We report a 2-ns constant pressure molecular dynamics simulation of halothane, at a mol fraction of 50%, in the hydrated liquid crystal bilayer phase of dipalmitoylphosphatidylcholine. Halothane molecules are found to preferentially segregate to the upper part of the lipid acyl chains, with a maximum probability near the C(5) methylene groups. However, a finite probability is also observed along the tail region and across the methyl trough. Over 95% of the halothane molecules are located below the lipid carbonyl carbons, in agreement with photolabeling experiments. Halothane induces lateral expansion and a concomitant contraction in the bilayer thickness. A decrease in the acyl chain segment order parameters, S(CD), for the tail portion, and a slight increase for the upper portion compared to neat bilayers, are in agreement with several NMR studies on related systems. The decrease in S(CD) is attributed to a larger accessible volume per lipid in the tail region. Significant changes in the electric properties of the lipid bilayer result from the structural changes, which include a shift and broadening of the choline headgroup dipole (P-N) orientation distribution. Our findings reconcile apparent controversial conclusions from experiments on diverse lipid systems.

We report the results of constant temperature and pressure molecular dynamics calculations carried out on the liquid crystal (Lalpha) phase of dipalmitoylphosphatidylcholine with a mole fraction of 6.5% halothane (2-3 MAC). The present results are compared with previous simulations for pure dipalmitoylphosphatidylcholine under the same conditions (Tu et al., 1995. Biophys. J. 69:2558-2562) and with various experimental data. We have found subtle structural changes in the lipid bilayer in the presence of the anesthetic compared with the pure lipid bilayer: a small lateral expansion is accompanied by a modest contraction in the bilayer thickness. However, the overall increase in the system volume is found to be comparable to the molecular volume of the added anesthetic molecules. No significant change in the hydrocarbon chain conformations is apparent. The observed structural changes are in fair agreement with NMR data corresponding to low anesthetic concentrations. We have found that halothane exhibits no specific binding to the lipid headgroup or to the acyl chains. No evidence is obtained for preferential orientation of halothane molecules with respect to the lipid/water interface. The overall dynamics of the lipid-bound halothane molecules appears to be reminiscent of that of other small solutes (Bassolino-Klimas et al., 1995. J. Am. Chem. Soc. 117:4118-4129).

The structural features of volatile anesthetic binding sites on proteins are being examined with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work has suggested that introducing a cavity into the hydrophobic core improves anesthetic binding affinity. The more polarizable methionine side chain was substituted for a leucine, in an attempt to enhance the dispersion forces between the ligand and the protein. The resulting bundle variant has an improved affinity (K(d) = 0.20 +/- 0.01 mM) for halothane binding, compared with the leucine-containing bundle (K(d) = 0.69 +/- 0.06 mM). Photoaffinity labeling with (14)C-halothane reveals preferential labeling of the W15 residue in both peptides, supporting the view that fluorescence quenching by bound anesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four-alpha-helix bundle proteins stabilized the native folded conformations. Molecular dynamics simulations of the bundles illustrate the existence of the hydrophobic core, containing both W15 residues. These results suggest that in addition to packing defects, enhanced dispersion forces may be important in providing higher affinity anesthetic binding sites. Alternatively, the effect of the methionine substitution on halothane binding energetics may reflect either improved access to the binding site or allosteric optimization of the dimensions of the binding pocket. Finally, preferential stabilization of folded protein conformations may represent a fundamental mechanism of inhaled anesthetic action.

The structural perturbations of the fully hydrated dimyristoyl-phosphatidylcholine bilayer induced by the presence of hexafluoroethane C(2F6), a "nonimmobilizer," have been examined by molecular dynamics simulations and compared with the effects produced by halothane CF3CHBrCl, an "anesthetic," on a similar bilayer (DPPC) (Koubi et al., Biophys. J. 2000. 78:800). We find that the overall structure of the lipid bilayer and the zwitterionic head-group dipole orientation undergo only a slight modification compared with the pure lipid bilayer, with virtually no change in the potential across the interface. This is in contrast to the anesthetic case in which the presence of the molecule led to a large perturbation of the electrostatic potential across to the membrane interface. Similarly, the analysis of the structural and dynamical properties of the lipid core are unchanged in the presence of the nonimmobilizer although there is a substantial increase in the microscopic viscosity for the system containing the anesthetic. These contrasting perturbations of the lipid membrane caused by those quite similarly sized molecules may explain the difference in their physiological effects as anesthetics and nonimmobilizers, respectively.

We use molecular dynamics simulations to investigate the effects of halothane, a volatile anesthetic, and hexafluoroethane (HFE), its nonimmobilizer analogue, on the physical properties of a fully hydrated polyunsaturated (1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine) lipid bilayer in the liquid crystalline fluid lamellar phase, L R . In addition, we discuss the results obtained comparing them to previous studies on saturated lipid bilayers with the same solutes. At the analyzed concentration (25% mole fraction), the halothane molecules are located preferentially near the upper part of the lipid acyl chains, while the HFE molecules prefer the hydrocarbon chains and methyl trough region. In the case of halothane in the polyunsaturated lipid bilayer, there is an additional density maximum at the membrane center not observed in saturated lipids. The subtle effect of the solutes on the structural properties of the highly unsaturated lipid bilayer and the properties of the membrane interior is somewhat different from that observed for saturated lipids. Here, these differences are interpreted in terms of the unique properties of the polyunsaturated lipid bilayers. The effect of anesthetic molecules on the electrostatic properties of the membrane interface is similar for saturated and polyunsaturated lipid bilayers and is characterized by a change in the most probable orientation of the lipid headgroup dipoles, which point toward the membrane interior for halothane and are basically unchanged for HFE, i.e., point toward the water phase. The different distributions of anesthetic and nonimmobilizer molecules within the lipid bilayer systems seem to be a generic feature in simple models of biological membranes and are similar to those observed in systems with saturated lipids. Since polyunsaturated and other unsaturated lipids are ubiquitous in cell membranes, it is plausible to generalize these features to the more complex biological membranes.