SGTx1, a Kv channel gating-modifier toxin, binds to the interfacial region of lipid bilayers

SGTx1 is a gating-modifier toxin that has been shown to inhibit the voltage-gated potassium channel Kv2.1. SGTx1 is thought to bind to the S3b-S4a region of the voltage-sensor, and is believed to alter the energetics of gating. Gating-modifier toxins such as SGTx1 are of interest as they can be used to probe the structure and dynamics of their target channels. Although there are experimental data for SGTx1, its interaction with lipid bilayer membranes remains to be characterized. We performed atomistic and coarse-grained molecular dynamics simulations to study the interaction of SGTx1 with a POPC and a 3:1 POPE/POPG lipid bilayer membrane. We reveal the preferential partitioning of SGTx1 into the water/membrane interface of the bilayer. We also show that electrostatic interactions between the charged residues of SGTx1 and the lipid headgroups play an important role in stabilizing SGTx1 in a bilayer environment.     

Is lipid bilayer binding a common property of inhibitor cysteine knot ion-channel blockers?

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (DeltaG) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of DeltaG dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep DeltaG dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed DeltaG shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels.     

Lipid bilayer deformation and the free energy of interaction of a Kv channel gating-modifier toxin

A number of membrane proteins act via binding at the water/lipid bilayer interface. An important example of such proteins is provided by the gating-modifier toxins that act on voltage-gated potassium (Kv) channels. They are thought to partition to the headgroup region of lipid bilayers, and so provide a good system for probing the nature of interactions of a protein with the water/bilayer interface. We used coarse-grained molecular dynamics simulations to compute the one-dimensional potential of mean force (i.e., free energy) profile that governs the interaction between a Kv channel gating-modifier toxin (VSTx1) and model phospholipid bilayers. The reaction coordinate sampled corresponds to the position of the toxin along the bilayer normal. The course-grained representation of the protein and lipids enabled us to explore extended time periods, revealing aspects of toxin/bilayer dynamics and energetics that would be difficult to observe on the timescales currently afforded by atomistic molecular dynamics simulations. In particular, we show for this model system that the bilayer deforms as it interacts with the toxin, and that such deformations perturb the free energy profile. Bilayer deformation therefore adds an additional layer of complexity to be addressed in investigations of membrane/protein systems. In particular, one should allow for local deformations that may arise due to the spatial array of charged and hydrophobic elements of an interfacially located membrane protein.     

Finding a Needle in a Haystack: The Role of Electrostatics in Target Lipid Recognition by PH Domains

Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH) domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs) in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition via a multiscale procedure combining Brownian dynamics (BD) and molecular dynamics (MD) simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)). The interaction of GRP1-PH with PI(3,4,5)P(3) in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic 'decoy' lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5)P(3)-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as 'decoys', disrupting the interaction of GRP1-PH with the PI(3,4,5)P(3) molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5)P(3) within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5)P(3), forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation.     

Lipid bilayer molecular dynamics study of lipid-derived agonists of the putative cannabinoid receptor, GPR55

Both L-α-lysophosphatidylinositol (LPI) and 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) have been reported to activate the putative cannabinoid receptor, GPR55. Recent microsecond time-scale molecular dynamics (MD) simulations and isothiocyanate covalent labeling studies have suggested that a transmembrane helix 6/7 (TMH6/7) lipid pathway for ligand entry may be necessary for interaction with cannabinoid receptors. Because LPI and 2-AGPI are lipid-derived ligands, conformations that each assumes in the lipid bilayer are therefore likely important for their interaction with GPR55. We report here the results of 70 ns NAMD molecular dynamics (MD) simulations of LPI and of 2-AGPI in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). These simulations are compared with a 70 ns simulation of the cannabinoid CB1 receptor endogenous ligand, N-arachidonoylethanolamine (anandamide, AEA) in a POPC bilayer. These simulations revealed that (1) LPI and 2-AGPI sit much higher in the bilayer than AEA, with inositol headgroups that can at times be solvated completely by water; (2) the behavior of the acyl chains of AEA and 2-AGPI are similar in their flexibilities in the bilayer, while the acyl chain of LPI has reduced flexibility; and (3) both 2-AGPI and LPI can adopt a tilted headgroup orientation by hydrogen bonding to the phospholipid phosphate/glycerol groups or via intramolecular hydrogen bonding. This tilted head group conformation (which represents over 40% of the conformer population of LPI (42.2 ± 3.3%) and 2-AGPI (43.7 ± 1.4%)) may provide a low enough profile in the lipid bilayer for LPI and 2-AGPI to enter GPR55 via the putative TMH6/7 entry port.     

Structure and Orientation of a Voltage-Sensor Toxin in Lipid Membranes

Amphipathic protein toxins from tarantula venom inhibit voltage-activated potassium (Kv) channels by binding to a critical helix-turn-helix motif termed the voltage sensor paddle. Although these toxins partition into membranes to bind the paddle motif, their structure and orientation within the membrane are unknown. We investigated the interaction of a tarantula toxin named SGTx with membranes using both fluorescence and NMR spectroscopy. Depth-dependent fluorescence-quenching experiments with brominated lipids suggest that Trp³⁰ in SGTx is positioned ∼9 Å from the center of the bilayer. NMR spectra reveal that the inhibitor cystine knot structure of the toxin does not radically change upon membrane partitioning. Transferred cross-saturation NMR experiments indicate that the toxin's hydrophobic protrusion contacts the hydrophobic core of the membrane, whereas most surrounding polar residues remain at interfacial regions of the bilayer. The inferred orientation of the toxin reveals a twofold symmetry in the arrangement of basic and hydrophobic residues, a feature that is conserved among tarantula toxins. These results have important implications for regions of the toxin involved in recognizing membranes and voltage-sensor paddles, and for the mechanisms by which tarantula toxins alter the activity of different types of ion channels.     

Membrane models of E. coli containing cyclic moieties in the aliphatic lipid chain

Nearly all molecular dynamics simulations of bacterial membranes simplify the lipid bilayer by composing it of only one or two lipids. Previous attempts of developing a model E. coli membrane have used only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) POPG lipids. However, an important constituent of bacterial membranes are lipids containing a cyclopropane ring within the acyl chain. We have developed a complex membrane that more accurately reflects the diverse population of lipids within E. coli cytoplasmic membranes, including lipids with a cyclic moiety. Differences between the deuterium order profile of cyclic lipids and monounsaturated lipids are observed. Furthermore, the inclusion of the cyclopropane ring decreases the surface density of the bilayer and produces a more rigid membrane as compared to POPE/POPG membranes. Additionally, the diverse acyl chain length creates a thinner bilayer which matches the hydrophobic thickness of E. coli transmembrane proteins better than the POPE/POPG bilayer. We believe that the complex lipid bilayer more accurately describes a bacterial membrane and suggest the use of it in molecular dynamic simulations rather than simple POPE/POPG membranes.     

Atomistic-level study of the interactions between hIAPP protofibrils and membranes: Influence of pH and lipid composition

The pathology of type 2 diabetes mellitus is associated with the aggregation of human islet amyloid polypeptide (hIAPP) and aggregation-mediated membrane disruption. The interactions of hIAPP aggregates with lipid membrane, as well as the effects of pH and lipid composition at the atomic level, remain elusive. Herein, using molecular dynamics simulations, we investigate the interactions of hIAPP protofibrillar oligomers with lipids, and the membrane perturbation that they induce, when they are partially inserted in an anionic dipalmitoyl-phosphatidylglycerol (DPPG) membrane or a mixed dipalmitoyl-phosphatidylcholine (DPPC)/DPPG (7:3) lipid bilayer under acidic/neutral pH conditions. We observed that the tilt angles and insertion depths of the hIAPP protofibril are strongly correlated with the pH and lipid composition. At neutral pH, the tilt angle and insertion depth of hIAPP protofibrils at a DPPG bilayer reach ~52° and ~1.62 nm with respect to the membrane surface, while they become ~77° and ~1.75 nm at a mixed DPPC/DPPG membrane. The calculated tilt angle of hIAPP at DPPG membrane is consistent with a recent chiral sum frequency generation spectroscopic study. The acidic pH induces a smaller tilt angle of ~40° and a shallower insertion depth (~1.24 nm) of hIAPP at the DPPG membrane surface, mainly due to protonation of His18 near the turn region. These differences mainly result from a combination of distinct electrostatic, van der Waals, hydrogen bonding and salt-bridge interactions between hIAPP and lipid bilayers. The hIAPP-membrane interaction energy analysis reveals that besides charged residues K1, R11 and H18, aromatic residues Phe15 and Phe23 also exhibit strong interactions with lipid bilayers, revealing the crucial role of aromatic residues in stabilizing the membrane-bound hIAPP protofibrils. hIAPP-membrane interactions disturb the lipid ordering and the local bilayer thickness around the peptides. Our results provide atomic-level information of membrane interaction of hIAPP protofibrils, revealing pH-dependent and membrane-modulated hIAPP aggregation at the early stage.     

Interactions of the Auxilin-1 PTEN-like Domain with Model Membranes Result in Nanoclustering of Phosphatidyl Inositol Phosphates

Auxilin-1 is a neuron-specific membrane-binding protein involved in a late stage of clathrin-mediated endocytosis. It recruits Hsc70, thus initiating uncoating of the clathrin-coated vesicles. Interactions of auxilin-1 with the vesicle membrane are crucial for this function and are mediated via an N-terminal PTEN-like domain. We have used multiscale molecular dynamics simulations to probe the interactions of the auxilin-1 PTEN-like domain with lipid bilayers containing differing phospholipid composition, including bilayers containing phosphatidyl inositol phosphates. Our results suggest a novel, to our knowledge, model for the auxilin/membrane encounter and subsequent interactions. Negatively charged lipids (especially PIP₂) enhance binding of auxilin to lipid bilayers and facilitate its correct orientation relative to the membrane. Mutations in three basic residues (R301E/R307E/K311E) of the C2 subdomain of the PTEN-like domain perturbed its interaction with the bilayer, changing its orientation. The interaction of membrane-bound auxilin-1 PTEN-like domain with negatively charged lipid headgroups results in nanoclustering of PIP₂ molecules in the adjacent bilayer leaflet.     

Interaction of protegrin-1 with lipid bilayers: membrane thinning effect

Protegrins (PG) are important in defending host tissues, preventing infection via an attack on the membrane surface of invading microorganisms. Protegrins have powerful antibiotic abilities, but the molecular-level mechanisms underlying the interactions of their beta-sheet motifs with the membrane are not known. Protegrin-1 (PG-1) is composed of 18 amino acids with a high content of basic residues and two disulfide bonds. Here we focused on the stability of PG-1 at the amphipathic interface in lipid bilayers and on the details of the peptide-membrane interactions. We simulated all-atom models of the PG-1 monomer with explicit water and lipid bilayers composed of both homogeneous POPC (palmitoyl-oleyl-phosphatidylcholine) lipids and a mixture of POPC/POPG (palmitoyl-oleyl-phosphatidylglycerol) (4:1) lipids. We observed that local thinning of the lipid bilayers mediated by the peptide is enhanced in the lipid bilayer containing POPG, consistent with experimental results of selective membrane targeting. The beta-hairpin motif of PG-1 is conserved in both lipid settings, whereas it is highly bent in aqueous solution. The conformational dynamics of PG-1, especially the highly charged beta-hairpin turn region, are found to be mostly responsible for disturbing the membrane. Even though the eventual membrane disruption requires PG-1 oligomers, our simulations clearly show the first step of the monomeric effects. The thinning effects in the bilayer should relate to pore/channel formation in the lipid bilayer and thus be responsible for further defects in the membrane caused by oligomer.     

Propagating Subterahertz Longitudinal Phonon-like Modes in a Lipid Bilayer in Aqueous Medium

It has been revealed recently that the subterahertz/terahertz vibrational motions in enzymes and DNA immersed in aqueous solutions can be underdamped. Importantly, these motions are associated with coherent delocalized modes that control functional processes. Analogous propagating phonon-like modes have been found in free hydrated lipid bilayers. In the present work, subterahertz (frequencies of the order of tens and hundreds of gigahertz) longitudinal acoustic oscillations in a bilayer lipid membrane immersed in aqueous medium are investigated theoretically. We consider driven oscillations excited by tangential mechanical tensions at the bilayer surfaces and thermally induced phonon modes. The analysis is based on: (i) a generalized hydrodynamic model of two-dimensional lipid bilayer in aqueous medium; (ii) known estimates of frequencies and lifetimes of longitudinal acoustic phonons in free hydrated lipid bilayer and in water, which were obtained in the experiments on non-elastic X-ray scattering and the molecular dynamics simulations. We show that the membrane phonon-like excitations are underdamped for the typical values of the system parameters, and the contribution of aqueous medium to the membrane mode damping is small compared to the contribution of the lipid bilayer. The obtained results suggest the possibility of realization of thermally induced longitudinal membrane phonons in physiological conditions, as well as the possibility of resonance amplification of the impact of subnanosecond electric impulses and impulses of subterahertz electromagnetic radiation on membrane dynamics.     

E.?coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

Interaction of the Mechanosensitive Channel,MscS, with the Membrane Bilayer through Lipid Intercalation into Grooves and Pockets

All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein–lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even “conservative” hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4.     

E. coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

Molecular dynamics simulations of pentapeptides at interfaces: salt bridge and cation-pi interactions

Peptide-membrane interactions are important for understanding the binding, partitioning, and folding of membrane proteins; the activity of antimicrobial and fusion peptides; and a number of other processes. We describe molecular dynamics simulations (10-25 ns) of two pentapeptides Ace-WLXLL (with X = Arg or Lys side chain) (White, S. H., and Wimley, W.C. (1996) Nat. Struct. Biol. 3, 842-848) in water and three different membrane mimetic systems: (i) a water/cyclohexane interface, (ii) water-saturated octanol, and (iii) a solvated dioleoylphosphatidylcholine bilayer. A salt bridge is found between the protonated Arg or Lys side chains with the carboxyl terminus at the three interfaces. In water/cyclohexane, the salt bridge is most exposed to the water phase and least stable. In water/octanol and the lipid bilayer systems, the salt bridge once formed persists throughout the simulations. In the lipid bilayer, the salt bridge is more stable when the peptide penetrates deeper into the bilayer. In one of two peptides, a cation-pi interaction between the Arg and the Trp side chains is stable in the lipid bilayer for about 15 ns before breaking. In all cases, the conformations of the peptides are restricted by their presence at the interface and can be assigned to a few major conformational clusters. Side chains facing the water phase are most mobile. In the lipid bilayer, the peptides remain in the interface area, where they overlap with the carbonyl area of the lipid bilayer and perturb the local density profile of the bilayer. The tryptophan side chain remains in the water-lipid interface, where it interacts with the lipid choline group and forms hydrogen bonds with the ester carbonyl of the lipid and with water in the interface.     

Coarse-grained molecular dynamics simulations of membrane proteins and peptides

Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.     

Setting up and running molecular dynamics simulations of membrane proteins

Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the protein(s) on a widely spaced grid and then 'shrinking' the grid until the bilayer with the protein has the desired density, with lipids neatly packed around the protein. When starting from a grid based on a single lipid structure, or several potentially different lipid structures (method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD simulations of membrane proteins.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.