Membrane structural perturbations caused by anesthetics and nonimmobilizers: a molecular dynamics investigation.

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.     

Concentration effects of volatile anesthetics on the properties of model membranes: a coarse-grain approach

To gain insights into the molecular level mechanism of drug action at the membrane site, we have carried out extensive molecular dynamics simulations of a model membrane in the presence of a volatile anesthetic using a coarse-grain model. Six different anesthetic (halothane)/lipid (dimyristoylphosphatidylcholine) ratios have been investigated, going beyond the low doses typical of medical applications. The volatile anesthetics were introduced into a preassembled fully hydrated 512-molecule lipid bilayer and each of the molecular dynamics simulations were carried out at ambient conditions, using the NPT ensemble. The area per lipid increases monotonically with the halothane concentration and the lamellar spacing decreases, whereas the lipid bilayer thickness shows no appreciable differences and only a slight increase upon addition of halothane. The density profiles of the anesthetic molecules display a bimodal distribution along the membrane normal with maxima located close to the lipid-water interface region. We have studied how halothane molecules fluctuate between the two maxima of the bimodal distribution and we observed a different mechanism at low and high anesthetic concentrations. Through the investigation of the reorientational motions of the lipid tails, we found that the anesthetic molecules increase the segmental order of the lipids close to the membrane surface.     

Distribution of halothane in a dipalmitoylphosphatidylcholine bilayer from molecular dynamics calculations

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.     

AFM study of interaction forces in supported planar DPPC bilayers in the presence of general anesthetic halothane

In spite of numerous investigations, the molecular mechanism of general anesthetics action is still not well understood. It has been shown that the anesthetic potency is related to the ability of an anesthetic to partition into the membrane. We have investigated changes in structure, dynamics and forces of interaction in supported dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of the general anesthetic halothane. In the present study, we measured the forces of interaction between the probe and the bilayer using an atomic force microscope. The changes in force curves as a function of anesthetic incorporation were analyzed. Force measurements were in good agreement with AFM imaging data, and provided valuable information on bilayer thickness, structural transitions, and halothane-induced changes in electrostatic and adhesive properties.     

AFM study of interaction forces in supported planar DPPC bilayers in the presence of general anesthetic halothane

In spite of numerous investigations, the molecular mechanism of general anesthetics action is still not well understood. It has been shown that the anesthetic potency is related to the ability of an anesthetic to partition into the membrane. We have investigated changes in structure, dynamics and forces of interaction in supported dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of the general anesthetic halothane. In the present study, we measured the forces of interaction between the probe and the bilayer using an atomic force microscope. The changes in force curves as a function of anesthetic incorporation were analyzed. Force measurements were in good agreement with AFM imaging data, and provided valuable information on bilayer thickness, structural transitions, and halothane-induced changes in electrostatic and adhesive properties.     

Partitioning of anesthetics into a lipid bilayer and their interaction with membrane-bound peptide bundles

Molecular dynamics simulations have been performed to investigate the partitioning of the volatile anesthetic halothane from an aqueous phase into a coexisting hydrated bilayer, composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids, with embedded alpha-helical peptide bundles based on the membrane-bound portions of the alpha- and delta-subunits, respectively, of nicotinic acetylcholine receptor. In the molecular dynamics simulations halothane molecules spontaneously partitioned into the DOPC bilayer and then preferentially occupied regions close to lipid headgroups. A single halothane molecule was observed to bind to tyrosine (Tyr-277) residue in the alpha-subunit, an experimentally identified specific binding site. The binding of halothane attenuated the local loop dynamics of alpha-subunit and significantly influenced global concerted motions suggesting anesthetic action in modulating protein function. Steered molecular dynamics calculations on a single halothane molecule partitioned into a DOPC lipid bilayer were performed to probe the free energy profile of halothane across the lipid-water interface and rationalize the observed spontaneous partitioning. Partitioned halothane molecules affect the hydrocarbon chains of the DOPC lipid, by lowering of the hydrocarbon tilt angles. The anesthetic molecules also caused a decrease in the number of peptide-lipid contacts. The observed local and global effects of anesthetic binding on protein motions demonstrated in this study may underlie the mechanism of action of anesthetics at a molecular level.     

Raman spectroscopic studies of the packing properties of mixed dihexadecyl- and dipalmitoylphosphatidylcholine bilayer dispersions

X-ray diffraction studies suggest the existence of two separate gel phases for mixed dihexadecylphosphatidylcholine (DHPC)/dipalmitoylphosphatidylcholine (DPPC) bilayers [Kim, J. T., Mattai, J., & Shipley, G. G. (1987) Biochemistry 26, 6599-6603; Lohner, K., Schuster, A., Degovics, G., Müller, K., & Laggner, P. (1987) Chem. Phys. Lipids 44, 61-70]. In one gel phase the lipid chains are interdigitated, while the other gel phase exhibits the conventional bilayer form. We use Raman spectroscopy to provide a detailed molecular analysis of the intermolecular and intramolecular interactions of the DHPC and DPPC molecules within these mixed bilayers. Observation of the methylene chain C-H stretching modes of DHPC and the methylene chain C-D stretching modes of DPPC-d62 for various mixed DHPC/DPPC-d62 bilayers enables the packing characteristics and conformational order of each lipid to be monitored separately. The spectral data indicate that the packing properties of DPPC-d62 in the mixed-lipid bilayers remain relatively unchanged, while the intramolecular and intermolecular properties of DHPC change dramatically as a function of the composition of the DHPC/DPPC-d62 mixed bilayer. This is consistent with a model based upon the existence of three characteristic lipid types for the mixed-lipid system, namely, domains of pure DPPC-d62 and pure DHPC species with interface lipids or boundary regions between the bulk domains.     

Molecular dynamics simulations of C2F6 effects on gramicidin A: implications of the mechanisms of general anesthesia

It was recently postulated that the effects of general anesthetics on protein global dynamics might underlie a unitary molecular mechanism of general anesthesia. To verify that the specific dynamics effects caused by general anesthetics were not shared by nonanesthetic molecules, two parallel 8-ns all-atom molecular dynamics simulations were performed on a gramicidin A (gA) channel in a fully hydrated dimyristoylphosphatidylcholine membrane in the presence and absence of hexafluoroethane (HFE), which structurally resembles the potent anesthetic molecule halothane but produces no anesthesia. Similar to halothane, HFE had no measurable effects on the gA channel structure. In contrast to halothane, HFE produced no significant changes in the gA channel dynamics. The difference between halothane and HFE on channel dynamics can be attributed to their distinctly different distributions within the lipid bilayer and consequently to the different interactions of the anesthetic and the nonanesthetic molecules with the channel-anchoring tryptophan residues. The study further supports the notion that anesthetic-induced changes in protein global dynamics may play an important role in mediating anesthetic actions on proteins.     

Interaction of anesthetics with open and closed conformations of a potassium channel studied via molecular dynamics and normal mode analysis

A variety of experiments suggest that membrane proteins are important targets of anesthetic molecules, and that ion channels interact differently with anesthetics in their open and closed conformations. The availability of an open and a closed structural model for the KirBac1.1 potassium channel has made it possible to perform a comparative analysis of the interactions of anesthetics with the same channel in its open and closed states. To this end, all-atom molecular dynamics simulations supplemented by normal mode analysis have been employed to probe the interactions of the inhalational anesthetic halothane with both an open and closed conformer of KirBac1.1 embedded in a lipid bilayer. Normal mode analysis on the closed and open channel, in the presence and absence of halothane, reveals that the anesthetic modulates the global as well as the local dynamics of both conformations differently. In the case of the open channel, the observed reduction of flexibility of residues in the inner helices suggests a functional modification action of anesthetics on ion channels. In this context, preferential quenching of the aromatic residue motion and modulation of global dynamics by halothane may be seen as steps toward potentiating or favoring open state conformations. These molecular dynamics simulations provide the first insights into possible specific interactions between anesthetic molecules and ion channels in different conformations.     

Constant-pressure molecular dynamics investigation of cholesterol effects in a dipalmitoylphosphatidylcholine bilayer.

We report a 1.4-ns constant-pressure molecular dynamics simulation of cholesterol at 12.5 mol% in a dipalmitoylphosphatidylcholine (DPPC) bilayer at 50 degrees C and compare the results to our previous simulation of a pure DPPC bilayer. The interlamellar spacing was increased by 2.5 A in the cholesterol-containing bilayer, consistent with x-ray diffraction results, whereas the bilayer thickness was increased by only 1 A. The bilayer/water interface was more abrupt because the lipid headgroups lie flatter to fill spaces left by the cholesterol molecules. This leads to less compensation by the lipid headgroups of the oriented water contribution to the membrane dipole potential and could explain the experimentally observed increase in the magnitude of the dipole potential by cholesterol. Our calculations suggested that 12.5 mol% cholesterol does not significantly affect the conformations and packing of the hydrocarbon chains and produces only a slight reduction in the empty free volume. However, cholesterol has a significant influence on the subnanosecond time scale lipid dynamics: the diffusion constant for the center-of-mass "rattling" motion was reduced by a factor of 3, and the reorientational motion of the methylene groups was slowed along the entire length of the hydrocarbon chains.     

Interactions of liquid crystal-forming molecules with phospholipid bilayers studied by molecular dynamics simulations

Recent experiments have shown that liquid crystals can be used to image mammalian cell membranes and to amplify structural reorganization in phospholipid-laden liquid crystal-aqueous interfaces. In this work, molecular dynamics simulations were employed to explore the interactions between commonly used liquid crystal-forming molecules and phospholipid bilayers. In particular, umbrella sampling was used to obtain the potential of mean force of 4-cyano-4'-pentylbiphenyl (5CB) and 4'-(3,4-difluor-phenyl)-4-pentyl-bicylohexyl (5CF) molecules partitioning into a dipalmitoylphosphatidylcholine bilayer. In addition, results of simulations are presented for systems consisting of a fully hydrated bilayer with 5CB or 5CF molecules at the lowest (4.5 mol %) and highest (20 mol %) concentrations used in recent laboratory experiments. It is found that mesogens preferentially partition from the aqueous phase into the membrane; the potential of mean force exhibits highly favorable free energy differences for partitioning (-18 k(B)T for 5CB and -26 k(B)T for 5CF). The location and orientation of mesogens associated with the most stable free energies in umbrella sampling simulations of dilute systems were found to be consistent with those observed in liquid-crystal-rich bilayers. It is found that the presence of mesogens in the bilayer enhances the order of lipid acyl tails, and changes the spatial and orientational arrangement of lipid headgroup atoms. These effects are more pronounced at higher liquid-crystal concentrations. In comparing the behavior of 5CB and 5CF, a stronger spatial correlation (i.e., possibly leading to aggregation) is observed between 5CB molecules within a bilayer than between 5CF molecules. Also, the range of molecular orientations and positions along the bilayer normal is larger for 5CB molecules. At the same time, 5CF molecules were found to bind more strongly to lipid headgroups, thereby slowing the lateral motion of lipid molecules.     

Lessons of slicing membranes: interplay of packing, free area, and lateral diffusion in phospholipid/cholesterol bilayers

We employ 100-ns molecular dynamics simulations to study the influence of cholesterol on structural and dynamic properties of dipalmitoylphosphatidylcholine bilayers in the fluid phase. The effects of the cholesterol content on the bilayer structure are considered by varying the cholesterol concentration between 0 and 50%. We concentrate on the free area in the membrane and investigate quantities that are likely to be affected by changes in the free area and free volume properties. It is found that cholesterol has a strong impact on the free area properties of the bilayer. The changes in the amount of free area are shown to be intimately related to alterations in molecular packing, ordering of phospholipid tails, and behavior of compressibility moduli. Also the behavior of the lateral diffusion of both dipalmitoylphosphatidylcholine and cholesterol molecules with an increasing amount of cholesterol can in part be understood in terms of free area. Summarizing, our results highlight the central role of free area in comprehending the structural and dynamic properties of membranes containing cholesterol.     

Molecular dynamics simulation of structural changes of lipid bilayers induced by shock waves: Effects of incident angles

Unsteady and nonequilibrium molecular dynamics simulations of the response of dipalmitoylphosphatidylcholine (DPPC) bilayers to the shock waves of various incident angles are presented. The action of an incident shock wave is modeled by adding a momentum in an oblique direction to water molecules adjacent to a bilayer. We thereby elucidate the effects of incident shock angles on (i) collapse and rebound of the bilayer, (ii) lateral displacement of headgroups, (iii) tilts of lipid molecules, (iv) water penetration into the hydrophobic region of the bilayer, and (v) momentum transfer across the bilayer. The number of water molecules delivered into the hydrophobic region is found to be insensitive to incident shock angles. The most important structural changes are the lateral displacement of headgroups and tilts of lipid molecules, which are observed only in the half of the bilayer directly exposed to a shock wave for all incident shock angles studied here. As a result, only the normal component of the added oblique momentum is substantially transferred across the bilayer. This also suggests that the irradiation by shock waves may induce a jet-like streaming of the cytoplasm toward the nucleus.     

Molecular dynamics simulation of structural changes of lipid bilayers induced by shock waves: Effects of incident angles

Unsteady and nonequilibrium molecular dynamics simulations of the response of dipalmitoylphosphatidylcholine (DPPC) bilayers to the shock waves of various incident angles are presented. The action of an incident shock wave is modeled by adding a momentum in an oblique direction to water molecules adjacent to a bilayer. We thereby elucidate the effects of incident shock angles on (i) collapse and rebound of the bilayer, (ii) lateral displacement of headgroups, (iii) tilts of lipid molecules, (iv) water penetration into the hydrophobic region of the bilayer, and (v) momentum transfer across the bilayer. The number of water molecules delivered into the hydrophobic region is found to be insensitive to incident shock angles. The most important structural changes are the lateral displacement of headgroups and tilts of lipid molecules, which are observed only in the half of the bilayer directly exposed to a shock wave for all incident shock angles studied here. As a result, only the normal component of the added oblique momentum is substantially transferred across the bilayer. This also suggests that the irradiation by shock waves may induce a jet-like streaming of the cytoplasm toward the nucleus.     

Revisiting the bilayer structures of fluid phase phosphatidylglycerol lipids: Accounting for exchangeable hydrogens

We recently published two papers detailing the structures of fluid phase phosphatidylglycerol (PG) lipid bilayers (Ku?erka et al., 2012 J. Phys. Chem. B 116: 232–239; Pan et al., 2012 Biochim. Biophys. Acta Biomembr. 1818: 2135–2148), which were determined using the scattering density profile model. This hybrid experimental/computational technique utilizes molecular dynamics simulations to parse a lipid bilayer into components whose volume probabilities follow simple analytical functional forms. Given the appropriate scattering densities, these volume probabilities are then translated into neutron scattering length density (NSLD) and electron density (ED) profiles, which are used to jointly refine experimentally obtained small angle neutron and X-ray scattering data. However, accurate NSLD and ED profiles can only be obtained if the bilayer's chemical composition is known. Specifically, in the case of neutron scattering, the lipid's exchangeable hydrogens with aqueous D₂O must be accounted for, as they can have a measureable effect on the resultant lipid bilayer structures. This was not done in our above-mentioned papers. Here we report on the molecular structures of PG lipid bilayers by appropriately taking into account the exchangeable hydrogens. Analysis indicates that the temperature-averaged PG lipid areas decrease by 1.5 to 3.8 Å², depending on the lipid's acyl chain length and unsaturation, compared to PG areas when hydrogen exchange was not taken into account.     

Molecular dynamics simulation of a synthetic four-alpha-helix bundle that binds the anesthetic halothane.

The structural features of binding sites for volatile anesthetics are examined by performing a molecular dynamics simulation study of the synthetic four-alpha-helix bundles (Aalpha2)2, which are formed by association of two 62-residue di-alpha-helical peptides. The peptide bundle (Aalpha2)2 was designed by Johansson et al. [Biochemistry 37 (1998) 1421-1429] and was shown experimentally to have a high affinity for the binding of the anesthetic halothane (CF3CBrCIH) in a hydrophobic cavity. Since (Aalpha2)2 can exhibit either the anti or syn topologies, the two distinct bundles are simulated both in the presence and in the absence of halothane. Nanosecond length molecular dynamics trajectories were generated for each system at room temperature (T = 298 K). The structural and dynamic effects of the inclusion of halothane are compared, illustrating that the structures are stable over the course of the simulation; that the (Aalpha2)2 bundles have suitable pockets that can accommodate halothane; that the halothane remains in the designed hydrophobic cavity in close proximity to the Trp residues with a preferred orientation; and that the dimensions of the peptide are perturbed by the inclusion of an anesthetic molecule.     

An alamethicin channel in a lipid bilayer: molecular dynamics simulations

We present the results of 2-ns molecular dynamics (MD) simulations of a hexameric bundle of Alm helices in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer. These simulations explore the dynamic properties of a model of a helix bundle channel in a complete phospholipid bilayer in an aqueous environment. We explore the stability and conformational dynamics of the bundle in a phospholipid bilayer. We also investigate the effect on bundle stability of the ionization state of the ring of Glu18 side chains. If all of the Glu18 side chains are ionised, the bundle is unstable; if none of the Glu18 side chains are ionized, the bundle is stable. pKA calculations suggest that either zero or one ionized Glu18 is present at neutral pH, correlating with the stable form of the helix bundle. The structural and dynamic properties of water in this model channel were examined. As in earlier in vacuo simulations (Breed et al., 1996 .Biophys. J. 70:1643-1661), the dipole moments of water molecules within the pore were aligned antiparallel to the helix dipoles. This contributes to the stability of the helix bundle.     

Dantrolene inhibits halothane-induced membrane reorganization A study using 31P-NMR and differential scanning calorimetry

The action of the relaxing agent dantrolene on dipalmitoylphosphatidylcholine (DPPC) model membranes in the presence and absence of the general anesthetic halothane has been investigated by DSC and 31P-NMR. Dantrolene has a weak effect on both the thermodynamic and NMR parameters of the pure model membrane. When halothane is present in the system, the relaxing agent acts to counterbalance the strong anesthetic-induced membrane perturbation. This is reflected in DSC experiments by a change of the enthalpy variation (delta H) and of the main gel-to-fluid phase transition temperature (Tc) towards the values of the pure lipid system. The amount of halothane-induced small tumbling vesicles, as detected by 31P-NMR by the superposition of an isotropic line on a lamellar-type powder spectrum, is considerably reduced upon dantrolene addition. This means that the relaxing agent "cures" the membrane de-structuring action promoted by halothane. Membranes first treated with dantrolene are also protected from the halothane perturbation. So, the relaxing agent is both "curative" and "preventative" against halothane. The optimum effect is obtained for 1 dantrolene molecule per ca 34 halothane molecules. The mechanisms of action were discussed in relation to membrane fluidity.     

Unrestrained stochastic dynamics simulations of the UUCG tetraloop using an implicit solvation model.

Three unrestrained stochastic dynamics simulations have been carried out on the RNA hairpin GGAC[UUCG] GUCC, using the AMBER94 force field (Cornell et al., 1995. J. Am. Chem. Soc. 117:5179-5197) in MacroModel 5.5 (Mohamadi et al., 1990. J. Comp. Chem. 11:440-467) and either the GB/SA continuum solvation model (Still et al., 1990. J. Am. Chem. Soc. 112:6127-6129) or a linear distance-dependent dielectric (1/R) treatment. The linear distance-dependent treatment results in severe distortion of the nucleic acid structure, restriction of all hydroxyl dihedrals, and collapse of the counterion atmosphere over the course of a 5-ns simulation. An additional vacuum simulation without counterions shows somewhat improved behavior. In contrast, the two GB/SA simulations (1.149 and 3.060 ns in length) give average structures within 1.2 A of the initial NMR structure and in excellent agreement with results of an earlier explicit solvent simulation (Miller and Kollman, 1997. J. Mol. Biol. 270:436-450). In a 3-ns GB/SA simulation starting with the incorrect UUCG tetraloop structure (Cheong et al., 1990. Nature. 346:680-682), this loop conformation converts to the correct loop geometry (Allain and Varani, 1995. J. Mol. Biol. 250:333-353), suggesting enhanced sampling relative to the previous explicit solvent simulation. Thermodynamic effects of 2'-deoxyribose substitutions of loop nucleotides were experimentally determined and are found to correlate with the fraction of time the ribose 2'-OH is hydrogen bonded and the distribution of the hydroxyl dihedral is observed in the GB/SA simulations. The GB/SA simulations thus appear to faithfully represent structural features of the RNA without the computational expense of explicit solvent.