We report molecular dynamics simulations of water hydrating a lipid (dimyristoylphosphatidylcholine) monolayer under conditions chosen to eliminate simulation artifacts. These simulations provide a description of the behavior of the membrane–water interface that agrees with recent experimental studies. In particular, we find that the hydrating water orients to contribute the positive end of its dipole to the substantially positive electrostatic potential of the membrane interior, consistent with interpretations of recent experiments. In addition, recent experiments show that this water reorients rapidly on the NMR time scale. Our results concur, however the relatively rapid water motion does not preclude the preferential ordering that we observe. The limiting behavior of the system shows three hydration shells about the lipid PC headgroups and significant hydrogen bonding of water to the phosphate groups. The choline group experiences different environments, and the structure of the first hydration shell clearly corresponds to a clathrate. The motion of the hydrating water was found to be slower than that of bulk water, and the computed residence times for water about the lipids (20 ps about choline, 10 ps about phosphate) were in excellent agreement with results of NMR experiments. This further shows that water resides in a clathrate shell longer than in a shell about ions. In addition, we show that the structure and dynamics of water hydrating the lipids are very sensitive to the treatment of the long-range interactions. In particular, the radial structure sharpens considerably, a third hydration shell about the phosphate was observed only with large cutoffs, and hydrogen bonding of water to the lipids increased by 25%. The water moved more slowly than bulk when large cutoffs were employed but moved faster than bulk water when small cutoffs were used and the residence times for water about the lipids were twofold–fivefold larger using large cutoffs. In general it was found that the lipids significantly influence water out to several hydration shells, and that water hydrating the lipids behaves differently than bulk water.

To elucidate the mechanism of solute diffusion through lipid bilayer membranes, nearly 4 ns of molecular dynamics simulations of solutes in phospholipid bilayers was conducted. The study, the first atomic level study of solute diffusion in a lipid bilayer, involved four simulations of an all-atom representation of a fully solvated dimyristoylphosphatidylcholine (DMPC) bilayer in the L alpha phase with benzene molecules as solutes, totaling over 7100 atoms. These simulations agree with experimental evidence that the presence of small solutes does not affect bilayer thickness but does result in slight perturbations in the ordering of the hydrocarbon chains. At room temperature, the benzene molecules have essentially isotropic motion and rotate freely. The rate of translational diffusion varies with position within the bilayer and is faster in the center than near the zwitterionic headgroups and is in excellent agreement with experimental values for the diffusion of small solutes in a bilayer. These simulations have elucidated the mechanism of diffusion in a bilayer to be similar to the "hopping" mechanism found for the diffusion of gases through soft polymers. Jumps of up to 8 A can occur in as little as 5 ps whereas average motions for that time period are only approximately 1.5 A. In many cases, the jumps are moderated by torsional changes in the hydrocarbon chains which serve as "gates" between voids through which the benzene molecules move. Comparison of these simulations with another 1000-ps simulation of benzene in a pure alkane provides evidence that lipid bilayers should not be treated as a homogeneous bulk hydrocarbon phase.

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This study extends previous studies of the mechanism of small molecule diffusion through lipid membranes. Atomic level molecular dynamics simulations of over 4 ns of benzene in fully hydrated dimyristoylphosphatidylcholine (DMPC) bilayers were performed at four different temperatures above the gel-to-La phase transition temperature.These studies confirm previous observations that small solutes diffuse at different rates in different locations in the bilayer. This difference in diffusion is likely to be due to "jumps" (single, large movements) between voids which are most common in the center of the bilayer. The benzene molecules appear to favor different regions of the bilayer at different temperatures. Although at 320 K the solutes show no regional preference, at 310 K they migrate to the center of the bilayer, while at 340 K they reside mostly near the head group region. This correlates with the distribution of free volume which concentrates at the bilayer center at low temperature but becomes more diffuse at higher temperatures. The mechanism of the diffusional process was found to be complex. Not only does the rate of diffusion depend on location within the bilayer, but the characteristics of this process appear to respond to temperature changes differently in the different regions of the bilayer. Only short time motions are dependent directly on the temperature. Longer time motions depend additionally on the size and availability of voids and the rate of torsional isomerization of the lipid molecules. It was found that an increase in kinetic energy was not always coincident with a jump; some jumps may be passive processes. This study provides further evidence that the interior of lipid bilayer membranes is not a homogeneous system analogous to pure alkane. Rather it is a structured system with different properties depending on the distance from the lipidwater interface.

The new functionality of the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168;Bassolino-Klimas D et al., 1996, Protein Sci5:593-603) has been applied for energy refinement of two previously determined solution NMR structures, murine epidermal growth factor (mEGF) and human type-a transforming growth factor (hTGFa). A summary of considerations used in converting experimental NMR data into distance constraints for CONGEN is presented. A general protocol for simulated annealing with restrained molecular dynamics is applied to generate NMR solution structures using CONGEN together with real experimental NMR data. A total of 730 NMR-derived constraints for mEGF and 424 NMR-derived constraints for hTGFa were used in these energy-refinement calculations. Different weighting schemes and starting conformations were studied to check and/or improve the sampling of the low-energy conformational space that is consistent with all constraints. The results demonstrate that loosened (i.e., "relaxed") sets of the EGF and hTGFa internuclear distance constraints allow molecules to overcome local minima in the search for a global minimum with respect to both distance restraints and conformational energy. The resulting energy-refined structures of mEGF and hTGFa are compared with structures determined previously and with structures of homologous proteins determined by NMR and X-ray crystallography.