re-form several times during their lifetime.

Construction and optimisation of a computer model for a bacterial membrane.

The main steps in the construction of a computer model for a bacterial membrane are described. The membrane has been built of 72 lipid molecules, 54 of which being 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine (POPE) and 18--1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidyl-rac-glycerol (POPG) molecules (thus in the proportion of 3:1). The membrane was hydrated with 1955 water molecules (approximately 27 water molecules per lipid). To neutralise the electronic charge (-e) on each POPG molecule, 18 sodium ions (Na+) were added to the membrane close to the POPG phosphate groups. The atomic charges on the POPE and POPG headgroups were obtained from ab initio quantum mechanical restrained electrostatic potential fitting (RESP) (Bayly et al., 1993, J. Phys. Chem. 97, 10269) using the GAMESS program at the 6-31G* level (Schmidt et al., 1993, J. Comput. Chem. 14, 1347). The model constructed in this way provided an initial structure for subsequent molecular dynamics simulation studies intended to elucidate the atomic level interactions responsible for the structure and dynamics of the bacterial membrane.

Conformations, orientations and time scales characterising dimyristoylphosphatidylcholine bilayer membrane. Molecular dynamics simulation studies.

The results of molecular dynamics simulation of fully hydrated dimyristoylphosphatidylcholine (DMPC) bilayer membrane in the liquid-crystalline phase are presented. They show that the probability of a gauche conformation varies periodically along the chain with only a slight increase towards the end of the chain. However, the frequency of transition between conformations increases, due to a decrease in the lifetime of the trans conformation, along the chain. The average lifetimes for trans conformations are in the range of 1-2 x 10(-10) s and for gauche conformations in the range of 4-7 x 10(-11) s. The alpha-chain of the DMPC head group has mainly an extended conformation, due to predominantly trans conformation of alpha5 torsion. The rotational correlation time for the P-N vector is 3.7 ns. The C2-C1-O11-P fragment of the DMPC head group (theta1, alpha1, alpha2 torsions) is rigid while the P-O12-C11-C12 fragment (alpha3, alpha4, alpha5 torsions) is flexible. The lateral diffusion coefficient for DMPC self-diffusion in the membrane is 2 x 10(-7) cm2/s; the rate of transverse diffusion is the same. Large differences in the calculated rotational correlation times for the alpha-, beta-, gamma-chains and for the O21-C1 vector indicate that in the liquid-crystalline bilayer each segment of the DMPC molecule exhibits its own rotational freedom, in addition to its internal flexibility resulting from rotational isomerism. The results obtained in these calculations, although in general agreement with some experimental data, shed new light on the dynamical behaviour of phosphatidylcholine molecules in the bilayer membrane in the liquid-crystalline phase.

Computer modelling of human alpha 1-antitrypsin reactive site loop behaviour under mild conditions.

Human alpha 1-antitrypsin (alpha 1-PI) is a member of the serpin superfamily of proteins. The reactive site loop (RSL) of the serpin binds to the active site of its target proteinase. Deficiency of alpha 1-antitrypsin is associated with a spontaneous conformational transition in the molecule which leads to a polymer formation. Mild conditions (1 M guanidinium.HCl), temperature and point mutations within the RSL are the factors that induce polymerisation. Initiation of this process has been associated with the disruption of a salt bridge Glu342-->Lys290. In this paper the interaction of guanidinium ion with Glu342 and Lys290 as well as the effect of this interaction on the mobility of RSL is studied by molecular modelling.

Influence of phospholipid unsaturation on the cholesterol distribution in membranes.

Over the last half decade, we have studied saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes, with special attention paid to fluid-phase immiscibility in cis-unsaturated PC-cholesterol membranes. The investigations were carried out with fatty acid and sterol analogue spin labels for which reorientational diffusion of the nitroxide was measured using conventional ESR technique. We also used saturation recovery ESR technique where dual probes were utilized. Bimolecular collision rates between a membrane-soluble square-planar copper complex,3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato)copper(II) (CuKTMS2) and one of several nitroxide radical lipid-type spin labels were determined by measuring the nitroxide spin-lattice relaxation time (T1). The results obtained in all these studies can be explained if the following model is assumed: 1) at physiological temperatures, fluid-phase micro-immiscibility takes place in cis-unsaturated PC-cholesterol membranes, which induces cholesterol-rich domains in the membrane due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the cis double bond of the alkyl chains of unsaturated PC. 2) The cholesterol-rich domains are small and/or of short lifetime (10(-9) s to less than 10(-7) s). Our results also suggest that the extra space that is available for conformational disorder and accommodation of small molecules is created in the central part of the bilayer by intercalation of cholesterol in cis-unsaturated PC membrane due to the mismatch in the hydrophobic length and nonconformability between cis-unsaturated PC alkyl chains and the bulky tetracyclic ring of cholesterol.

Rotational diffusion of a steroid molecule in phosphatidylcholine-cholesterol membranes: fluid-phase microimmiscibility in unsaturated phosphatidylcholine-cholesterol membranes.

Rotational diffusion of androstane spin-label (ASL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of the chain length and unsaturation of the alkyl chains, cholesterol mole fraction, and temperature for a better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. Special attention was paid to the differences in the cholesterol effects on ASL motion between saturated and unsaturated PC membranes. ASL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines were obtained for various PC-cholesterol membranes in the liquid-crystalline phase. Cholesterol decreases both the cone angle and the wobbling rotational diffusion constant for ASL in all PC membranes studied in this work. The cholesterol effects are the largest in DMPC membranes. An increase of cholesterol mole fraction from 0 to 30% decreases the rotational diffusion constant by a factor of 9-15 (depending on temperature) and the cone angle by a factor of about 2. In dioleoyl-PC membranes, addition of 30 mol % cholesterol reduces both the rotational diffusion constant and the cone angle of ASL by factors of approximately 2.5 and approximately 1.3, respectively, while it was previously found to cause only modest effects on the motional freedom of phospholipid analogue spin probes [Kusumi, A., Subczynski, W. K., Pasenkiewicz-Gierula, M., Hyde, J. S., & Merkle, H. (1986) Biochim. Biophys. Acta 854, 307-317]. It is proposed that fluid-phase microimmiscibility takes place in dioleoyl-PC-cholesterol membranes at physiological temperatures, which induces cholesterol-rich domains in the membrane, partially due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the C9-C10 cis double bond of the alkyl chains of dioleoyl-PC. The mechanism by which cholesterol influences the lipid dynamics in the membrane is different between saturated and unsaturated PC membranes.

Type 2-depleted fungal laccase.

Although copper is quantitatively removed from fungal laccase (Polyporus versicolor) by extended dialysis against high concentrations of cyanide, we have been unable to reconstitute the protein by addition of Cu(I) ions. However, two new methods for reversibly removing the type 2 Cu centre have been developed. The visible absorption at 610 nm, which is attributable to type 1 Cu, is unaffected by the procedure, but the absorbance of the type 3 Cu at 330 nm is decreased by 60 +/- 10%. The decrease is due, at least in part, to partial reduction of the binuclear type 3 centre, although there may be some change in the molar absorptivity of the oxidized chromophore as well. The change in the c.d. spectrum that occurs at approx. 350 nm may be explained in the same way, but it may also reflect the loss of a signal due to the type 2 Cu. Upon removal of the type 2 Cu an absorbance increase appears at approx. 435 nm, and it is assigned to the semi-reduced form of the type 3 pair. In the e.p.r. spectrum of the type 2-depleted enzyme the type 1 Cu signal exhibits well-resolved ligand hyperfine splitting, which can be simulated on the basis of contributions from two N and two H nuclei (AH congruent to AN congruent to 25 MHz). The H atoms are assumed to be attached to the beta-carbon of the covalently bonded cysteine ligand. A signal from a semi-reduced form(s) of the type 3 site can also be resolved in the spectrum of the type 2-depleted enzyme, and on the basis of the second integral of the e.p.r. spectrum 40% of the type 3 pairs are believed to be in a partially reduced state. The semi-reduced type 3 site is remarkably stable and is not readily oxidized by H2O2 or IrCl6(2-) or reduced by Fe(CN)6(4-). Intramolecular electron transfer is apparently quite slow in at least some forms of the type 2-depleted enzyme, and this may explain why the activity is at best 5% of that of the native enzyme. Full activity returns when type 2 copper is restored.

Rotational diffusion of a steroid molecule in phosphatidylcholine membranes: effects of alkyl chain length, unsaturation, and cholesterol as studied by a spin-label method.

Rotational diffusion of cholestane spin-label (CSL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of chain length and unsaturation of alkyl chains, cholesterol mole fraction, and temperature for better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. CSL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines are obtained for various membranes in the liquid-crystalline phase. The wobbling diffusion constant decreases in the order dilauroyl-PC greater than dimyristoyl-PC greater than dioleoyl-PC approximately dipalmitoyl-PC greater than distearoyl-PC greater than dioleoyl-PC/cholesterol = 3/1 greater than dioleoyl-PC/cholesterol = 1/1 membranes. Activation energy for the wobbling diffusion of the long axis of CSL is strongly dependent on alkyl chain length, unsaturation, and cholesterol mole fraction. It decreases with decrease in alkyl chain length and by introduction of unsaturation in the alkyl chains. In dioleoylphosphatidylcholine membranes, activation energy decreases by a factor of approximately 3 in the presence of 50 mol % cholesterol. Activation energy for wobbling diffusion of CSL in phosphatidylcholine membranes is smaller than the activation energy for translational diffusion of a phospholipid. The former is more dependent on alkyl chain length and unsaturation.(ABSTRACT TRUNCATED AT 250 WORDS)