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.     

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.     

Vstx1, a modifier of Kv channel gating, localizes to the interfacial region of lipid bilayers

VSTx1 is a tarantula venom toxin which binds to the archaebacterial voltage-gated potassium channel KvAP. VSTx1 is thought to access the voltage sensor domain of the channel via the lipid bilayer phase. In order to understand its mode of action and implications for the mechanism of channel activation, it is important to characterize the interactions of VSTx1 with lipid bilayers. Molecular dynamics (MD) simulations (for a total simulation time in excess of 0.2 micros) have been used to explore VSTx1 localization and interactions with zwitterionic (POPC) and with anionic (POPE/POPG) lipid bilayers. In particular, three series of MD simulations have been used to explore the net drift of VSTx1 relative to the center of a bilayer, starting from different locations of the toxin. The preferred location of the toxin is at the membrane/water interface. Although there are differences between POPC and POPE/POPG bilayers, in both cases the toxin forms favorable interactions at the interface, maximizing H-bonding to lipid headgroups and to water molecules while retaining interactions with the hydrophobic core of the bilayer. A 30 ns unrestrained simulation reveals dynamic partitioning of VSTx1 into the interface of a POPC bilayer. The preferential location of VSTx1 at the interface is discussed in the context of Kv channel gating models and provides support for a mode of action in which the toxin interacts with the Kv voltage sensor "paddle" formed by the S3 and S4 helices.     

Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation

Liposomal formulation of curcumin is an important therapeutic agent for the treatment of various cancers. Despite extensive studies on the biological effects of this formulation in cancer treatment, much remains unknown about curcumin–liposome interactions. Understanding how different lipid bilayers respond to curcumin molecule may help us to design more effective liposomal curcumin. Here, we used molecular dynamics simulation method to investigate the behavior of curcumin in two lipid bilayers commonly used in preparation of liposomal curcumin, namely dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG). First, the free energy barriers for translocation of one curcumin molecule from water to the lipid bilayer were determined by using the potential of mean force (PMF). The computed free energy profile exhibits a global minimum at the solvent–headgroup interface (LH region) for both lipid membranes. We also evaluated the free energy difference between the equilibrium position of curcumin in the lipid bilayer and bulk water as the excess chemical potential. Our results show that curcumin has the higher affinity in DMPG compared to DPPC lipid bilayer (?8.39 vs. ?1.69 k_BT) and this is related to more hydrogen bond possibility for curcumin in DMPG lipid membrane. Next, using an unconstrained molecular dynamic simulation with curcumin initially positioned at the center of lipid bilayer, we studied various properties of each lipid bilayer system in the presence of curcumin molecule that was in full agreement with PMF and experimental data. The results of these simulation studies suggest that membrane composition could have a large effect on interaction of curcumin–lipid bilayer.     

How Does a Voltage Sensor Interact with a Lipid Bilayer? Simulations of a Potassium Channel Domain

The nature of voltage sensing by voltage-activated ion channels is a key problem in membrane protein structural biology. The way in which the voltage-sensor (VS) domain interacts with its membrane environment remains unclear. In particular, the known structures of Kv channels do not readily explain how a positively charged S4 helix is able to stably span a lipid bilayer. Extended (2 x 50 ns) molecular dynamics simulations of the high-resolution structure of the isolated VS domain from the archaebacterial potassium channel KvAP, embedded in zwitterionic and in anionic lipid bilayers, have been used to explore VS/lipid interactions at atomic resolution. The simulations reveal penetration of water into the center of the VS and bilayer. Furthermore, there is significant local deformation of the lipid bilayer by interactions between lipid phosphate groups and arginine side chains of S4. As a consequence of this, the electrostatic field is "focused" across the center of the bilayer.     

Molecular dynamics simulation studies of lipid bilayer systems

The main structural element of biological membranes is a liquid-crystalline lipid bilayer. Other constituents, i.e. proteins, sterols and peptides, either intercalate into or loosely attach to the bilayer. We applied a molecular dynamics simulation method to study membrane systems at various levels of compositional complexity. The studies were started from simple lipid bilayers containing a single type phosphatidylcholine (PC) and water molecules (PC bilayers). As a next step, cholesterol (Chol) molecules were introduced to the PC bilayers (PC-Chol bilayers). These studies provided detailed information about the structure and dynamics of the membrane/water interface and the hydrocarbon chain region in bilayers built of various types of PCs and Chol. This enabled studies of membrane systems of higher complexity. They included the investigation of an integral membrane protein in its natural environment of a PC bilayer, and the antibacterial activity of magainin-2. The latter study required the construction of a model bacterial membrane which consisted of two types of phospholipids and counter ions. Whenever published experimental data were available, the results of the simulations were compared with them.     

Energetics of inclusion-induced bilayer deformations.

The material properties of lipid bilayers can affect membrane protein function whenever conformational changes in the membrane-spanning proteins perturb the structure of the surrounding bilayer. This coupling between the protein and the bilayer arises from hydrophobic interactions between the protein and the bilayer. We analyze the free energy cost associated with a hydrophobic mismatch, i.e., a difference between the length of the protein's hydrophobic exterior surface and the average thickness of the bilayer's hydrophobic core, using a (liquid-crystal) elastic model of bilayer deformations. The free energy of the deformation is described as the sum of three contributions: compression-expansion, splay-distortion, and surface tension. When evaluating the interdependence among the energy components, one modulus renormalizes the other: e.g., a change in the compression-expansion modulus affects not only the compression-expansion energy but also the splay-distortion energy. The surface tension contribution always is negligible in thin solvent-free bilayers. When evaluating the energy per unit distance (away from the inclusion), the splay-distortion component dominates close to the bilayer/inclusion boundary, whereas the compression-expansion component is more prominent further away from the boundary. Despite this complexity, the bilayer deformation energy in many cases can be described by a linear spring formalism. The results show that, for a protein embedded in a membrane with an initial hydrophobic mismatch of only 1 A, an increase in hydrophobic mismatch to 1.3 A can increase the Boltzmann factor (the equilibrium distribution for protein conformation) 10-fold due to the elastic properties of the bilayer.     

The influence of 1-alkanols and external pressure on the lateral pressure profiles of lipid bilayers

The suggestion by Robert Cantor, that drug-induced pressure changes in lipid bilayers can change the conformational equilibrium between open and closed states of membrane proteins and thereby cause anesthesia, attracted much attention lately. Here, we studied the effect of both large external pressure and of 1-alkanols of different chain lengths—some of them anesthetics, others not—on the lateral pressure profiles across dimyristoylphosphatidylcholine (DMPC) bilayers by molecular dynamics simulations. For a pure DMPC bilayer, high pressure both reduced and broadened the tension at the interface hydrophobic/hydrophilic and diminished the repulsion between the phospholipid headgroups. Whereas the effect of ethanol on the lateral pressure profile was similar to the effect of a large external pressure on a DMPC bilayer, long-chain 1-alkanols significantly amplified local maxima and minima in the lateral pressure profile. For most 1-alkanols, external pressure had moderate effects and did not reverse the changes 1-alkanols exerted on the pressure profile. Nevertheless, assuming the bent helix model as a simple geometric model for the transmembrane region of a membrane protein, protein conformational equilibria were shifted in opposite directions by addition of 1-alkanols and additional application of external pressure.     

Protein shape change has a major effect on the gating energy of a mechanosensitive channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k_BT to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k_BT on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.     

Coarse-grained molecular dynamics simulations of the energetics of helix insertion into a lipid bilayer

Experimental and computational studies have indicated that hydrophobicity plays a key role in driving the insertion of transmembrane alpha-helices into lipid bilayers. Molecular dynamics simulations allow exploration of the nature of the interactions of transmembrane alpha-helices with their lipid bilayer environment. In particular, coarse-grained simulations have considerable potential for studying many aspects of membrane proteins, ranging from their self-assembly to the relation between their structure and function. However, there is a need to evaluate the accuracy of coarse-grained estimates of the energetics of transmembrane helix insertion. Here, three levels of complexity of model system have been explored to enable such an evaluation. First, calculated free energies of partitioning of amino acid side chains between water and alkane yielded an excellent correlation with experiment. Second, free energy profiles for transfer of amino acid side chains along the normal to a phosphatidylcholine bilayer were in good agreement with experimental and atomistic simulation studies. Third, estimation of the free energy profile for transfer of an arginine residue, embedded within a hydrophobic alpha-helix, to the center of a lipid bilayer gave a barrier of approximately 15 kT. Hence, there is a substantial barrier to membrane insertion for charged amino acids, but the coarse-grained model still underestimates the corresponding free energy estimate (approximately 29 kT) from atomistic simulations (Dorairaj, S., and Allen, T. W. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 4943-4948). Coarse-grained simulations were then used to predict the free energy profile for transfer of a simple model transmembrane alpha-helix (WALP23) across a lipid bilayer. The results indicated that a transmembrane orientation was favored by about -70 kT.     

Life at the border: Adaptation of proteins to anisotropic membrane environment

This review discusses main features of transmembrane (TM) proteins which distinguish them from water‐soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co‐translational and post‐translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large‐scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen‐bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side‐chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding.     

Exploring peptide-membrane interactions with coarse-grained MD simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers.     

An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation

Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins.     

含有长亲水链团的膜糖蛋白与脂类双分子层的相互作用—脂类-蛋白相互作用的一种模型

基于作者已经发表的实验结果,本文认为脂类—蛋白相互作用自由能可来自脂双层内疏水区的相互作用,也可来自膜表面的吸附作用.本文对含有长亲水链团的膜糖蛋白(如血型糖蛋白和胰岛素受体)与脂双层的相互作用的热力学估计(正则溶液近似)表明,脂类—蛋白相互作用自由能可能主要来自胰蛋白的亲水链团在膜表而吸附的贡献.本文理论分析的结论与已经发表的实验结果相一致.     

Insertion and pore formation driven by adsorption of proteins onto lipid bilayer membrane-water interfaces

We describe the binding of proteins to lipid bilayers in the case for which binding can occur either by adsorption to the lipid bilayer membrane-water interface or by direct insertion into the bilayer itself. We examine in particular the case when the insertion and pore formation are driven by the adsorption process using scaled particle theory. The adsorbed proteins form a two-dimensional "surface gas" at the lipid bilayer membrane-water interface that exerts a lateral pressure on the lipid bilayer membrane. Under conditions of strong intrinsic binding and a high degree of interfacial converge, this pressure can become high enough to overcome the energy barrier for protein insertion. Under these conditions, a subtle equilibrium exists between the adsorbed and inserted proteins. We propose that this provides a control mechanism for reversible insertion and pore formation of proteins such as melittin and magainin. Next, we discuss experimental data for the binding isotherms of cytochrome c to charged lipid membranes in the light of our theory and predict that cytochrome c inserts into charged lipid bilayers at low ionic strength. This prediction is supported by titration calorimetry results that are reported here. We were furthermore able to describe the observed binding isotherms of the pore-forming peptides endotoxin (alpha 5-helix) and of pardaxin to zwitterionic vesicles from our theory by assuming adsorption/insertion equilibrium.     

Atomic-scale structure and electrostatics of anionic palmitoyloleoylphosphatidylglycerol lipid bilayers with Na+ counterions

Anionic palmitoyloleoylphosphatidylglycerol (POPG) is one of the most abundant lipids in nature, yet its atomic-scale properties have not received significant attention. Here we report extensive 150-ns molecular dynamics simulations of a pure POPG lipid membrane with sodium counterions. It turns out that the average area per lipid of the POPG bilayer under physiological conditions is approximately 19% smaller than that of a bilayer built from its zwitterionic phosphatidylcholine analog, palmitoyloleoylphosphatidylcholine. This suggests that there are strong attractive interactions between anionic POPG lipids, which overcome the electrostatic repulsion between negative charges of PG headgroups. We demonstrate that interlipid counterion bridges and strong intra- and intermolecular hydrogen bonding play a key role in this seemingly counterintuitive behavior. In particular, the substantial strength and stability of ion-mediated binding between anionic lipid headgroups leads to complexation of PG molecules and ions and formation of large PG-ion clusters that act in a concerted manner. The ion-mediated binding seems to provide a possible molecular-level explanation for the low permeability of PG-containing bacterial membranes to organic solvents: highly polar interactions at the water/membrane interface are able to create a high free energy barrier for hydrophobic molecules such as benzene.     

A method to determine dielectric constants in nonhomogeneous systems: application to biological membranes

Continuum electrostatic models have had quantitative success in describing electrostatic-mediated phenomena on atomistic scales; however, there continues to be significant disagreement about how to assign dielectric constants in mixed, nonhomogeneous systems. We introduce a method for determining a position-dependent dielectric profile from molecular dynamics simulations. In this method, the free energy of introducing a test charge is computed two ways: from a free energy perturbation calculation and from a numerical solution to Poisson's Equation. The dielectric profile of the system is then determined by minimizing the discrepancy between these two calculations simultaneously for multiple positions of the test charge. We apply this method to determine the dielectric profile of a lipid bilayer surrounded by water. We find good agreement with dielectric models for lipid bilayers obtained by other approaches. The free energy of transferring an ion from bulk water to the lipid bilayer computed from the atomistic simulations indicates that large errors are introduced when the bilayer is represented as a single slab of low dielectric embedded in the higher-dielectric solvent. Significant improvement results from introducing an additional layer of intermediate dielectric (∼3) on each side of the low dielectric core extending from ∼12 Å to 18 Å. A small dip in transfer free energy just outside the lipid headgroups indicates the presence of a very high dielectric. These results have implications for the design of implicit membrane models and our understanding of protein-membrane interactions.     

Structure refinement of membrane proteins via molecular dynamics simulations

A refinement protocol based on physics‐based techniques established for water soluble proteins is tested for membrane protein structures. Initial structures were generated by homology modeling and sampled via molecular dynamics simulations in explicit lipid bilayer and aqueous solvent systems. Snapshots from the simulations were selected based on scoring with either knowledge‐based or implicit membrane‐based scoring functions and averaged to obtain refined models. The protocol resulted in consistent and significant refinement of the membrane protein structures similar to the performance of refinement methods for soluble proteins. Refinement success was similar between sampling in the presence of lipid bilayers and aqueous solvent but the presence of lipid bilayers may benefit the improvement of lipid‐facing residues. Scoring with knowledge‐based functions (DFIRE and RWplus) was found to be as good as scoring using implicit membrane‐based scoring functions suggesting that differences in internal packing is more important than orientations relative to the membrane during the refinement of membrane protein homology models.     

Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths.