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BBA - Biomembranes (v.1818, #2)
Outer membrane phospholipase A in phospholipid bilayers: A model system for concerted computational and experimental investigations of amino acid side chain partitioning into lipid bilayers by Patrick J. Fleming; J. Alfredo Freites; C. Preston Moon; Douglas J. Tobias; Karen G. Fleming (pp. 126-134).
Understanding the forces that stabilize membrane proteins in their native states is one of the contemporary challenges of biophysics. To date, estimates of side chain partitioning free energies from water to the lipid environment show disparate values between experimental and computational measures. Resolving the disparities is particularly important for understanding the energetic contributions of polar and charged side chains to membrane protein function because of the roles these residue types play in many cellular functions. In general, computational free energy estimates of charged side chain partitioning into bilayers are much larger than experimental measurements. However, the lack of a protein-based experimental system that uses bilayers against which to vet these computational predictions has traditionally been a significant drawback. Moon & Fleming recently published a novel hydrophobicity scale that was derived experimentally by using a host–guest strategy to measure the side chain energetic perturbation due to mutation in the context of a native membrane protein inserted into a phospholipid bilayer. These values are still approximately an order of magnitude smaller than computational estimates derived from molecular dynamics calculations from several independent groups. Here we address this discrepancy by showing that the free energy differences between experiment and computation become much smaller if the appropriate comparisons are drawn, which suggests that the two fields may in fact be converging. In addition, we present an initial computational characterization of the Moon & Fleming experimental system used for the hydrophobicity scale: OmpLA in DLPC bilayers. The hydrophobicity scale used OmpLA position 210 as the guest site, and our preliminary results demonstrate that this position is buried in the center of the DLPC membrane, validating its usage in the experimental studies. We further showed that the introduction of charged Arg at position 210 is well tolerated in OmpLA and that the DLPC bilayers accommodate this perturbation by creating a water dimple that allows the Arg side chain to remain hydrated. Lipid head groups visit the dimple and can hydrogen bond with Arg, but these interactions are transient. Overall, our study demonstrates the unique advantages of this molecular system because it can be interrogated by both computational and experimental practitioners, and it sets the stage for free energy calculations in a system for which there is unambiguous experimental data. This article is part of a Special Issue entitled: Membrane protein structure and function.Display Omitted► We simulated OmpLA in DLPC to elucidate molecular events in response to charged residues in the bilayer. ► An Arg mutation on the lipid surface of OmpLA induces water dimples that maintain the hydration of the guanidinium group. ► A Leu mutation on the lipid surface of OmpLA is in a dehydrated environment. ► We validate OmpLA in DLPC membranes as a molecular system for experimental hydrophobicity scales.
Keywords: Abbreviations; DLPC; dilaurylphosphatidylcholine; MF; refers to the Moon & Fleming hydrophobicity scale [24]; OmpLA; outer membrane phospholipase A; PMF; potential of mean force; POPC; palmitoyl-oleoyl-phosphatidylcholine; WW; refers to the Wimley & White octanol scale [16,17]Membrane proteins; Protein stability; Thermodynamics; Computation; Solvation; Phospholipid bilayers
The role of membrane thickness in charged protein–lipid interactions by Libo B. Li; Igor Vorobyov; Toby W. Allen (pp. 135-145).
Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26kcal/mol for 18 carbons to 6kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pKa shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein–lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.► Simulations reveal ion-induced defect mechanism for charge movement in membranes. ► Translocation free energies are linearly dependent on bilayer thickness. ► The cost of burying arginine in a thin membrane is small, consistent with experiments. ► Pores form in thinner bilayers, solubility-diffusion predicted in thicker bilayers. ► Small pKa shifts observed, but with deprotonation predicted in thick bilayers.
Keywords: Protein–lipid interaction; Arginine; Ion-induced defect; Membrane thickness; Ion permeation
Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy by Martin Gustavsson; Nathaniel J. Traaseth; Gianluigi Veglia (pp. 146-153).
In this paper, we analyzed the ground and excited states of phospholamban (PLN), a membrane protein that regulates sarcoplasmic reticulum calcium ATPase (SERCA), in different membrane mimetic environments. Previously, we proposed that the conformational equilibria of PLN are central to SERCA regulation. Here, we show that these equilibria detected in micelles and bicelles are also present in native sarcoplasmic reticulum lipid membranes as probed by MAS solid-state NMR. Importantly, we found that the kinetics of conformational exchange and the extent of ground and excited states in detergent micelles and lipid bilayers are different, revealing a possible role of the membrane composition on the allosteric regulation of SERCA. Since the extent of excited states is directly correlated to SERCA inhibition, these findings open up the exciting possibility that calcium transport in the heart can be controlled by the lipid bilayer composition. This article is part of a Special Issue entitled: Membrane protein structure and function.Display Omitted► MAS NMR probes ground and excited states of phospholamban. ► The conformational equilibria are detected in native lipid bilayers. ► Populations and exchange rates of the conformational states depend on membrane composition.
Keywords: Abbreviations; PLN; Phospholamban; SERCA; sarcoplasmic reticulum Ca; 2+; -ATPase; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DHPC; 1,2-dihexanoyl-sn-glycero-3-phosphocholine; DPC; dodecylphosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phospho-(1′-rac-glycerol); MLVs; multi-lamellar lipid vesiclesMembrane protein; NMR; Phospholamban; Excited states; Lipid bilayers; Magic angle spinning
Förster resonance energy transfer as a probe of membrane protein folding by Guipeun Kang; Lopez-Pena Ignacio López-Peña; Vanessa Oklejas; Cyril S. Gary; Weihan Cao; Judy E. Kim (pp. 154-161).
The folding reaction of a β-barrel membrane protein, outer membrane protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.► Membrane protein folding of OmpA is investigated using FRET measurements. ► FRET pairs are tryptophan as donor and naphthalene derivative as acceptor. ► Different timescales for protein pore formation and membrane traversal are observed.
Keywords: Fluorescence; Tryptophan; OmpA; IAEDANS; Vesicle; Bilayer
Biophysics of α-synuclein membrane interactions by Candace M. Pfefferkorn; Zhiping Jiang; Jennifer C. Lee (pp. 162-171).
Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.► We provide a synopsis of biophysical probes of membrane protein structure. ► We review the role of α-synuclein, a neuronal membrane-associated protein, in Parkinson's disease. ► We discuss the interactions of α-synuclein with model membranes and current structural models of the membrane-bound form. ► We present different mechanisms of how α-synuclein and membrane interactions are linked to Parkinson's disease.
Keywords: Protein structure; Parkinson's disease; Amyloid; Vesicles
Lipid–protein interactions in biological membranes: A dynamic perspective by Adam W. Smith (pp. 172-177).
Though an increasing number of biological functions at the membrane are attributed to direct associations between lipid head groups and protein side chains or lipid protein hydrophobic attractive forces, surprisingly limited information is available about the dynamics of these interactions. The static in vitro representation provided by membrane protein structures, including very insightful lipid–protein binding geometries, still fails to recapitulate the dynamic behavior characteristic of lipid membranes. Experimental measures of the interaction time of lipid–protein association are very rare, and have only provided order-of-magnitude estimates in an extremely limited number of systems. In this review, a brief outline of the experimental approaches taken in this area to date is given. The bulk of the review will focus on two methods that are promising techniques for measuring lipid–protein interactions: time-resolved fluorescence microscopy, and two-dimensional infrared (2D IR) spectroscopy. Time-resolved fluorescence microscopy is the name given to a sophisticated toolbox of measurements taken using pulsed laser excitation and time-correlated single photon counting (TCSPC). With this technique the dynamics of interaction can be measured on the time scale of nanoseconds to milliseconds. 2D IR is a femtosecond nonlinear spectroscopy that can resolve vibrational coupling between lipids and proteins at molecular-scale distances and at time scales from femtoseconds to picoseconds. These two methods are poised to make significant advances in our understanding of the dynamic properties of biological membranes. This article is part of a Special Issue entitled: Membrane protein structure and function.► Review of methods to probe lipid–protein interaction dynamics. ► Time-resolved fluorescence microscopy measures correlated lipid–protein diffusion. ► 2D IR spectroscopy probes lipid–protein vibrational coupling and dynamics.
Keywords: Lipid protein dynamics; Two-dimensional infrared spectroscopy; Fluorescence cross-correlation spectroscopy
Hydrogen-bond energetics drive helix formation in membrane interfaces by Paulo F. Almeida; Alexey S. Ladokhin; Stephen H. White (pp. 178-182).
The free energy cost Δ G of partitioning many unfolded peptides into membrane interfaces is unfavorable due to the cost of partitioning backbone peptide bonds. The partitioning cost is dramatically reduced if the peptide bonds participate in hydrogen bonds. The reduced cost underlies secondary structure formation by amphiphilic peptides partitioned into membrane interfaces through a process referred to as partitioning–folding coupling. This coupling is characterized by the free energy reduction per residue, ∆ G res that drives folding. There is some debate about the correct value of ∆ G res and its dependence on the hydrophobic moment (μH) of amphiphilic α-helical peptides. We show how to compute ∆ G res correctly. Using published data for two families of peptides with different hydrophobic moments and charges, we find that ∆ G res does not depend upon μH. The best estimate of ∆ G res is −0.37±0.02kcalmol−1. This article is part of a Special Issue entitled: Membrane protein structure and function.► Partitioning of unfolded peptides to the membrane interface is generally unfavorable ► Helix formation reduces the partitioning free energy by Δ G res per residue ► Δ G res is independent of the hydrophobic moment of amphipathic α-helical peptides ► Δ G res is ascribed to formation of backbone hydrogen bonds ► The best estimate Δ G res is −0.37±0.02 (SEM) kcalmol−1
Keywords: Membrane-active peptide; Membrane protein folding; Antimicrobial peptide; Thermodynamics
Transmembrane helix dimerization: Beyond the search for sequence motifs by Edwin Li; William C. Wimley; Kalina Hristova (pp. 183-193).
Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.► Transmembrane helix dimerization studies shed light on membrane protein folding. ► Helix dimerization in membranes has been described within the sequence motif paradigm. ► The sequence motif paradigm in membrane protein folding is incomplete. ► The search for sequence motifs cannot solve the membrane protein folding problem.
Keywords: Membrane protein folding; Transmembrane helix dimerization; Sequence motif
Mechanism of structural transformations induced by antimicrobial peptides in lipid membranes by Kin Lok H. Lam; Hao Wang; Ting Ann Siaw; Matthew R. Chapman; Alan J. Waring; James T. Kindt; Ka Yee C. Lee (pp. 194-204).
It has long been suggested that pore formation is responsible for the increase in membrane permeability by antimicrobial peptides (AMPs). To better understand the mechanism of AMP activity, the disruption of model membrane by protegrin-1 (PG-1), a cationic antimicrobial peptide, was studied using atomic force microscopy. We present here the direct visualization of the full range of structural transformations in supported lipid bilayer patches induced by PG-1 on zwitterionic 1,2-dimyristoyl-snglycero-phospho-choline (DMPC) membranes. When PG-1 is added to DMPC, the peptide first induces edge instability at low concentrations, then pore-like surface defects at intermediate concentrations, and finally wormlike structures with a specific length scale at high concentrations. The formation of these structures can be understood using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent. Atomistic molecular dynamics simulations on lipid bilayer ribbons with PG-1 molecules placed at the edge or interior positions are carried out to calculate the effect of PG-1 in reducing line tension. Further investigation of the placement of PG-1 and its association with defects in the bilayer is carried out using unbiased assembly of a PG-1 containing bilayer from a random mixture of PG-1, DMPC, and water. A generalized model of AMP induced structural transformations is also presented in this work. This article is part of a Special Issue entitled: Membrane protein structure and function.►Direct visualization of membrane structural transformations induced by PG-1 via AFM. ►Structural transformations: edge instability -> pores -> wormlike micelles. ►Simulation work shows PG-1 lowering line tension at DMPC bilayer ribbon edges. ►Disruption process understood via a lipid/line-active peptide mesophase framework.
Keywords: Abbreviations; AMP; AntiMicrobial Peptide; PG-1; Protegrin-1; AFM; Atomic Force Microscopy; DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phospho-choline; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phospho-choline; SDS; sodium-dodecyl-sulfate; DPC; dodecyl-phospho-choline; POPC; palmitoyl-oleoylphosphatidyl-choline; POPG; palmitoyl-oleoylphosphatidyl-glycerol; NMR; Nuclear Magnetic Resonance; C; b; bulk peptide concentration; C; b; *; critical bulk peptide concentration; GROMACS; Groningen Machine for Chemical Simulations; MD; Molecular DynamicsMembrane disruption mechanism; Phospholipid; Protegrin-1; Atomic force microscopy; Molecular dynamics; Pore formation
Lipid composition regulates the conformation and insertion of the antimicrobial peptide maculatin 1.1 by Marc-Antoine Sani; Thomas C. Whitwell; Frances Separovic (pp. 205-211).
Antimicrobial peptides interact with cell membranes and their selectivity is contingent on the nature of the constituent lipids. Eukaryotic and bacterial membranes are comprised of different proportions of a range of lipid species with different physical properties. Hence, characterisation of antimicrobial peptides with respect to the magnitude of their interactions with model membranes of different lipid types is needed. Maculatin 1.1 is a short antimicrobial peptide secreted from the skin of several Australian tree-frog species. Circular dichroism spectroscopy (CD) was used to explore the interaction of maculatin 1.1 with a wide range of model membrane systems of different head group and acyl chain characteristics. For neutral phosphatidylcholine (PC), unlike anionic phospholipids, the magnitude of the peptide interactions was dependent on the length and degree of saturation of the constituent acyl chains. Oriented circular dichroism (OCD) data indicated that helical structure was likely promoted by peptide insertion into the hydrophobic core of PC bilayers. The addition of cholesterol (30% mol/mol) tended to decrease the membrane interaction of maculatin 1.1. Anionic lipids locked maculatin 1.1 via electrostatic interactions onto the surface of oriented bilayers as seen in OCD spectra. Furthermore, increasing the membrane curvature by reducing the vesicle radii only slightly reduced the proportion of helical structure in all systems by approximately 10%. The peptide–lipid interaction was strongly dependent on both the lipid chain length and head group, which highlights the importance of the lipid composition used to mimic different cell types. This article is part of a Special Issue entitled: Membrane protein structure and function.► Different phospholipid model membranes affect the structure of an antimicrobial peptide. ► Conformation of the peptide maculatin 1.1 depends on the lipid chain length and head group. ► Peptide distinguishes between prokaryotic and eukaryotic lipid membranes.
Keywords: Abbreviations; AMP; Antimicrobial peptide; CD; Circular dichroism; Chol; Cholesterol; CL; Cardiolipin; DD; Didecanoyl (C10:0); DH; Dihexanoyl (C6:0); DL; Dilauroyl (12:0); DM; Dimyristoyl (C14:0); DO; Dioleoyl (C18:1); DP; Dipalmitoyl (C16:0); DS; Distearoyl (C18:0); LPG; Lysophosphatidylglycerol; LUV; Large unilamellar vesicle; OCD; Oriented circular dichroism; PA; Phosphatidic acid; PC; Phosphatidylcholine; PE; Phosphatidylethanolamine; PG; Phosphatidylglycerol; PO; Palmitoyloleoyl (C16:0, 18:1); PS; Phosphatidylserine; SDS; Sodium dodecylsulfate; SUV; Small unilamellar vesicle; TFE; Trifluorethanol; TM; Tetramyristoyl (C14:0)Circular dichroism; Oriented circular dichroism; Phospholipids; Antimicrobial peptide; Hydrophobic mismatch; Curvature
Characterization of a potent antimicrobial lipopeptide via coarse-grained molecular dynamics by Joshua N. Horn; Jesse D. Sengillo; Dejun Lin; Tod D. Romo; Alan Grossfield (pp. 212-218).
The prevalence of antibiotic-resistant pathogens is a major medical concern, prompting increased interest in the development of novel antimicrobial compounds. One such set of naturally occurring compounds, known as antimicrobial peptides (AMPs), have broad-spectrum activity, but come with many limitations for clinical use. Recent work has resulted in a set of antimicrobial lipopeptides (AMLPs) with micromolar minimum inhibitory concentrations and excellent selectivity for bacterial membranes. To characterize a potent, synthetic lipopeptide, C16-KGGK, we used multi-microsecond coarse-grained simulations with the MARTINI forcefield, with a total simulation time of nearly 46μs. These simulations show rapid binding of C16-KGGK, which forms micelles in solution, to model bacterial lipid bilayers. Furthermore, upon binding to the surface of the bilayer, these lipopeptides alter the local lipid organization by recruiting negatively charged POPG lipids to the site of binding. It is likely that this drastic reorganization of the bilayer has major effects on bilayer dynamics and cellular processes that depend on specific bilayer compositions. By contrast, the simulations revealed no association between the lipopeptides and model mammalian bilayers. These simulations provide biophysical insights into lipopeptide selectivity and suggest a possible mechanism for antimicrobial action. This article is part of a Special Issue entitled: Membrane protein structure and function.► C16-KGGK is a potent antimicrobial lipopeptide. ► Coarse-grained molecular dynamics simulations yield detailed insights into peptide-membrane interactions. ► Selectivity for bacterial membranes is due to favorable electrostatic interactions. ► Lipopeptide binding reorganizes the membrane by recruiting anionic lipids to the binding site.
Keywords: Antimicrobial peptides; Lipopeptides; Molecular dynamics
A reinterpretation of neutron scattering experiments on a lipidated Ras peptide using replica exchange molecular dynamics by Alexander Vogel; Matthew Roark; Scott E. Feller (pp. 219-224).
The Ras family of proteins plays crucial roles in a variety of cell signaling networks where they have the function of a molecular switch. Their particular medical relevance arises from mutations in these proteins that are implicated in ~30% of human cancers. The various Ras proteins exhibit a high degree of homology in their soluble domains but extremely high variability in the membrane anchoring regions that are crucial for protein function and are the focus of this study. We have employed replica exchange molecular dynamics computer simulations to study a doubly lipidated heptapeptide, corresponding to the C-terminus of the human N-Ras protein, incorporated into a dimyristoylphosphatidylcholine lipid bilayer. This same system has previously been investigated experimentally utilizing a number of techniques, including neutron scattering. Here we present results of well converged simulations that describe the subtle changes in scattering density in terms of the location of the peptide and its lipid modifications and in terms of changes in phospholipid density arising from the incorporation of the peptide into the membrane bilayer. The detailed picture that emerges from the combination of experimental and computational data exemplifies the power of combining isotopic substitution neutron scattering with atomistic molecular dynamics simulation. This article is part of a Special Issue entitled: Membrane protein structure and function.► Lipid–peptide interactions in peripheral membrane protein. ► Combined experimental/simulation approach. ► Replica exchange molecular dynamics. ► Interpretation of neutron scattering experiments.
Keywords: Molecular dynamics; Ras protein; Neutron scattering; Replica exchange
Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors by Eugene Serebryany; Gefei Alex Zhu; Elsa C.Y. Yan (pp. 225-233).
Functional reconstitution of transmembrane proteins remains a significant barrier to their biochemical, biophysical, and structural characterization. Studies of seven-transmembrane G-protein coupled receptors (GPCRs) in vitro are particularly challenging because, ideally, they require access to the receptor on both sides of the membrane as well as within the plane of the membrane. However, understanding the structure and function of these receptors at the molecular level within a native-like environment will have a large impact both on basic knowledge of cell signaling and on pharmacological research. The goal of this article is to review the main classes of membrane mimics that have been, or could be, used for functional reconstitution of GPCRs. These include the use of micelles, bicelles, lipid vesicles, nanodiscs, lipidic cubic phases, and planar lipid membranes. Each of these approaches is evaluated with respect to its fundamental advantages and limitations and its applications in the field of GPCR research. This article is part of a Special Issue entitled: Membrane protein structure and function.Display Omitted► Reconstitution into native membrane-like environments is needed for quantitative biophysical studies of GPCRs. ► Six classes of membrane mimics are reviewed and evaluated for GPCR reconstitution. ► Classes reviewed here include micelles, bicelles, nanodiscs, lipid vesicles, planar lipid membranes, and lipidic cubic phases. ► Theoretical advantages and limitations for each reconstitution system are discussed. ► Examples of applications are provided.
Keywords: Abbreviations; GPCR; G-protein coupled receptor; SUV; small unilamellar vesicle; GUV; giant unilamellar vesicle; MSP; membrane scaffold protein; PLM; planar lipid membraneGPCR reconstitution; Bicelle; Nanodisc; Giant unilamellar vesicle; Planar lipid membrane; Lipidic cubic phase
The role of the lipid matrix for structure and function of the GPCR rhodopsin by Olivier Soubias; Klaus Gawrisch (pp. 234-240).
Photoactivation of rhodopsin in lipid bilayers results within milliseconds in a metarhodopsin I (MI)–metarhodopsin II (MII) equilibrium that is very sensitive to the lipid composition. It has been well established that lipid bilayers that are under negative curvature elastic stress from incorporation of lipids like phosphatidylethanolamines (PE) favor formation of MII, the rhodopsin photointermediate that is capable of activating G protein. Furthermore, formation of the MII state is favored by negatively charged lipids like phosphatidylserine and by lipids with longer hydrocarbon chains that yield bilayers with larger membrane hydrophobic thickness. Cholesterol and rhodopsin–rhodopsin interactions from crowding of rhodopsin molecules in lipid bilayers shift the MI–MII equilibrium towards MI. A variety of mechanisms seems to be responsible for the large, lipid-induced shifts between MI and MII: adjustment of the thickness of lipid bilayers to rhodopsin and adjustment of rhodopsin helicity to the thickness of bilayers, curvature elastic deformations in the lipid matrix surrounding the protein, direct interactions of PE headgroups and polyunsaturated hydrocarbon chains with rhodopsin, and direct or lipid-mediated interactions between rhodopsin molecules. This article is part of a Special Issue entitled: Membrane protein structure and function.► Adjustment of the thickness of lipid bilayers to rhodopsin. ► Adjustment of rhodopsin helicity to the thickness of bilayers. ► Curvature elastic deformations in the lipid matrix surrounding the protein. ► Direct interactions of PE headgroups and polyunsaturated hydrocarbon chains with rhodopsin. ► Direct or lipid-mediated interactions between rhodopsin molecules.
Keywords: Abbreviations; GPCR; G protein-coupled membrane receptors; TM; transmembrane helix; G; t; G protein transducin; MI; metarhodopsin I; MII; metarhodopsin II; ROS; rod outer segments; CD; circular dichroism; FRET; Förster resonance energy transfer; EPR; electron paramagnetic resonance; EM; electron microscopy; CG-MD; coarse-grained molecular dynamic simulations; FSM; Flexible Surface Model; H; II; inverse hexagonal phase; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PS; phosphatidylserine; DHA; docosahexaenoic acid (22:6n-3); POPC; 1-palmitoyl-2-oleolyl-; sn; -glycero-3-phosphocholine (16:0–18:1n-9-PC); DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine (18:1n-9-18:1n-9-PC); SDPC; 1-stearoyl-2-docosahexaenoyl-; sn; -glycero-3-phosphocholine (18:0–22:6n-3-PC); DOPE-M; e1; 1,2-dioleoyl-; sn; -glycero-3-monomethyl-phosphoethanolamine (18:1n-9-18:1n-9-PE-Me; 1; ); DOPE-M; e2; 1; 2-dioleoyl-; sn; -glycero-3-dimethyl-phosphoethanolamine (18:1n-9-18:1n-9-PE-Me; 2; ); DOPE 1; 2-dioleoyl-; sn; -glycero-3-phosphoethanolamine (18:1n-9-18:1n-9-PE); SDPE; 1-stearoyl-2-docosahexaenoyl-; sn; -glycero-3-phosphoethanolamine (18:0–22:6n-3-PE); MMPC; 1-myristoyl-2-myristoleoyl-; sn; -glycero-3-phosphocholine (14:0–14:1n-5-PC); EEPC; 1-eicosanoyl-2-eicosenoyl-; sn; -glycero-3-phosphocholine (20:0–20:1n-9-PC); DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine (16:0–16:0-PC); DDPC; 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6n-3-22:6n-3-PC)Rhodopsin; Lipid–protein interaction; Hydrophobic mismatch; Docosahexaenoic acid; NMR; Protein oligomerization
Molecular simulations and solid-state NMR investigate dynamical structure in rhodopsin activation by Blake Mertz; Andrey V. Struts; Scott E. Feller; Michael F. Brown (pp. 241-251).
Rhodopsin has served as the primary model for studying G protein-coupled receptors (GPCRs)—the largest group in the human genome, and consequently a primary target for pharmaceutical development. Understanding the functions and activation mechanisms of GPCRs has proven to be extraordinarily difficult, as they are part of a complex signaling cascade and reside within the cell membrane. Although X-ray crystallography has recently solved several GPCR structures that may resemble the activated conformation, the dynamics and mechanism of rhodopsin activation continue to remain elusive. Notably solid-state2H NMR spectroscopy provides key information pertinent to how local dynamics of the retinal ligand change during rhodopsin activation. When combined with molecular mechanics simulations of proteolipid membranes, a new paradigm for the rhodopsin activation process emerges. Experiment and simulation both suggest that retinal isomerization initiates the rhodopsin photocascade to yield not a single activated structure, but rather an ensemble of activated conformational states. This article is part of a Special Issue entitled: Membrane protein structure and function.► Solid-state2H NMR probes dynamics of retinal ligand. ► Molecular simulations test specific counterion models for rhodopsin. ► NMR relaxation theory and molecular dynamics are complementary. ► Quantum mechanical calculations address the retinal force field. ► Rhodopsin activation is described by ensemble mechanism.
Keywords: Abbreviations; CHARMM; Chemistry at Harvard Macromolecular Mechanics; EPR; electron paramagnetic resonance; FTIR; Fourier transform infrared; GPCR; G protein-coupled receptor; MD; molecular dynamics; Meta I; metarhodopsin I; Meta II; metarhodopsin II; 2MBD; 2-methyl-butadiene; 3MHT; 3-methyl-hexatriene; MM; molecular mechanics; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSB; protonated Schiff base; RDC; residual dipolar coupling; RQC; residual quadrupolar coupling; SDPC; 1-stearoyl-2-docosahexaenoyl-; sn; -glycero-3-phosphocholine; SDPE; 1-stearoyl-2-docosahexaenoyl-; sn; -glycero-3-phosphoethanolamine; QM; quantum mechanicsG protein-coupled receptor; Membrane; Molecular dynamics; Solid-state NMR; Rhodopsin; Vision
An ensemble dynamics approach to decipher solid-state NMR observables of membrane proteins by Wonpil Im; Sunhwan Jo; Taehoon Kim (pp. 252-262).
Solid-state NMR (SSNMR) is an invaluable tool for determining orientations of membrane proteins and peptides in lipid bilayers. Such orientational descriptions provide essential information about membrane protein functions. However, when a semi-static single conformer model is used to interpret various SSNMR observables, important dynamics information can be missing, and, sometimes, even orientational information can be misinterpreted. In addition, over the last decade, molecular dynamics (MD) simulation and semi-static SSNMR interpretation have shown certain levels of discrepancies in terms of transmembrane helix orientation and dynamics. Dynamic fitting models have recently been proposed to resolve these discrepancies by taking into account transmembrane helix whole body motions using additional parameters. As an alternative approach, we have developed SSNMR ensemble dynamics (SSNMR-ED) using multiple conformer models, which generates an ensemble of structures that satisfies the experimental observables without any fitting parameters. In this review, various computational methods for determining transmembrane helix orientations are discussed, and the distributions of VpuTM (from HIV-1) and WALP23 (a synthetic peptide) orientations from SSNMR-ED simulations are compared with those from MD simulations and semi-static/dynamic fitting models. Such comparisons illustrate that SSNMR-ED can be used as a general means to extract both membrane protein structure and dynamics from the SSNMR measurements. This article is part of a Special Issue entitled: Membrane protein structure and function.Display Omitted►A review of solid-state NMR (SSNMR) structure calculation methods. ►Illustration of TM helix dynamics' effects in interpretation of SSNMR observables. ►An introduction of SSNMR ensemble dynamics (SSNMR-ED). ►Comparison of SSNMR-ED with MD and semi-static/dynamic fitting methods.
Keywords: Abbreviations; CSA; chemical shift anisotropy; DC; dipolar coupling; DQS; deuterium quadrupolar splitting; EPR; electron paramagnetic resonance; GALA; geometric analysis of labeled alanine; PISA; polar index slant angle; PISEMA; polarization inversion spin exchange at the magic angle; SSNMR; solid-state nuclear magnetic resonance; SSNMR-ED; SSNMR ensemble dynamicsChemical shift anisotropy; Dipolar coupling; Deuterium quadrupolar splitting; Semi-static fitting model; Dynamic fitting model; Potential of mean force
A gate-free pathway for substrate release from the inward-facing state of the Na+-galactose transporter by Jing Li; Emad Tajkhorshid (pp. 263-271).
Employing molecular dynamics (MD) simulations, the pathway and mechanism of substrate unbinding from the inward-facing state of the Na+-coupled galactose transporter, vSGLT, have been investigated. During a 200-ns equilibrium simulation, repeated spontaneous unbinding events of the substrate from its binding site have been observed. In contrast to the previously proposed gating role of a tyrosine residue (Y263), the unbinding mechanism captured in the present equilibrium simulation does not rely on the displacement and/or rotation of this side chain. Rather, the unbinding involves an initial lateral displacement of the substrate out of the binding site which allows the substrate to completely emerge from the region covered by the side chain of Y263 without any noticeable conformational changes of the latter. Starting with the snapshots taken from this equilibrium simulation with the substrate outside the binding site, steered MD (SMD) simulations were then used to probe the translocation of the substrate along the remaining of the release pathway within the protein's lumen and to characterize the nature of protein–substrate interactions involved in the process. Combining the results of the equilibrium and SMD simulations, we provide a description of the full translocation pathway for the substrate release from the binding site into the cytoplasm. Residues E68, N142, T431, and N267 facilitate the initial substrate's displacement out of the binding site, while the translocation of the substrate along the remainder of the exit pathway formed between TM6 and TM8 is facilitated by H-bond interactions between the substrate and a series of conserved, polar residues (Y138, N267, R273, S365, S368, N371, S372, and T375). The observed molecular events indicate that no gating is required for the release of the substrate from the crystallographically captured structure of the inward-facing state of SGLT, suggesting that this conformation might represent an open, rather than occluded, state of the transporter. This article is part of a Special Issue entitled: Membrane protein structure and function.► Equilibrium dynamics of substrate unbinding from the galactose transporter. ► Characterizing the complete release pathway and the role of conserved residues. ► Proposing a gate-free mechanism and pathway for substrate unbinding. ► Proposing that the crystal structure is an open, rather than an occluded state.
Keywords: Membrane transporters; LeuT-fold secondary transporter; Substrate unbinding; Molecular dynamics simulation; Inward-facing state; Sodium-coupled transporter
Structural correlates of selectivity and inactivation in potassium channels by Jason G. McCoy; Crina M. Nimigean (pp. 272-285).
Potassium channels are involved in a tremendously diverse range of physiological applications requiring distinctly different functional properties. Not surprisingly, the amino acid sequences for these proteins are diverse as well, except for the region that has been ordained the “selectivity filter”. The goal of this review is to examine our current understanding of the role of the selectivity filter and regions adjacent to it in specifying selectivity as well as its role in gating/inactivation and possible mechanisms by which these processes are coupled. Our working hypothesis is that an amino acid network behind the filter modulates selectivity in channels with the same signature sequence while at the same time affecting channel inactivation properties. This article is part of a Special Issue entitled: Membrane protein structure and function.► We review the role of the selectivity filter region in selectivity and inactivation. ► We focus on three different families of potassium channels: Kv, Kir, and KCa. ► Residues near the filter modulate selectivity and inactivation.
Keywords: Selectivity; Inactivation; Potassium channel; KCSA
Water wires in atomistic models of the Hv1 proton channel by Mona L. Wood; Eric V. Schow; J. Alfredo Freites; Stephen H. White; Francesco Tombola; Douglas J. Tobias (pp. 286-293).
The voltage-gated proton channel (Hv1) is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels but lacks a separate pore domain. The Hv1 monomer has dual functions: it gates the proton current and also serves as the proton conduction pathway. To gain insight into the structure and dynamics of the yet unresolved proton permeation pathway, we performed all-atom molecular dynamics simulations of two different Hv1 homology models in a lipid bilayer in excess water. The structure of the Kv1.2–Kv2.1 paddle-chimera VSD was used as template to generate both models, but they differ in the sequence alignment of the S4 segment. In both models, we observe a water wire that extends through the membrane, whereas the corresponding region is dry in simulations of the Kv1.2–Kv2.1 paddle-chimera. We find that the kinetic stability of the water wire is dependent upon the identity and location of the residues lining the permeation pathway, in particular, the S4 arginines. A measurement of water transport kinetics indicates that the water wire is a relatively static feature of the permeation pathway. Taken together, our results suggest that proton conduction in Hv1 may occur via Grotthuss hopping along a robust water wire, with exchange of water molecules between inner and outer ends of the permeation pathway minimized by specific water–protein interactions. This article is part of a Special Issue entitled: Membrane protein structure and function.► Two homology models of the Hv1 proton channel are presented. ► The models are based on two alignments with the voltage-sensing domain of a potassium channel of known structure. ► Both models support water wires, but the wire is more stable in one of the models. ► No water flux is observed in the model with the most robust water wire. ► The model with the most robust wire is supported by experimental studies and modeling of a mutant.
Keywords: Voltage-gated ion channels; Voltage-sensing domains; Membrane proteins; Molecular dynamics simulations
Constant electric field simulations of the membrane potential illustrated with simple systems by James Gumbart; Fatemeh Khalili-Araghi; Marcos Sotomayor; Benoît Roux (pp. 294-302).
Advances in modern computational methods and technology make it possible to carry out extensive molecular dynamics simulations of complex membrane proteins based on detailed atomic models. The ultimate goal of such detailed simulations is to produce trajectories in which the behavior of the system is as realistic as possible. A critical aspect that requires consideration in the case of biological membrane systems is the existence of a net electric potential difference across the membrane. For meaningful computations, it is important to have well validated methodologies for incorporating the latter in molecular dynamics simulations. A widely used treatment of the membrane potential in molecular dynamics consists of applying an external uniform electric field E perpendicular to the membrane. The field acts on all charged particles throughout the simulated system, and the resulting applied membrane potential V is equal to the applied electric field times the length of the periodic cell in the direction perpendicular to the membrane. A series of test simulations based on simple membrane-slab models are carried out to clarify the consequences of the applied field. These illustrative tests demonstrate that the constant-field method is a simple and valid approach for accounting for the membrane potential in molecular dynamics studies of biomolecular systems. This article is part of a Special Issue entitled: Membrane protein structure and function.► The electric potential difference across the membrane must be incorporated in computer simulation using a well-validated methodology. ► A widely used treatment of the membrane potential in molecular dynamics consists in applying a constant electric field throughout the system. ► A series of test simulations based on simple models are carried out to clarify and illustrate the constant field method.
Keywords: Electrostatics; Electrodes; Free energy; Patch clamp
Coarse grained model for exploring voltage dependent ion channels by Anatoly Dryga; Suman Chakrabarty; Spyridon Vicatos; Arieh Warshel (pp. 303-317).
The relationship between the membrane voltage and the gating of voltage activated ion channels and other systems have been a problem of great current interest. Unfortunately, reliable molecular simulations of external voltage effects present a major challenge, since meaningful converging microscopic simulations are not yet available and macroscopic treatments involve major uncertainties in terms of the dielectric used and other key features. This work extends our coarse grained (CG) model to simulations of membrane/protein systems under external potential. Special attention is devoted to a consistent modeling of the effect of external potential due to the electrodes, emphasizing semimacroscopic description of the electrolytes in the solution regions between the membranes and the electrodes, as well as the coupling between the combined potential from the electrodes plus the electrolytes and the protein ionized groups. We also provide a clear connection to microscopic treatment of the electrolytes and thus can explore possible conceptual problems that are hard to resolve by other current approaches. For example, we obtain a clear description of the charge distribution in the entire electrolyte system, including near the electrodes in membrane/electrodes systems (where continuum models do not seem to provide the relevant results). Furthermore, the present treatment provides an insight on the distribution of the electrolyte charges before and after equilibration across the membrane, and thus on the nature of the gating charge. The different aspects of the model have been carefully validated by considering problems ranging for the simple Debye–Huckel, and the Gouy–Chapman models to the evaluation of the electrolyte distribution between two electrodes, as well as the effect of extending the simulation system by periodic replicas. Overall the clear connection to microscopic descriptions combined with the power of the CG modeling seems to offer a powerful tool for exploring the balance between the protein conformational energy and the interaction with the external potential in voltage activated channels. To illustrate these features we present a preliminary study of the gating charge in the voltage activated Kv1.2 channel, using the actual change in the electrolyte charge distribution rather than the conventional macroscopic estimate. We also discuss other special features of the model, which include the ability to capture the effect of changes in the protonation states of the protein residues during the close to open voltage induced transition. This article is part of a Special Issue entitled: Membrane protein structure and function.► This work develops a CG model for membrane/protein systems. ► Provides strategy for consistent simulations of the effect of membrane potential on proteins. ► Alternative micro and macroscopic approaches do not yet provide a reliable picture. ► The model allows one to simulate the gating current. ► The balance between the protein energy and the external potential can be assessed.
Keywords: Membrane potential; Kv1.2; Gating charge
Charge equilibration force fields for molecular dynamics simulations of lipids, bilayers, and integral membrane protein systems by Timothy R. Lucas; Brad A. Bauer; Sandeep Patel (pp. 318-329).
With the continuing advances in computational hardware and novel force fields constructed using quantum mechanics, the outlook for non-additive force fields is promising. Our work in the past several years has demonstrated the utility of polarizable force fields, those based on the charge equilibration formalism, for a broad range of physical and biophysical systems. We have constructed and applied polarizable force fields for lipids and lipid bilayers. In this review of our recent work, we discuss the formalism we have adopted for implementing the charge equilibration (CHEQ) method for lipid molecules. We discuss the methodology, related issues, and briefly discuss results from recent applications of such force fields. Application areas include DPPC-water monolayers, potassium ion permeation free energetics in the gramicidin A bacterial channel, and free energetics of permeation of charged amino acid analogs across the water-bilayer interface. This article is part of a Special Issue entitled: Membrane protein structure and function.► Polarizable force field improves modeling of monolayer dipole potential. ► Polarizable force field shows water and headgroups help stabilize methyl guanidinium in bilayer center. ► Polarizable force field reduces equilibrium potential of mean force for potassium permeation in gramicidin A.
Keywords: Charge equilibration; Polarizable force field; Molecular dynamics simulation; Lipid; Bilayer
Side-chain oxysterols: From cells to membranes to molecules by Brett N. Olsen; Paul H. Schlesinger; Daniel S. Ory; Nathan A. Baker (pp. 330-336).
This review discusses the application of cellular biology, molecular biophysics, and computational simulation to understand membrane-mediated mechanisms by which oxysterols regulate cholesterol homeostasis. Side-chain oxysterols, which are produced enzymatically in vivo, are physiological regulators of cholesterol homeostasis and primarily serve as cellular signals for excess cholesterol. These oxysterols regulate cholesterol homeostasis through both transcriptional and non-transcriptional pathways; however, many molecular details of their interactions in these pathways are still not well understood. Cholesterol trafficking provides one mechanism for regulation. The current model of cholesterol trafficking regulation is based on the existence of two distinct cholesterol pools in the membrane: a low and a high availability/activity pool. It is proposed that the low availability/activity pool of cholesterol is integrated into tightly packing phospholipids and relatively inaccessible to water or cellular proteins, while the high availability cholesterol pool is more mobile in the membrane and is present in membranes where the phospholipids are not as compressed. Recent results suggest that oxysterols may promote cholesterol egress from membranes by shifting cholesterol from the low to the high activity pools. Furthermore, molecular simulations suggest a potential mechanism for oxysterol “activation” of cholesterol through its displacement in the membrane. This review discusses these results as well as several other important interactions between oxysterols and cholesterol in cellular and model lipid membranes. This article is part of a Special Issue entitled: Membrane protein structure and function.Display Omitted► A review of oxysterol action in cholesterol homeostasis with particular emphasis on molecular details of interaction. ► Cellular, molecular, and computational approaches provide multiscale insight into oxysterol interaction with lipid bilayers. ► Potential mechanisms for oxysterol “activation” of cholesterol are discussed.
Keywords: Abbreviations; ACAT; acyl-CoA cholesterol acyl transferase; CF; carboxyfluorescein; DOPC; dioleolyphosphatidylcholine; DPPC; dipalmitoylphosphatidylcholine; HC; hydroxycholesterol; LXR; liver X receptor; POPC; 1-palmitoyl-2-oleoyl-phosphatidylcholine; Scap; SREBP cleavage-activating protein; SREBP; sterol response element binding proteinOxysterol; Cholesterol; Sterol; Homeostasis; Molecular dynamics
Atomistic models of ion and solute transport by the sodium-dependent secondary active transporters by Igor Zdravkovic; Chunfeng Zhao; Bogdan Lev; Javier Eduardo Cuervo; Sergei Yu. Noskov (pp. 337-347).
The recent determination of high-resolution crystal structures of several transporters offers unprecedented insights into the structural mechanisms behind secondary transport. These proteins utilize the facilitated diffusion of the ions down their electrochemical gradients to transport the substrate against its concentration gradient. The structural studies revealed striking similarities in the structural organization of ion and solute binding sites and a well-conserved inverted-repeat topology between proteins from several gene families. In this paper we will overview recent atomistic simulations applied to study the mechanisms of selective binding of ion and substrate in LeuT, Glt, vSGLT and hSERT as well as its consequences for the transporter conformational dynamics. This article is part of a Special Issue entitled: Membrane protein structure and function.► Molecular Mechanism of Secondary Transport Regulation by Binding of Small. ► Molecules Studied by Atomistic MD simulations and Free Energy Computations.
Keywords: Secondary transporter; Molecular mechanism; Substrate specificity; Ion selectivity; Water permeation