Bilayer conformation of fusion peptide of influenza virus hemagglutinin: a molecular dynamics simulation study

Unraveling the conformation of membrane-bound viral fusion peptides is essential for understanding how those peptides destabilize the bilayer topology of lipids that is important for virus-cell membrane fusion. Here, molecular dynamics (MD) simulations were performed to investigate the conformation of the 20 amino acids long fusion peptide of influenza hemagglutinin of strain X31 bound to a dimyristoyl phosphatidylcholine (DMPC) bilayer. The simulations revealed that the peptide adopts a kinked conformation, in agreement with the NMR structures of a related peptide in detergent micelles. The peptide is located at the amphipathic interface between the headgroups and hydrocarbon chains of the lipid by an energetically favorable arrangement: The hydrophobic side chains of the peptides are embedded into the hydrophobic region and the hydrophilic side chains are in the headgroup region. The N-terminus of the peptide is localized close to the amphipathic interface. The molecular dynamics simulations also revealed that the peptide affects the surrounding bilayer structure. The average hydrophobic thickness of the lipid phase close to the N-terminus is reduced in comparison with the average hydrophobic thickness of a pure dimyristoyl phosphatidylcholine bilayer.     

Transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations.

To probe the fundamentals of membrane/protein interactions, all-atom multi-nanosecond molecular dynamics simulations were conducted on a single transmembrane poly(32)alanine helix in a fully solvated dimyristoyphosphatidylcholine (DMPC) bilayer. The central 12 residues, which interact only with the lipid hydrocarbon chains, maintained a very stable helical structure. Helical regions extended beyond these central 12 residues, but interactions with the lipid fatty-acyl ester linkages, the lipid headgroups, and water molecules made the helix less stable in this region. The C and N termini, exposed largely to water, existed as random coils. As a whole, the helix tilted substantially, from perpendicular to the bilayer plane (0 degree) to a 30 degrees tilt. The helix experienced a bend at its middle, and the two halves of the helix at times assumed substantially different tilts. Frequent hydrogen bonding, of up to 0.7 ns in duration, occurred between peptide and lipid molecules. This resulted in correlated translational diffusion between the helix and a few lipid molecules. Because of the large variation in lipid conformation, the lipid environment of the peptide was not well defined in terms of "annular" lipids and on average consisted of 18 lipid molecules. When compared with a "neat" bilayer without peptide, no significant difference was seen in the bilayer thickness, lipid conformations or diffusion, or headgroup orientation. However, the lipid hydrocarbon chain order parameters showed a significant decrease in order, especially in those methylene groups closest to the headgroup.     

Peptide models of the helical hydrophobic transmembrane segments of membrane proteins: interactions of acetyl-K2-(LA)12-K2-amide with phosphatidylethanolamine bilayer membranes

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, acetyl-Lys(2)-(Leu-Ala)(12)-Lys(2)-amide [(LA)(12)], and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mole fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the protein/lipid ratio. However, the fractional contribution of this component to the total enthalpy changes decreases with increases in peptide concentration, and this component completely disappears at higher protein mole fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher protein concentrations. These two components of the DSC endotherm have been assigned to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in (LA)(12) concentration, the magnitude of this downward shift is progressively greater as the length of the PE hydrocarbon chain decreases. As well, mixtures of (LA)(12) with the longer chain PEs exhibit unusual biomodal enthalpy variations, suggesting peptide immiscibility in thicker gel state bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to zero even at high peptide concentrations, indicating that (LA)(12) attenuates but does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our FTIR spectroscopic data indicate that (LA)(12) remains in a predominantly alpha-helical conformation in liquid-crystalline PE bilayers of various hydrophobic thickness but that the helical conformation is altered in gel-state PE bilayers generally, probably due to peptide lateral aggregation. These data also suggest that (LA)(12) significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states, relatively independently of lipid hydrocarbon chain length. Many aspects of PE/(LA)(12) interactions exhibit a different dependence on the hydrophobic thickness of the host bilayer than was observed in our previous study of (LA)(12)-phosphatidylcholine (PC) model membranes [Zhang et al. (1995) Biochemistry 34, 2362-2371]. The differing effects of (LA)(12) incorporation on PE and PC bilayers is ascribed primarily to the much stronger lipid polar headgroup interactions characteristic of the former system. Finally, the considerable differences observed in the behavior of (LA)(12) and the related polyleucine-based peptide P(24) in both PC and PE bilayers indicate that the structure of the hydrophobic core of alpha-helical transmembrane peptides can affect their conformational plasticity and state of aggregation and thus the nature of their interactions with different phospholipid bilayers.     

Effects of anesthetics on the structure of a phospholipid bilayer: molecular dynamics investigation of halothane in the hydrated liquid crystal phase of dipalmitoylphosphatidylcholine.

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).     

Molecular dynamics simulation of a phospholipid membrane

We present the results of molecular dynamics (MD) simulations of a phospholipid membrane in water, including full atomic detail. The goal of the simulations was twofold: first we wanted to set up a simulation system which is able to reproduce experimental results and can serve as a model membrane in future simulations. This goal being reached it is then further possible to gain insight in to those properties that are experimentally more difficult to access. The system studied is dipalmitoylphosphatidylcholine/water, consisting of 5408 atoms. Using original force field parameters the membrane turned out to approach a gel-like state. With slight changes of the parameters, the system adopted a liquid-crystalline state. Separate 80 ps runs were performed on both the gel and liquid-crystalline systems. Comparison of MD results with reliable experimental data (bilayer repeat distance, surface area per lipid, tail order parameters, atom distributions) showed that our simulations, especially the one in the liquid-crystalline phase, can serve as a realistic model for a phospholipid membrane. Further analysis of the trajectories revealed valuable information on various properties. In the liquid-crystalline phase, the interface turns out to be quite diffuse, with water molecules penetrating into the bilayer to the position of the carbonyl groups. The 10–90% width of the interface turns out to be 1.3 nm and the width of the hydrocarbon interior 3.0 nm. The headgroup dipoles are oriented at a small angle with respect to the bilayer plane. The resulting charge distribution is almost completely cancelled by the water molecules. The electron density distribution shows a large dip in the middle of the membrane. In this part the tails are more flexible. The mean life time between dihedral transitions is 20 ps. The average number of gauche angles per tail is 3.5. The occurrence of kinks is not a significant feature.Abbreviations MD molecular dynamics - DPPC dipalmitoylphosphatidylcholine - SPC simple point charges - DPPE dipalmitoylphosphatidylethanolamine Correspondence to: H. J. C. Berendsen     

Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.     

The effect of cholesterol on short- and long-chain monounsaturated lipid bilayers as determined by molecular dynamics simulations and X-ray scattering

We investigate the structure of cholesterol-containing membranes composed of either short-chain (diC14:1PC) or long-chain (diC22:1PC) monounsaturated phospholipids. Bilayer structural information is derived from all-atom molecular dynamics simulations, which are validated via direct comparison to x-ray scattering experiments. We show that the addition of 40 mol % cholesterol results in a nearly identical increase in the thickness of the two different bilayers. In both cases, the chain ordering dominates over the hydrophobic matching between the length of the cholesterol molecule and the hydrocarbon thickness of the bilayer, which one would expect to cause a thinning of the diC22:1PC bilayer. For both bilayers there is substantial headgroup rearrangement for lipids directly in contact with cholesterol, supporting the so-called umbrella model. Importantly, in diC14:1PC bilayers, a dynamic network of hydrogen bonds stabilizes long-lived reorientations of some cholesterol molecules, during which they are found to lie perpendicular to the bilayer normal, deep within the bilayer’s hydrophobic core. Additionally, the simulations show that the diC14:1PC bilayer is significantly more permeable to water. These differences may be correlated with faster cholesterol flip-flop between the leaflets of short-chain lipid bilayers, resulting in an asymmetric distribution of cholesterol molecules. This asymmetry was observed experimentally in a case of unilamellar vesicles (ULVs), and reproduced through a set of novel asymmetric simulations. In contrast to ULVs, experimental data for oriented multilamellar stacks does not show the asymmetry, suggesting that it results from the curvature of the ULV bilayers.     

Influence of stigmastanol and stigmastanyl-phosphorylcholine, two plasma cholesterol lowering substances, on synthetic phospholipid membranes. A 2H- and 31P-NMR study.

Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase.     

Interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylethanolamine bilayers: differential scanning calorimetric and Fourier transform infrared spectroscopic studies.

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, Acetyl-Lys2-Gly-Leu24-Lys2-Ala-Amide, and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mol fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher-temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the peptide/lipid ratio. However, the fractional contribution of this component to the total enthalpy change decreases with increases in peptide concentration, and this component completely disappears at higher peptide mol fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher peptide concentrations. These two components of the DSC endotherm can be attributed to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in peptide concentration, the magnitude of this shift is independent of the length of the PE hydrocarbon chain. In addition, the width of the phase transition observed at higher peptide concentrations is also relatively insensitive to PE hydrocarbon chain length, except that peptide gel-phase immiscibility occurs in very short- or very long-chain PE bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to 0 even at high peptide concentrations, suggesting that this peptide does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. The FTIR spectroscopic data indicate that the peptide remains in a predominantly alpha-helical conformation, but that the peptide alpha-helix is subject to small distortions coincident with the changes in hydrophobic thickness that accompany the chain-melting phase transition of the PE bilayer. These data also indicate that the peptide significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states relatively independently of lipid hydrocarbon chain length. The relative independence of many aspects of PE-peptide interactions on the hydrophobic thickness of the host bilayer observed in the present study is in marked contrast to the results of our previous study of peptide-phosphatidylcholine (PC) model membranes (Zhang et al. (1992) Biochemistry 31:11579-11588), where strong hydrocarbon chain length-dependent effects were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylcholine bilayers: differential scanning calorimetric and FTIR spectroscopic studies.

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic model hydrophobic peptide, Lys2-Gly-Leu24-Lys2-Ala-amide, and members of the homologous series of n-saturated diacylphosphatidylcholines. In the low range of peptide mole fractions, the DSC thermograms exhibited by the lipid/peptide mixtures are resolvable into two components. One of these components is fairly narrow, highly cooperative, and exhibits properties which are similar to but not identical with those of the pure lipid. In addition, the fractional contribution of this component to the total enthalpy change, the peak transition temperature, and cooperativity decrease with an increase in peptide concentration, more or less independently of acyl chain length. The other component is very broad and predominates in the high range of peptide concentration. These two components have been assigned to the chain-melting phase transitions of populations of bulk lipid and peptide-associated lipid, respectively. Moreover, when the mean hydrophobic thickness of the PC bilayer is less than the peptide hydrophobic length, the peptide-associated lipid melts at higher temperatures than does the bulk lipid and vice versa. In addition, the chain-melting enthalpy of the broad endotherm does not decrease to zero even at high peptide concentrations, suggesting that this peptide reduces but do not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our DSC results indicate that the width of the phase transition observed at high peptide concentration is inversely but discontinuously related to hydrocarbon chain length and that gel phase immiscibility occurs when the hydrophobic thickness of the bilayer greatly exceeds the hydrophobic length of the peptide. The FTIR spectroscopic data indicate that the peptide forms a very stable alpha-helix under all of our experimental conditions but that small distortions of its alpha-helical conformation are induced in response to any mismatch between peptide hydrophobic length and bilayer hydrophobic thickness. These results also indicate that the peptide alters the conformational disposition of the acyl chains in contact with it and that the resultant conformational changes in the lipid hydrocarbon chains tend to minimize the extent of mismatch of peptide hydrophobic length and bilayer hydrophobic thickness.     

Influence of Hydrophobic Mismatch and Amino Acid Composition on the Lateral Diffusion of Transmembrane Peptides

We investigated the effect of amino acid composition and hydrophobic length of α-helical transmembrane peptides and the role of electrostatic interactions on the lateral diffusion of the peptides in lipid membranes. Model peptides of varying length and composition, and either tryptophans or lysines as flanking residues, were synthesized. The peptides were labeled with the fluorescent label Alexa Fluor 488 and incorporated into phospholipid bilayers of different hydrophobic thickness and composition. Giant unilamellar vesicles were formed by electroformation, and the lateral diffusion of the transmembrane peptides (and lipids) was determined by fluorescence correlation spectroscopy. In addition, we performed coarse-grained molecular-dynamics simulations of single peptides of different hydrophobic lengths embedded in planar membranes of different thicknesses. Both the experimental and simulation results indicate that lateral diffusion is sensitive to membrane thickness between the peptides and surrounding lipids. We did not observe a difference in the lateral diffusion of the peptides with respect to the presence of tryptophans or lysines as flanking residues. The specific lipid headgroup composition of the membrane has a much less pronounced impact on the diffusion of the peptides than does the hydrophobic thickness.     

Influence of hydrophobic mismatch on structures and dynamics of gramicidin a and lipid bilayers

Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-D sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA dimers and monomers in all-atom, explicit dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4–5 Å from the channel, which was followed by an increase in DOPC and POPC or a further decrease in DLPC and DMPC bilayers. The bilayer thickness varied little in the monomer simulations—except one of three independent simulations for DMPC and all three DLPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA dimers due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA dimers are more complex than predicted from a simple mismatch model, which has implications for understanding the energetics of membrane protein-lipid interactions.     

Structural change in lipid bilayers and water penetration induced by shock waves: molecular dynamics simulations

The structural change of a phospholipid bilayer in water under the action of a shock wave is numerically studied with unsteady nonequilibrium molecular dynamics simulations. The action of shock waves is modeled by the momentum change of water molecules, and thereby we demonstrate that the resulting collapse and rebound of the bilayer are followed by the penetration of water molecules into the hydrophobic region of the bilayer. The high-speed phenomenon that occurs during the collapse and rebound of the bilayer is analyzed in detail, particularly focusing on the change of bilayer thickness, the acyl chain bend angles, the lateral fluidity of lipid molecules, and the penetration rate of water molecules. The result shows that the high-speed phenomenon can be divided into two stages: in the first stage the thickness of bilayer and the order parameter are rapidly reduced, and then in the second stage they are recovered relatively slowly. It is in the second stage that water molecules are steadily introduced into the hydrophobic region. The penetration of water molecules is enhanced by the shock wave impulse and this qualitatively agrees with a recent experimental result.     

Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes.

Deuterium (2H) NMR was used to study bilayer hydrophobic thickness and mechanical properties when cholesterol and/or synthetic amphiphillic polypeptides were added to deuterated POPC lipid bilayer membranes in the liquid-crystalline (fluid) phase. Smoothed acyl chain orientational order profiles were used to calculate bilayer hydrophobic thickness. Addition of 30 mol% cholesterol to POPC at 25 degrees C increased the bilayer thickness from 2.58 to 2.99 nm. The peptides were chosen to span the bilayers with more or less mismatch between the hydrophobic peptide length and membrane hydrophobic thickness. The average thickness of the pure lipid bilayers was significantly perturbed upon addition of peptide only in cases of large mismatch, being increased (decreased) when the peptide hydrophobic length was greater (less) than that of the pure bilayer, consistent with the "mattress" model of protein lipid interactions (Mouritsen, O.G., and M. Bloom. 1984. Biophys. J. 46:141-153). The experimental results were also used to examine the combined influence of the polypeptides and cholesterol on the orientational order profile and thickness expansivity of the membranes. A detailed model for the spatial distribution of POPC and cholesterol molecules in the bilayers was proposed to reconcile the general features of these measurements with micromechanical measurements of area expansivity in closely related systems. Experiments to test the model were proposed.     

Examining the effects of cholesterol on model membranes at high temperatures: Laurdan and Patman see it differently

At high temperature, the presence of cholesterol in phospholipid membranes alters the influence of membrane dipoles, including water molecules, on naphthalene-based fluorescent probes such as Laurdan and Patman (solvatochromism). Although both of these probes report identical changes to their emission spectra as a function of temperature in pure phosphatidylcholine bilayers, they differ in their response to cholesterol. Computer simulations of the spectra based on a simple model of solvatochromism indicated that the presence of cholesterol reduces the probability of bilayer dipole relaxation and also blunts the tendency of heat to enhance that probability. While the overall effect of cholesterol on membrane dipoles was detected identically by the two probes, Laurdan was influenced much more by the additional effect on temperature sensitivity than was Patman. A comparison of the fluorescence data with simulations using a coarse-grained bilayer model (de Meyer et al., 2010) suggested that these probes may be differentially sensitive to two closely related properties distinguishable in the presence of cholesterol. Specifically, Patman fluorescence correlated best with the average phospholipid acyl chain order. On the other hand, Laurdan fluorescence tracked more closely with the area per lipid molecule which, although affected generally by chain order, is also impacted by additional membrane-condensing effects of cholesterol. We postulate that this difference between Laurdan and Patman may be attributed to the bulkier charged headgroup of Patman which may cause the probe to preferentially locate in juxtaposition to the diminutive headgroup of cholesterol as the membrane condenses.     

Structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies

Cell-signaling peptides have been extensively used to transport functional molecules across the plasma membrane into living cells. These peptides consist of a hydrophobic sequence and a cationic nuclear localization sequence (NLS). It has been assumed that the hydrophobic region penetrates the hydrophobic lipid bilayer and delivers the NLS inside the cell. To better understand the transport mechanism of these peptides, in this study, we investigated the structure, orientation, tilt of the peptide relative to the bilayer normal, and the membrane interaction of two cell-signaling peptides, SA and SKP. Results from CD and solid-state NMR experiments combined with molecular dynamics simulations suggest that the hydrophobic region is helical and has a transmembrane orientation with the helical axis tilted away from the bilayer normal. The influence of the hydrophobic mismatch, between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, on the tilt angle of the peptides was investigated using thicker POPC and thinner DMPC bilayers. NMR experiments showed that the hydrophobic domain of each peptide has a tilt angle of 15 +/- 3 degrees in POPC, whereas in DMPC, 25 +/- 3 degree and 30 +/- 3 degree tilts were observed for SA and SKP peptides, respectively. These results are in good agreement with molecular dynamics simulations, which predict a tilt angle of 13.3 degrees (SA in POPC), 16.4 degrees (SKP in POPC), 22.3 degrees (SA in DMPC), and 31.7 degrees (SKP in DMPC). These results and simulations on the hydrophobic fragment of SA or SKP suggest that the tilt of helices increases with a decrease in bilayer thickness without changing the phase, order, and structure of the lipid bilayers.     

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.     

Structural Thermodynamics of myr-Src(2–19) Binding to Phospholipid Membranes

Many proteins are anchored to lipid bilayer membranes through a combination of hydrophobic and electrostatic interactions. In the case of the membrane-bound nonreceptor tyrosine kinase Src from Rous sarcoma virus, these interactions are mediated by an N-terminal myristoyl chain and an adjacent cluster of six basic amino-acid residues, respectively. In contrast with the acyl modifications of other lipid-anchored proteins, the myristoyl chain of Src does not match the host lipid bilayer in terms of chain conformation and dynamics, which is attributed to a tradeoff between hydrophobic burial of the myristoyl chain and repulsion of the peptidic moiety from the phospholipid headgroup region. Here, we combine thermodynamic information obtained from isothermal titration calorimetry with structural data derived from ²H, ¹³C, and ³¹P solid-state nuclear magnetic resonance spectroscopy to decipher the hydrophobic and electrostatic contributions governing the interactions of a myristoylated Src peptide with zwitterionic and anionic membranes made from lauroyl (C12:0) or myristoyl (C14:0) lipids. Although the latter are expected to enable better hydrophobic matching, the Src peptide partitions more avidly into the shorter-chain lipid analog because this does not require the myristoyl chain to stretch extensively to avoid unfavorable peptide/headgroup interactions. Moreover, we find that Coulombic and intrinsic contributions to membrane binding are not additive, because the presence of anionic lipids enhances membrane binding more strongly than would be expected on the basis of simple Coulombic attraction.     

Mean-field calculations of chain packing and conformational statistics in lipid bilayers: comparison with experiments and molecular dynamics studies.

A molecular, mean-field theory of chain packing statistics in aggregates of amphiphilic molecules is applied to calculate the conformational properties of the lipid chains comprising the hydrophobic cores of dipalmitoyl-phosphatidylcholine (DPPC), dioleoyl-phosphatidylcholine (DOPC), and palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers in their fluid state. The central quantity in this theory, the probability distribution of chain conformations, is evaluated by minimizing the free energy of the bilayer assuming only that the segment density within the hydrophobic region is uniform (liquidlike). Using this distribution we calculate chain conformational properties such as bond orientational order parameters and spatial distributions of the various chain segments. The lipid chains, both the saturated palmitoyl (-(CH2)14-CH3) and the unsaturated oleoyl (-(CH2)7-CH = CH-(CH2)7-CH3) chains are modeled using rotational isomeric state schemes. All possible chain conformations are enumerated and their statistical weights are determined by the self-consistency equations expressing the condition of uniform density. The hydrophobic core of the DPPC bilayer is treated as composed of single (palmitoyl) chain amphiphiles, i.e., the interactions between chains originating from the same lipid headgroup are assumed to be the same as those between chains belonging to different molecules. Similarly, the DOPC system is treated as a bilayer of oleoyl chains. The POPC bilayer is modeled as an equimolar mixture of palmitoyl and oleoyl chains. Bond orientational order parameter profiles, and segment spatial distributions are calculated for the three systems above, for several values of the bilayer thickness (or, equivalently, average area/headgroup) chosen, where possible, so as to allow for comparisons with available experimental data and/or molecular dynamics simulations. In most cases the agreement between the mean-field calculations, which are relatively easy to perform, and the experimental and simulation data is very good, supporting their use as an efficient tool for analyzing a variety of systems subject to varying conditions (e.g., bilayers of different compositions or thicknesses at different temperatures).