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The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide [(LA)(12)] on the molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes were investigated using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels, and the results were compared with our earlier, similar study of Ac-K(2)L(24)K(2)-amide (L(24)) [Subczynski, W. K., Lewis, R. N. A. H., McElhaney, R. N., Hodges, R. S., Hyde, J. S., and Kusumi, A. (1998) Biochemistry 37, 3156-3164]. At peptide-to-POPC ratios between 1/10 and 1/40, both methods (covering a time scale of 100 ps-10 micros) detect the presence of a single homogeneous membrane environment for both peptides, suggesting that these peptides are both well dispersed and that POPC is exchanging rapidly between the boundary and the bulk domains. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane, studied by saturation-recovery EPR, decreases by a factor of about 2 by including 10 mol % (LA)(12) whereas incorporating L(24) has practically no effect. (LA)(12) increases the alkyl chain order of POPC more than L(24). L(24) increases hydrophobicity (decreases the degree of water penetration into the hydrophobic region of the membrane) more than does (LA)(12). We ascribe the much stronger effects of (LA)(12) on membrane order and dynamics to the increased roughness of its hydrophobic surface and also to the increased motional freedom of its leucine side chains. In L(24), the leucine side chains are packed tightly, giving a smooth hydrophobic surface. In (LA)(12), they are separated by the small methyl groups of the alanine side chains, giving them additional motional freedom and the ability to protrude between the phospholipid hydrocarbon chains. The frequency of gauche-trans isomerization of hydrocarbon chains and concentration of vacant pockets (voids) in the lipid bilayer are thus reduced, which decreases oxygen transport. This explanation was confirmed by calculating the orientational order of leucine side chains in (LA)(12) and L(24) from molecular dynamics simulation studies.

Epicholesterol (Echol) is an epimeric form of cholesterol (Chol). A molecular dynamics simulation of the fully hydrated dimyristoylphosphatidylcholine-Echol (DMPC-Echol) bilayer membrane containing approximately 22 mol % of Echol was carried out for 5 ns. A 3-ns trajectory generated between 2 and 5 ns of molecular dynamics simulation was used for analyses to determine the effects of Echol on the membrane properties. As reference systems, pure DMPC and mixed DMPC-Chol bilayers were used. The study shows that Echol, like Chol, changes the organization of the bilayer/water interface and increases membrane order and condensation, but to a lesser degree. Effects of both sterols are based on the same atomic level mechanisms; their different strength arises from different vertical localizations of Echol and Chol hydroxyl groups in the membrane/water interface.

Molecular dynamics (MD) simulations complement experimental methods in studies of the structure and dynamics of lipid bilayers. The choice of algorithms employed in this computational method represents a trade-off between the accuracy and real calculation time. The largest portion of the simulation time is devoted to calculation of long-range electrostatic interactions. To speed-up evaluation of these interactions, various approximations have been used. The most common ones are the truncation of long-range interactions with the use of cut-offs, and the particle-mesh Ewald (PME) method. In this study, several multi-nanosecond cut-off and PME simulations were performed to establish the influence of the simulation protocol on the bilayer properties. Two bilayers were used. One consisted of neutral phosphatidylcholine molecules. The other was a mixed lipid bilayer consisting of neutral phosphatidylethanolamine and negatively charged phosphatidylglycerol molecules. The study shows that the cut-off simulation of a bilayer containing charge molecules generates artefacts; in particular the mobility and order of the charged molecules are vastly different from those determined experimentally. In the PME simulation, the bilayer properties are in general agreement with experimental data. The cut-off simulation of bilayers containing only uncharged molecules does not generate artefacts, nevertheless, the PME simulation gives generally better agreement with experimental data.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.

The interaction of pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum with the cofactor analogue 5-deaza-6-methyltetrahydropterin and the cofactor 6,7-dimethyltetrahydropterin (DMPH4) has been investigated by multifrequency electron spin resonance (ESR) spectroscopy. 5-Deaza-6-methyltetrahydropterin, which lacks the N-5 nitrogen present in the pyrazine ring of DMPH4, binds tightly to the cupric form of the enzyme; however, no changes are observed in the ESR parameters of the copper center. In contrast, the binding of DMPH4 (or 6-methyltetrahydropterin) shifts the ESR parameters (g and A) associated with the cupric enzyme. In addition, superhyperfine transitions were resolved and assigned to hyperfine splitting from nitrogen ligands. ESR spectra of the enzyme recorded in the presence of [5-14N]DMPH4 or [5-15N]DMPH4 were computer simulated and found to be consistent with pterin serving as a direct donor ligand to the copper center through the N-5 position.

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)