Reversible Effects of Peptide Concentration and Lipid Composition on H-Ras Lipid Anchor Clustering

Dynamic clusters of lipid-anchored Ras proteins are important for high-fidelity signal transduction in cells. The average size of Ras nanoclusters was reported to be independent of protein expression levels, and cholesterol depletion is commonly used to test the raft-preference of nanoclusters. However, whether protein concentration and membrane domain stability affect Ras clustering in a reversible manner is not well understood. We used coarse-grained molecular dynamics simulations to examine the reversibility of the effects of peptide and cholesterol concentrations as well as a lipid domain-perturbing nanoparticle (C₆₀) on the dynamics and stability of H-Ras lipid-anchor nanoclusters. By comparing results from these simulations with previous observations from the literature, we show that effects of peptide/cholesterol concentrations on the dynamics and stability of H-Ras peptide nanoclusters are reversible. Our results also suggest a correlation between the stabilities of lipid domains and Ras nanoclusters, which is supported by our finding that C₆₀ penetrates into the liquid-disordered domain of the bilayer, destabilizing lipid domains and thereby the stability of the nanoclusters.     

Short-range order and collective dynamics of DMPC bilayers: a comparison between molecular dynamics simulations, X-ray, and neutron scattering experiments

We present an extensive comparison of short-range order and short wavelength dynamics of a hydrated phospholipid bilayer derived by molecular dynamics simulations, elastic x-ray, and inelastic neutron scattering experiments. The quantities that are compared between simulation and experiment include static and dynamic structure factors, reciprocal space mappings, and electron density profiles. We show that the simultaneous use of molecular dynamics and diffraction data can help to extract real space properties like the area per lipid and the lipid chain ordering from experimental data. In addition, we assert that the interchain distance can be computed to high accuracy from the interchain correlation peak of the structure factor. Moreover, it is found that the position of the interchain correlation peak is not affected by the area per lipid, while its correlation length decreases linearly with the area per lipid. This finding allows us to relate a property of the structure factor quantitatively to the area per lipid. Finally, the short wavelength dynamics obtained from the simulations and from inelastic neutron scattering are analyzed and compared. The conventional interpretation in terms of the three-effective-eigenmode model is found to be only partly suitable to describe the complex fluid dynamics of lipid chains.     

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.     

Structure and Dynamics of the Central Lipid Pool and Proteins of the Bacterial Holo-Translocon

The bacterial Sec translocon, SecYEG, associates with accessory proteins YidC and the SecDF-YajC subcomplex to form the bacterial holo-translocon (HTL). The HTL is a dynamic and flexible protein transport machine capable of coordinating protein secretion across the membrane and efficient lateral insertion of nascent membrane proteins. It has been hypothesized that a central lipid core facilitates the controlled passage of membrane proteins into the bilayer, ensuring the efficient formation of their native state. By performing small-angle neutron scattering on protein solubilized in “match-out” deuterated detergent, we have been able to interrogate a “naked” HTL complex, with the scattering contribution of the surrounding detergent micelle rendered invisible. Such an approach has allowed the confirmation of a lipid core within the HTL, which accommodates between 8 and 29 lipids. Coarse-grained molecular dynamics simulations of the HTL also demonstrate a dynamic, central pool of lipids. An opening at this lipid-rich region between YidC and the SecY lateral gate may provide an exit gateway for newly synthesized, correctly oriented, membrane protein helices, or even small bundles of helices, to emerge from the HTL.     

Molecular dynamics simulations of discoidal bilayers assembled from truncated human lipoproteins

Human apolipoprotein A-1 (apo A-1) is the major protein component of high-density lipoproteins. The apo A-1 lipid-binding domain was used as a template for the synthesis of amphipathic helical proteins termed membrane scaffold proteins, employed to self-assemble soluble monodisperse discoidal particles called Nanodiscs. In these particles, membrane scaffold proteins surround a lipid bilayer in a belt-like fashion forming bilayer disks of discrete size and composition. Here we investigate the structure of Nanodiscs through molecular dynamics simulations in which Nanodiscs were built from scaffold proteins of various lengths. The simulations showed planar or deformed Nanodiscs depending on optimal length and alignment of the scaffold proteins. Based on mean surface area per lipid calculations, comparison of small-angle x-ray scattering curves, and the relatively planar shape of Nanodiscs made from truncated scaffold proteins, one can conclude that the first 17 to 18 residues of the 200-residue apo A-1 lipid-binding domain are not involved in formation of the protein "belts" surrounding the lipid bilayer. To determine whether the addition of an integral membrane protein has an effect on the overall structure of a Nanodisc, bacteriorhodopsin was embedded into a Nanodisc and simulated using molecular dynamics, revealing a planar disk with a slightly rectangular shape.     

Determining Peptide Partitioning Properties via Computer Simulation

The transfer of polypeptide segments into lipid bilayers to form transmembrane helices represents the crucial first step in cellular membrane protein folding and assembly. This process is driven by complex and poorly understood atomic interactions of peptides with the lipid bilayer environment. The lack of suitable experimental techniques that can resolve these processes both at atomic resolution and nanosecond timescales has spurred the development of computational techniques. In this review, we summarize the significant progress achieved in the last few years in elucidating the partitioning of peptides into lipid bilayer membranes using atomic detail molecular dynamics simulations. Indeed, partitioning simulations can now provide a wealth of structural and dynamic information. Furthermore, we show that peptide-induced bilayer distortions, insertion pathways, transfer free energies, and kinetic insertion barriers are now accurate enough to complement experiments. Further advances in simulation methods and force field parameter accuracy promise to turn molecular dynamics simulations into a powerful tool for investigating a wide range of membrane active peptide phenomena.     

Membrane-bound ARF1 peptide: interpretation of neutron diffraction data by molecular dynamics simulation methods

Adenosine diphosphate ribosylation factor-1 (ARF1) is activated by cell membrane binding of a self-folding N-terminal domain. We have previously presented four possible conformations of the membrane bound, human ARF1 N-terminal peptide in planar lipid bilayers of DOPC and DOPG (7:3 molar ratio), determined from lamellar neutron diffraction and circular dichroism data. In this paper we analyse the four possible conformations by molecular dynamics simulations. The aim of these simulations was to use MD to distinguish which of the four possible membrane bound structures was the most likely. The most likely conformation was determined according to the following criteria: (a) location of label positions on the peptide in relation to the bilayer, (b) lowest mean square displacement from the initial structure, (c) lowest system energy, (d) most peptide-lipid headgroup hydrogen bonding, (e) analysis of phi/psi angles of the peptide. These findings demonstrate the application of molecular dynamics simulations to explore neutron diffraction data.     

Membrane-bound ARF1 peptide: interpretation of neutron diffraction data by molecular dynamics simulation methods

Adenosine diphosphate ribosylation factor-1 (ARF1) is activated by cell membrane binding of a self-folding N-terminal domain. We have previously presented four possible conformations of the membrane bound, human ARF1 N-terminal peptide in planar lipid bilayers of DOPC and DOPG (7:3 molar ratio), determined from lamellar neutron diffraction and circular dichroism data. In this paper we analyse the four possible conformations by molecular dynamics simulations. The aim of these simulations was to use MD to distinguish which of the four possible membrane bound structures was the most likely. The most likely conformation was determined according to the following criteria: (a) location of label positions on the peptide in relation to the bilayer, (b) lowest mean square displacement from the initial structure, (c) lowest system energy, (d) most peptide-lipid headgroup hydrogen bonding, (e) analysis of phi/psi angles of the peptide. These findings demonstrate the application of molecular dynamics simulations to explore neutron diffraction data.     

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.     

Aggregation of Lipid-Anchored Full-Length H-Ras in Lipid Bilayers: Simulations with the MARTINI Force Field

Lipid-anchored Ras oncoproteins assemble into transient, nano-sized substructures on the plasma membrane. These substructures, called nanoclusters, were proposed to be crucial for high-fidelity signal transmission in cells. However, the molecular basis of Ras nanoclustering is poorly understood. In this work, we used coarse-grained (CG) molecular dynamics simulations to investigate the molecular mechanism by which full-length H-ras proteins form nanoclusters in a model membrane. We chose two different conformations of H-ras that were proposed to represent the active and inactive state of the protein, and a domain-forming model bilayer made up of di16:0-PC (DPPC), di18:2-PC (DLiPC) and cholesterol. We found that, irrespective of the initial conformation, Ras molecules assembled into a single large aggregate. However, the two binding modes, which are characterized by the different orientation of the G-domain with respect to the membrane, differ in dynamics and organization during and after aggregation. Some of these differences involve regions of Ras that are important for effector/modulator binding, which may partly explain observed differences in the ability of active and inactive H-ras nanoclusters to recruit effectors. The simulations also revealed some limitations in the CG force field to study protein assembly in solution, which we discuss in the context of proposed potential avenues of improvement.     

The determinants of hydrophobic mismatch response for transmembrane helices

Hydrophobic mismatch arises from a difference in the hydrophobic thickness of a lipid membrane and a transmembrane protein segment, and is thought to play an important role in the folding, stability and function of membrane proteins. We have investigated the possible adaptations that lipid bilayers and transmembrane α-helices undergo in response to mismatch, using fully-atomistic molecular dynamics simulations totaling 1.4 μs. We have created 25 different tryptophan-alanine-leucine transmembrane α-helical peptide systems, each composed of a hydrophobic alanine–leucine stretch, flanked by 1–4 tryptophan side chains, as well as the β-helical peptide dimer, gramicidin A. Membrane responses to mismatch include changes in local bilayer thickness and lipid order, varying systematically with peptide length. Adding more flanking tryptophan side chains led to an increase in bilayer thinning for negatively mismatched peptides, though it was also associated with a spreading of the bilayer interface. Peptide tilting, bending and stretching were systematic, with tilting dominating the responses, with values of up to ~ 45° for the most positively mismatched peptides. Peptide responses were modulated by the number of tryptophan side chains due to their anchoring roles and distributions around the helices. Potential of mean force calculations for local membrane thickness changes, helix tilting, bending and stretching revealed that membrane deformation is the least energetically costly of all mismatch responses, except for positively mismatched peptides where helix tilting also contributes substantially. This comparison of energetic driving forces of mismatch responses allows for deeper insight into protein stability and conformational changes in lipid membranes.     

Lateral dynamics of proteins with polybasic domain on anionic membranes: a dynamic Monte-Carlo study

Positively charged polybasic domains are essential for recruiting multiple signaling proteins, such as Ras GTPases and Src kinase, to the negatively charged cellular membranes. Much less, however, is known about the influence of electrostatic interactions on the lateral dynamics of these proteins. We developed a dynamic Monte-Carlo automaton that faithfully simulates lateral diffusion of the adsorbed positively charged oligopeptides as well as the dynamics of mono- (phosphatidylserine) and polyvalent (PIP₂) anionic lipids within the bilayer. In agreement with earlier results, our simulations reveal lipid demixing that leads to the formation of a lipid shell associated with the peptide. The computed association times and average numbers of bound lipids demonstrate that tetravalent PIP₂ interacts with the peptide much more strongly than monovalent lipid. On the spatially homogeneous membrane, the lipid shell affects the behavior of the peptide only by weakly reducing its lateral mobility. However, spatially heterogeneous distributions of monovalent lipids are found to produce peptide drift, the velocity of which is determined by the total charge of the peptide-lipid complex. We hypothesize that this predicted phenomenon may affect the spatial distribution of proteins with polybasic domains in the context of cell-signaling events that alter the local density of monovalent anionic lipids.     

Nanoscale structural and mechanical effects of beta-amyloid (1-42) on polymer cushioned membranes: a combined study by neutron reflectometry and AFM Force Spectroscopy

The interaction of beta-amyloid peptides with lipid membranes is widely studied as trigger agents in Alzheimer's disease. Their mechanism of action at the molecular level is unknown and their interaction with the neural membrane is crucial to elucidate the onset of the disease. In this study we have investigated the interaction of water soluble forms of beta-amyloid Aβ(1-42) with lipid bilayers supported by polymer cushion. A reproducible protocol for the preparation of a supported phospholipid membrane with composition mimicking the neural membrane and in physiological condition (PBS buffer, pH=7.4) was refined by neutron reflectivity. The change in structure and local mechanical properties of the membrane in the presence of Aβ(1-42) was investigated by neutron reflectivity and Atomic Force Microscopy (AFM) Force Spectroscopy. Neutron reflectivity evidenced that Aβ(1-42) interacts strongly with the supported membrane, causing a change in the scattering length density profile of the lipid bilayer, and penetrates into the membrane. Concomitantly, the local mechanical properties of the bilayer are deeply modified by the interaction with the peptide as seen by AFM Force Spectroscopy. These results may be of great importance for the onset of the Alzheimer's disease, since a simultaneous change in the structural and mechanical properties of the lipid matrix could influence all membrane based signal cascades.     

Interactions of the Anticancer Drug Tamoxifen with Lipid Membranes

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer area compressibility modulus K_A is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.     

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

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

The helix-to-sheet transition of an HIV-1 fusion peptide derivative changes the mechanical properties of lipid bilayer membranes

A short sequence on the gp41 envelope protein of HIV-1 is integral to infection by the virus. Without this sequence, termed the fusion peptide (FP), the virus is far less effective at fusing with the cellular membrane. One of the interesting features of the isolated FP is that it transitions between an α-helical conformation and a β-sheet conformation in lipid bilayer membranes as a function of lipid composition and concentration, and the transition correlates with fusion. To better understand how the conformations of the FP impact lipid bilayer membranes, a variant of the FP that does not strongly promote fusion, termed gp41rk, was studied. Circular dichroism spectroscopy, dynamic light scattering, small-angle neutron scattering (SANS) and neutron spin echo spectroscopy (NSE) were used to relate the conformation of gp41rk to the structure and mechanical properties of lipid bilayer membrane vesicles composed of a 7:3 molar ratio mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol). At a peptide-to-lipid ratio (P/L) of 1/200, it adopts an α-helical conformation, while gp41rk is a β-sheet at a P/L of 1/50 in the unilamellar vesicles. SANS reveals that the lipid bilayer membrane becomes thicker when gp41rk adopts a β-sheet conformation, which indicates that the high-concentration state of the peptide increases the order of the lipid acyl chains. At the same time, NSE demonstrates that the bilayer becomes more rigid, demonstrating that the β-sheet conformation, which correlates with fusion for the native FP sequence, stiffens the bilayer. The results have implications for the function of the FP.     

Transmembrane helix-helix interactions: comparative simulations of the glycophorin a dimer

The glycophorin helix dimer is a paradigm for the exploration of helix-helix interactions in integral membrane proteins. Two NMR structures of the dimer are known, one in a detergent micelle and one in a lipid bilayer. Multiple (4 x 50 ns) molecular dynamics simulations starting from each of the two NMR structures, with each structure in either a dodecyl phosphocholine (DPC) micelle or a dimyristoyl phosphatidylcholine (DMPC) bilayer, have been used to explore the conformational dynamics of the helix dimer. Analysis of the helix-helix interaction, mediated by the GxxxG sequence motif, suggests convergence of the simulations to a common model. This is closer to the NMR structure determined in a bilayer than to micelle structure. The stable dimer interface in the final simulation model is characterized by (i) Gly/Gly packing and (ii) Thr/Thr interhelix H-bonds. These results demonstrate the ability of extended molecular dynamics simulations in a lipid bilayer environment to refine membrane protein structures or models derived from experimental data obtained in protein/detergent micelles.     

Membrane simulations of OpcA: gating in the loops?

Mobility of extracellular loops may play an important role in the function of outer membrane proteins from Gram-negative bacteria. Molecular dynamics simulations of OpcA from Neisseria meningitidis, embedded in a lipid bilayer, have been used to explore the relationship between the crystal structure and dynamic function of this protein. The results suggest that the crystal environment may constrain the membrane protein structure in a nonphysiological state. The presence of lipids and physiological salt concentrations result in changes in the conformation of the extracellular loops of OpcA, leading to opening of a pore, and to modulation of the molecular surface implicated in recognition of proteoglycan. These changes may be related to the role of OpcA in pathogenesis via modulation of the conformation of a possible sialic acid binding site.     

Coarse-grained MD simulations of membrane protein-bilayer self-assembly

Complete determination of a membrane protein structure requires knowledge of the protein position within the lipid bilayer. As the number of determined structures of membrane proteins increases so does the need for computational methods which predict their position in the lipid bilayer. Here we present a coarse-grained molecular dynamics approach to lipid bilayer self-assembly around membrane proteins. We demonstrate that this method can be used to predict accurately the protein position in the bilayer for membrane proteins with a range of different sizes and architectures.     

Solid-state NMR spectroscopy to study protein–lipid interactions

The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid–protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly contributed to the understanding of lipid–protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid–protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K⁺ channel KcsA are discussed. This article is part of a Special Issue entitled Tools to study lipid functions.