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Cardiolipin (CL) is a unique phospholipid with a dimeric structure having four acyl chains and two phosphate groups found almost exclusively in certain membranes of bacteria and of mitochondria of eukaryotes. CL interacts with numerous proteins and has been implicated in function and stabilization of several integral membrane proteins (IMPs). While both functional and stabilization roles of CL in IMPs has been generally acknowledged, there are, in fact, only limited number of quantitative analysis that support this function of CL. This is likely caused by relatively complex determination of parameters characterizing stability of IMPs and particularly intricate assessment of role of specific phospholipids such as CL in IMPs stability. This review aims to summarize quantitative findings regarding stabilization role of CL in IMPs reported up to now.

Membrane proteins are one of the most challenging and attractive objects in modern structural biology, as they are targets for the majority of medicines. However, studies of membrane proteins are hindered by several obstacles, including their low ability to crystallize, highly dynamic behavior of some of their domains, and need for membrane-like environment. Although solution nuclear magnetic resonance (NMR) is a very powerful technique of structural biology in terms of the amount of provided data, it imposes several limitations on the object under investigation, with the main constraint being related to the size of the object. For this reason, the membrane mimetic has to form particles of small size and simultaneously to properly simulate the bilayer membrane to be applicable for solution NMR spectroscopy. Here we review the recent advances in the field of membrane mimetics for solution NMR studies, discuss the advantages and drawbacks of specific membrane-like environments, and formulate the criteria for the selection of proper environment for a particular membrane protein or domain.

International audience; Understanding how membrane proteins interact with their environment is fundamental to the understanding of their structure, function and interactions. We have performed coarse-grained molecular dynamics simulations on a series of membrane proteins in a membrane environment to examine the perturbations of the lipids by the presence of protein. We analyze these perturbations in terms of elastic membrane deformations and local lipid protein interactions. However these two factors are insufficient to describe the variety of effects that we observe and the changes caused by membranes proteins to the structure and dynamics of their lipid environment. Other factors that change the conformation available to lipid molecules are evident and are able to modify lipid structure far from the protein surface, and thus mediate long-range interactions between membrane proteins. We suggest that these multiple modifications to lipid behavior are responsible, at the molecular level, for the lipophobic effect we have proposed to account for our observations of membrane protein organization.

Lipid droplets (LDs), or oil bodies in plants, are specialized organelles that primarily serve as hubs of cellular metabolic energy storage and consumption. These ubiquitous cytoplasmic organelles are derived from the endoplasmic reticulum (ER) and consist of a hydrophobic neutral lipid core - mainly consisting of triglycerides and sterol esters - that is encircled by a phospholipid monolayer. The dynamic metabolic functions of the LDs are mainly executed and regulated by proteins on the monolayer surface. However, its unique architecture puts some structural constraints on the types of proteins that can associate with LDs. The lipid monolayer is decorated with either peripheral proteins or with integral membrane proteins that adopt a monotopic topology. Due to its oil-water interface, which is energetically costly, the LD surface happens to be favorable to the recruitment of many proteins involved in metabolic but also non-metabolic functions. We only started very recently to understand biophysical and biochemical principles controlling protein targeting to LDs. This review aims to summarize the most recent findings regarding this topic and proposes directions that will potentially lead to a better understanding of LD surface characteristics, as compared to bilayer membranes, and how that impacts protein-LD interactions.

Although membrane proteins are crucial participants in photosynthesis and other biological processes, many lack high-resolution structures. Prior to achieving a high-resolution structure, we are investigating whether MS-based footprinting can provide coarse-grained protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding. Our platform probes topology and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochemical oxidation of proteins (FPOP), and lipid Nanodiscs, which more similar to the native membrane environment than are the widely used detergent micelles. We describe here results that show a protein’s outer membrane regions are more heavily footprinted by OH radicals whereas the regions spanning the lipid bilayer remain inert to the labeling. Nanodiscs generally exhibit more protection of membrane proteins compared to detergent micelles and less shielding to those protein residues that exist outside the membrane. The combination of immobilizing the protein in Nanodiscs and footprinting with the FPOP approach is a feasible approach to map extra-membrane protein surfaces, even at the amino-acid level, and to illuminate intrinsic membrane protein topology.

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method.

Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state.

The integration of membrane proteins into “lipid raft” membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

Lipids are critical components of membranes and yet their precise composition and effects on membrane-embedded protein complexes are often difficult to define. Using mass spectrometry I will show how we can add lipids and quantify their effects on the stability of membrane protein complexes using unfolding experiments. I will also show how results from these experiments inform about suitable solution conditions for crystallography and function. Turning to the endogenous lipid cohort that surrounds a membrane protein I will show how we can define these lipids and demonstrate their importance for function.

New technologies for the purification of stable membrane proteins have emerged in recent years, in particular methods that allow the preparation of membrane proteins with their native lipid environment. Here, we look at the progress achieved with the use of styrene-maleic acid copolymers (SMA) which are able to insert into biological membranes forming nanoparticles containing membrane proteins and lipids. This technology can be applied to membrane proteins from any host source, and, uniquely, allows purification without the protein ever being removed from a lipid bilayer. Not only do these SMA lipid particles (SMALPs) stabilise membrane proteins, allowing structural and functional studies, but they also offer opportunities to understand the local lipid environment of the host membrane. With any new or different method, questions inevitably arise about the integrity of the protein purified: does it retain its activity; its native structure; and ability to perform its function? How do membrane proteins within SMALPS perform in existing assays and lend themselves to analysis by established methods? We outline here recent work on the structure and function of membrane proteins that have been encapsulated like this in a polymer-bound lipid bilayer, and the potential for the future with this approach. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids.

Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein structure databank. Solid-state Nuclear Magnetic Resonance (SSNMR) has long been considered as an attractive alternative because it allows for studies of membrane proteins in both native-like membranes composed of synthetic lipids and in cell membranes. Over the past decade, SSNMR has been rapidly developing into a major structural method, and a growing number of membrane protein structures obtained by this technique highlights its potential. Here we discuss membrane protein sample requirements, review recent progress in SSNMR methodologies, and describe recent advances in characterizing membrane proteins in the environment of a cellular membrane.

The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Membrane proteins depend on their natural lipid environment for function, which makes them more difficult to study in isolation. A number of approaches that mimic the lipid bilayer of biological membranes have been described (nanodiscs, SMALPs), enabling novel ways to assay activity and elucidate structures of this important class of proteins. More recently, the use of saposin A, a protein that is involved in lipid transport, to form Salipro (saposin–lipid–protein) complexes was demonstrated for a range of membrane protein targets (Frauenfeld et al., 2016). The method is fast and requires few resources. The saposin–lipid–scaffold adapts to various sizes of transmembrane regions during self-assembly, forming a minimal lipid nanoparticle. This results in the formation of a well-defined membrane protein–lipid complex, which is desirable for structural characterization. Here, we describe a protocol to reconstitute the sarco-endoplasmic reticulum calcium ATPase (SERCA) into Salipro nanoparticles. The complex formation is analyzed using negative stain electron microscopy (EM), allowing to quickly determine an initial structure of the membrane protein and to evaluate sample conditions for structural studies using single-particle cryo-EM in a detergent-free environment.

Transmembrane proteins inhabit a highly asymmetric environment that is, to a first approximation, two-dimensional. Many of them serve to transmit information between aqueous compartments, while others serve as conduits for the transport of material between compartments. In serving these functions they have to adopt at least two stable structures and rapidly interconvert between them. The paucity of atomic resolution structures has been limiting in elucidating the mechanisms by which these proteins carry out their functions. However, this century has seen the determination of the three-dimensional structures of a number of membrane proteins, leading to the start of an understanding of the dynamics displayed by them within the bilayer. Without attempting to be exhaustive, we provide illustrative examples of dynamics in membrane proteins and review their underlying mechanisms as they insert, fold and function in biological membranes.

Summary Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact membrane proteins natively anchored with the correct topology in genuine biological membranes. This approach circumvents the need to conduct tedious detergent screens for solubilization, purification, and reconstitution required in classical membrane protein studies. We have applied this method to three integral type I membrane proteins, namely the Caenorhabditis elegans cell-cell fusion proteins AFF-1 and EFF-1 and the glycoprotein B (gB) from Herpes simplex virus type 1 (HSV1). Electron cryotomography followed by subvolume averaging allowed the 3D reconstruction of EFF-1 and HSV1 gB in the membrane as well as an analysis of the spatial distribution and interprotein interactions on the membrane. MPEEVs have many applications beyond structural/functional investigations, such as facilitating the raising of antibodies, for protein-protein interaction assays or for diagnostics use, as biomarkers, and possibly therapeutics., Graphical Abstract, Highlights • Intact membrane proteins with correct topology in genuine biological membranes • Display system circumvents need for detergent solubilization and reconstitution • 3D reconstruction of proteins in the membrane by subvolume averaging • Mapping spatial distribution and relationships between proteins on the membrane, An elegant approach to display target membrane proteins on cell-derived vesicles in the topologically correct orientation is presented. Structural analysis by cryo-EM of those vesicles allowed reconstruction of the target proteins and mapping the spatial distribution on the membrane.

Remorins are well-established marker proteins for plasma membrane microdomains. They specifically localize to the inner membrane leaflet despite an overall hydrophilic amino acid composition. Here, we determined amino acids and post-translational lipidations that are required for membrane association of remorin proteins. We used a combination of cell biological and biochemical approaches to localize remorin proteins and truncated variants of those in living cells and determined S-acylation on defined residues in these proteins. S-acylation of cysteine residues in a C-terminal hydrophobic core contributes to membrane association of most remorin proteins. While S-acylation patterns differ between members of this multi-gene family, initial membrane association is mediated by protein-protein or protein-lipid interactions. However, S-acylation is not a key determinant for the localization of remorins in membrane microdomains. Although remorins bind via a conserved mechanism to the plasma membrane, other membrane-resident proteins may be involved in the recruitment of remorins into membrane domains. S-acylation probably occurs after an initial targeting of the proteins to the plasma membrane and locks remorins in this compartment. As S-acylation is a reversible post-translational modification, stimulus-dependent intracellular trafficking of these proteins can be envisioned.

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function. However only relatively recently has high resolution structure determination of membrane proteins progressed to the point of providing atomic level structure of lipid binding sites on membrane proteins. Analysis of X-ray diffraction, electron crystallography and NMR data over 100 specific lipid binding sites on membrane proteins. These data demonstrate tight lipid binding of both phospholipids and cholesterol to membrane proteins. Membrane lipids bind to membrane proteins by their headgroups, or by their acyl chains, or binding is mediated by the entire lipid molecule. When headgroups bind, binding is stabilized by polar interactions between lipid headgroups and the protein. When acyl chains bind, van der Waals effects dominate as the acyl chains adopt conformations that complement particular sites on the rough protein surface. No generally applicable motifs for binding have yet emerged. Previously published biochemical and biophysical data link this binding with function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Cellular membranes are densely crowded with a diverse population of integral and membrane-associated proteins. In this complex environment, lipid rafts, which are phase-separated membrane domains enriched in cholesterol and saturated lipids, are thought to organize the membrane surface. Specifically, rafts may help to concentrate proteins and lipids locally, enabling cellular processes such as assembly of caveolae, budding of enveloped viruses, and sorting of lipids and proteins in the Golgi. However, the ability of rafts to concentrate protein species has not been quantified experimentally. Here we show that when membrane-bound proteins become densely crowded within liquid-ordered membrane regions, steric pressure arising from collisions between proteins can destabilize lipid phase separations, resulting in a homogeneous distribution of proteins and lipids over the membrane surface. Using a reconstituted system of lipid vesicles and recombinant proteins, we demonstrate that protein-protein steric pressure creates an energetic barrier to the stability of phase-separated membrane domains that increases in significance as the molecular weight of the proteins increases. Comparison with a simple analytical model reveals that domains are destabilized when the steric pressure exceeds the approximate enthalpy of membrane mixing. These results suggest that a subtle balance of free energies governs the stability of phase-separated cellular membranes, providing a new perspective on the role of lipid rafts as concentrators of membrane proteins.

The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane — monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro.

Evidence that membrane proteins respond conformationally and functionally to their environment is gaining pace. Structural models, by necessity, have been characterized in preparations where the protein has been removed from its native environment. Different structural methods have used various membrane mimetics that have recently included lipid bilayers as a more native-like environment. Structural tools applied to lipid bilayer-embedded integral proteins are informing us about important generic characteristics of how membrane proteins respond to the lipid environment as compared with their response to other non-lipid environments. Here, we review the current status of the field, with specific reference to observations of some well-studied α-helical membrane proteins, as a starting point to aid the development of possible generic principals for model refinement.

The development of new methods and strategies for the investigation of membrane proteins is limited by poor solubility of these proteins in an aqueous environment and hindered by a number of other problems linked to the instability of the proteins outside lipid bilayers. Therefore, current research focuses on an analysis of membrane proteins incorporated into model lipid membrane, most frequently liposomes. In this work, we introduce a new electrochemical methodology for the analysis of transmembrane proteins reconstituted into a liposomal system. The proposed analytical approach is based on proteoliposomal sample adsorption on the surface of working electrodes followed by analysis of the anodic and cathodic signals of the reconstituted proteins. It works based on the fact that proteins are electroactive species, in contrast to the lipid components of the membranes under the given experimental conditions. Electroanalytical experiments were performed with two transmembrane proteins; the Na(+)/K(+)ATPase that contains transmembrane as well as large extramembraneous segments and the mitochondrial uncoupling protein 1, which is a transmembrane protein essentially lacking extramembraneous segments. Electrochemical analyses of proteoliposomes were compared with analyses of both proteins solubilized with detergents (C12E8 and octyl-PoE) and supported by the following complementary methods: microscopy techniques, protein activity testing, molecular model visualizations, and immunochemical identification of both proteins. The label-free electrochemical platform presented here enables studies of reconstituted transmembrane proteins at the nanomolar level. Our results may contribute to the development of new electrochemical sensors and microarray systems applicable within the field of poorly water-soluble proteins.

Integral membrane proteins constitute more than 30% of the human genome. Substantial lipid-protein interactions contribute to the structure and function of membrane proteins such as GPCRs and viral membrane ion channels. We address how physical properties of the membrane as well as positive charge on the lipid head group modulate protein activation. The flexible surface model (FSM) describes physical properties of the lipid environment that effect membrane protein activation [3]. According to the FSM, elastic coupling of the membrane lipids to integral membrane proteins define lipid-protein interactions through a balance of curvature and hydrophobic forces. Using UV-visible spectroscopy, FTIR spectroscopy [2], and molecular dynamics (MD) simulations, we discovered that lipid bilayer properties like head group and chain length modulate membrane protein functioning by influencing the conformational states. Using rhodopsin, a canonical G-protein-coupled receptor (GPCR) as a prototype, we show the effect of positively charged membrane lipid head group on rhodopsin activation using UV-visible spectroscopy and MD simulations. We discovered rhodopsin reconstituted in membrane lipids with a positively charged head group like DOTAP shifts the equilibrium to active Meta-II state. We propose a mechanism where membrane lipids with positively charged head group interact with Glu134 present on lipid-protein interface breaking the salt bridge with Arg135 in the conserved ERY motif. The interaction between Glu134 and the positively charged head group stabilizes the active Meta-II state. Our study gives insight on the role of membrane lipid-protein interactions in rhodopsin activation mechanism which can ultimately be extended to influenza virus membrane ion channels and other integral proteins. [1] A.V. Struts et al. (2015) Meth. Mol. Biol. 1271, 133-158. [2] E. Zaitseva et al. (2010) JACS 132, 4815-4821. [3] M.F. Brown (2012) Biochemistry 51, 9782-9795.

Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions on protein activity and the roles of membrane proteins in disease pathways.

Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

Cellular membrane deformation and the associated redistribution of membrane-bound proteins are important aspects of membrane function. Current model membrane approaches for studying curvature sensing are limited to positive curvatures and often require complex and delicate experimental setups. To overcome these challenges, we fabricated a wavy substrate by imposing a range of curvatures onto an adhering lipid bilayer membrane. We examined the curvature sorting of several peripheral proteins binding to the wavy membrane and observed them to partition into distinct regions of curvature. Furthermore, single-molecule imaging experiments suggested that the curvature sensing of proteins on low-curvature substrates requires cooperative interactions.

Eukaryotic cells contain many different membrane compartments with characteristic shapes, lipid compositions, and dynamics. A large fraction of cytoplasmic proteins associate with these membrane compartments. Such protein–lipid interactions, which regulate the subcellular localizations and activities of peripheral membrane proteins, are fundamentally important for a variety of cell biological processes ranging from cytoskeletal dynamics and membrane trafficking to intracellular signaling. Reciprocally, many membrane-associated proteins can modulate the shape, lipid composition, and dynamics of cellular membranes. Determining the exact mechanisms by which these proteins interact with membranes will be essential to understanding their biological functions. In this Technical Perspective, we provide a brief introduction to selected biochemical methods that can be applied to study protein–lipid interactions. We also discuss how important it is to choose proper lipid composition, type of model membrane, and biochemical assay to obtain reliable and informative data from the lipid-interaction mechanism of a protein of interest.

Critical to the use of solution NMR to describe the structure and flexibility of membrane proteins is the thorough understanding of the degree of perturbation induced by the detergent or other membrane mimetic. To develop a deeper understanding of the interaction between membrane proteins and micelles or bicelles, we will investigate the differences in structure and flexibility of a model membrane protein TM0026 from Thermotoga maritima using solution NMR. A comparison of the structural differences between TM0026 solubilized in different detergent combinations will provide important insight into the degree of modulation of membrane proteins by detergent physical properties. Here we report the nearly complete backbone and Cβ resonance assignments of the two transmembrane helical model protein TM0026. These assignments are the first step to using TM0026 to elucidate the interaction between membrane proteins and membrane mimetics.

Membrane proteins account for about 20–30 % of the protein-encoding genes in the genomes of all living organisms.2 Their fundamental importance in human health and disease is underlined by the fact that many drugs in current use act on them.3 Functional and structural studies of membrane proteins are therefore of increasing importance but more difficult and demanding than studies with soluble proteins, because membrane proteins function in the lipid bilayer of cell membranes. For in vitro studies, the proteins are first extracted from the membrane by detergent solubilization and then purified in detergent solution. Sensitive membrane proteins are often unstable in detergent solution, but quite stable once they are reconstituted into a lipid bilayer. Moreover, many membrane proteins require a lipid environment for activity. The reconstitution process, in which detergent is replaced by lipid, must be carefully controlled, as otherwise the proteins tend to denature and aggregate.4

We review the current state of membrane protein structure determination using solid-state nuclear magnetic resonance (NMR) spectroscopy. Multidimensional magic-angle-spinning correlation NMR combined with oriented-sample experiments has made it possible to measure a full panel of structural constraints of membrane proteins directly in lipid bilayers. These constraints include torsion angles, interatomic distances, oligomeric structure, protein dynamics, ligand structure and dynamics, and protein orientation and depth of insertion in the lipid bilayer. Using solid-state NMR, researchers have studied potassium channels, proton channels, Ca2+ pumps, G protein–coupled receptors, bacterial outer membrane proteins, and viral fusion proteins to elucidate their mechanisms of action. Many of these membrane proteins have also been investigated in detergent micelles using solution NMR. Comparison of the solid-state and solution NMR structures provides important insights into the effects of the solubilizing environment on membrane protein structure and dynamics.

Changes in inter-helical hydrogen bonding are associated with the conformational dynamics of membrane proteins. The function of the protein depends on the surrounding lipid membrane. Here we review through specific examples how dynamical hydrogen bonds can ensure an elegant and efficient mechanism of long-distance intra-protein and protein–lipid coupling, contributing to the stability of discrete protein conformational substates and to rapid propagation of structural perturbations. This article is part of a Special Issue entitled: Protein Folding in Membranes.

β-Barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They are important for pore formation, membrane anchoring, and enzyme activity. These proteins are also often responsible for bacterial virulence. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank. We have developed a computational method for predicting structures of the transmembrane (TM) domains of β-barrel membrane proteins. Based on physical principles, our method can predict structures of the TM domain of β-barrel membrane proteins of novel topology, including those from eukaryotic mitochondria. Our method is based on a model of physical interactions, a discrete conformational state space, an empirical potential function, as well as a model to account for interstrand loop entropy. We are able to construct three-dimensional atomic structure of the TM domains from sequences for a set of 23 nonhomologous proteins (resolution 1.8-3.0 Å). The median rmsd of TM domains containing 75-222 residues between predicted and measured structures is 3.9 Å for main chain atoms. In addition, stability determinants and protein-protein interaction sites can be predicted. Such predictions on eukaryotic mitochondria outer membrane protein Tom40 and VDAC are confirmed by independent mutagenesis and chemical cross-linking studies. These results suggest that our model captures key components of the organization principles of β-barrel membrane protein assembly.

Outer membrane β-barrel proteins differ from α-helical inner membrane proteins in lipid environment, secondary structure, and the proposed processes of folding and insertion. It is reasonable to expect that outer membrane proteins may contain primary sequence information specific for their folding and insertion behavior. In previous work, a depth-dependent insertion potential, Ez, was derived for α-helical inner membrane proteins. We have generated an equivalent potential for TM β-barrel proteins. The similarities and differences between these two potentials provide insight into unique aspects of the folding and insertion of β-barrel membrane proteins. This potential can predict orientation within the membrane and identify functional residues involved in intermolecular interactions.

Signals between a cell and its environment are often transmitted through membrane proteins; therefore, many membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels, are important drug targets. Structural information about membrane proteins remains limited owing to challenges in protein expression, purification and the selection of membrane-mimicking systems that will retain protein structure and function. This review describes recent advances in solution NMR applied to the structural study of integral membrane proteins. The examples herein demonstrate that solution NMR spectroscopy will play a unique role not only in structural analysis, but also drug discovery of membrane proteins.

The transfer free energies of the twenty natural amino acid side chains from water to phospholipid bilayers make a major contribution to the assembly and function of membrane proteins. Measurements of those transfer free energies will facilitate the identification of membrane protein sequences and aid in the understanding of how proteins interact with membranes during key biological events. We report the first water-to-bilayer transfer free energy scale (i.e., a “hydrophobicity scale”) for the twenty natural amino acid side chains measured in the context of a native transmembrane protein and a phospholipid bilayer. Our measurements reveal parity for apolar side-chain contributions between soluble and membrane proteins and further demonstrate that an arginine side-chain placed near the middle of a lipid bilayer is accommodated with much less energetic cost than predicted by molecular dynamics simulations.

The current view of the biological membrane is that in which lipids and proteins mutually interact to accomplish membrane functions. The lateral heterogeneity of the lipid bilayer can induce partitioning of membrane-associated proteins, favoring protein-protein interaction and influence signaling and trafficking. The Atomic Force Microscope allows to study the localization of membrane-associated proteins with respect to the lipid organization at the single molecule level and without the need for fluorescence staining. These features make AFM a technique of choice to study lipid/protein interactions in model systems or native membranes. Here we will review the technical aspects inherent to and the main results obtained by AFM in the study of protein partitioning in lipid domains concentrating in particular on GPI-anchored proteins, lipidated proteins, and transmembrane proteins. Whenever possible, we will also discuss the functional consequences of what has been imaged by Atomic Force Microscopy.

Previous studies have shown that cellular function depends on rod-like membrane proteins, among them Bin/Amphiphysin/Rvs (BAR) proteins may curve the membrane leading to physiologically important membrane invaginations and membrane protrusions. The membrane shaping induced by BAR proteins has a major role in various biological processes such as cell motility and cell growth. Different models of binding of BAR domains to the lipid bilayer are described. The binding includes hydrophobic insertion loops and electrostatic interactions between basic amino acids at the concave region of the BAR domain and negatively charged lipids. To shed light on the elusive binding dynamics, a novel experiment is proposed to expand the technique of single-molecule AFM for the traction of binding energy of a single BAR domain.

Phosphoinositides are a family of minority acidic phospholipids in cell membranes. Their principal role is instructional: they interact with proteins. Each cellular membrane compartment uses a characteristic species of phosphoinositide. This signature phosphoinositide attracts a specific complement of functionally important, loosely attached peripheral proteins to that membrane. For example, the phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane attracts phospholipase C, protein kinase C, proteins involved in membrane budding and fusion, proteins regulating the actin cytoskeleton, and others. Phosphoinositides also regulate the activity level of the integral membrane proteins. Many ion channels of the plasma membrane need the plasma-membrane-specific PIP2 to function. Their activity decreases when the abundance of this lipid falls, as for example after activation of phospholipase C. This behaviour is illustrated by the suppression of KCNQ K+ channel current by activation of M1 muscarinic receptors; KCNQ channels require PIP2 for their activity. In summary, phosphoinositides contribute to the selection of peripheral proteins for each membrane and regulate the activity of the integral proteins.

Integral membrane proteins play essential roles in many biological processes, such as energy transduction, transport of molecules, and signaling. The correct function of membrane proteins is likely to depend strongly on the chemical and physical properties of the membrane. However, membrane proteins are not accessible to many biophysical methods in their native cellular membrane. A major limitation for their functional and structural characterization is thus the requirement for an artificial environment that mimics the native membrane to preserve the integrity and stability of the membrane protein. Most commonly employed are detergent micelles, which can however be detrimental to membrane protein activity and stability. Here, we review recent developments for alternative, non-micellar solubilization techniques, with a particular focus on their application to solution NMR studies. We discuss the use of amphipols and lipid bilayer systems, such as bicelles and nanolipoprotein particles (NLPs). The latter show great promise for structural studies in near native membranes.