Surrounding Lipid Matrix Research Articles

Allosteric modulation of integral protein activity by differential stress in asymmetric membranes.

The activity of integral membrane proteins is tightly coupled to the properties of the surrounding lipid matrix. In particular, transbilayer asymmetry, a hallmark of all plasma membranes, might be exploited to control membrane-protein activity. Here, we hypothesized that the membrane-embedded enzyme outer membrane phospholipase A (OmpLA) is susceptible to the lateral pressure differences that build up between such asymmetric membrane leaflets. Upon reconstituting OmpLA into synthetic, chemically well-defined phospholipid bilayers exhibiting different lateral pressure profiles, we indeed observed a substantial decrease in the enzyme's hydrolytic activity with increasing membrane asymmetry. No such effects were observed in symmetric mixtures of the same lipids. To quantitatively rationalize how the differential stress in asymmetric lipid bilayers inhibits OmpLA, we developed a simple allosteric model within the lateral pressure framework. Thus, we find that membrane asymmetry can serve as the dominant factor in controlling membrane-protein activity, even in the absence of specific, chemical cues or other physical membrane determinants such as hydrophobic mismatch.

Alteration of lipid bilayer mechanics by volatile anesthetics: Insights from μs-long molecular dynamics simulations

SummaryVery few drugs in clinical practice feature the chemical diversity, narrow therapeutic window, unique route of administration, and reversible cognitive effects of volatile anesthetics. The correlation between their hydrophobicity and their potency and the increasing amount of evidence suggesting that anesthetics exert their action on transmembrane proteins, justifies the investigation of their effects on phospholipid bilayers at the molecular level, given the strong functional and structural link between transmembrane proteins and the surrounding lipid matrix. Molecular dynamics simulations of a model lipid bilayer in the presence of ethylene, desflurane, methoxyflurane, and the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (also called F6 or 2N) at different concentrations highlight the structural consequences of VA partitioning in the lipid phase, with a decrease of lipid order and bilayer thickness, an increase in overall lipid lateral mobility and area-per-lipid, and a marked reduction in the mechanical stiffness of the membrane, that strongly correlates with the compounds' hydrophobicity.

Exploring the in meso crystallization mechanism by characterizing the lipid mesophase microenvironment during the growth of single transmembrane α-helical peptide crystals.

The proposed mechanism for in meso crystallization of transmembrane proteins suggests that a protein or peptide is initially uniformly dispersed in the lipid self-assembly cubic phase but that crystals grow from a local lamellar phase, which acts as a conduit between the crystal and the bulk cubic phase. However, there is very limited experimental evidence for this theory. We have developed protocols to investigate the lipid mesophase microenvironment during crystal growth using standard procedures readily available in crystallography laboratories. This technique was used to characterize the microenvironment during crystal growth of the DAP12-TM peptide using synchrotron small angle X-ray scattering (SAXS) with a micro-sized X-ray beam. Crystal growth was found to occur from the gyroid cubic mesophase. For one in four crystals, a highly oriented local lamellar phase was observed, providing supporting evidence for the proposed mechanism for in meso crystallization. A new observation of this study was that we can differentiate diffraction peaks from crystals grown in meso, from peaks originating from the surrounding lipid matrix, potentially opening up the possibility of high-throughput SAXS analysis of in meso grown crystals.This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'.

Interaction of G protein coupled receptors and cholesterol

G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol. Some of them are functionally dependent on the specific presence of cholesterol. Over the last years, enormous progress on receptor structures has been achieved. While lipophilic ligands other than cholesterol have been shown to bind either inside the helix bundle or at the receptor-lipid interface, the binding site of cholesterol was either a single transmembrane helix or a groove between two or more transmembrane helices. A clear preference for one of the two membrane leaflets has not been observed. Not surprisingly, many hydrophobic residues (primarily leucine and isoleucine) were found to be involved in cholesterol binding. In most cases, the rough β-face of cholesterol contacted the transmembrane helix bundle rather than the surrounding lipid matrix. The polar hydroxy group of cholesterol was localized near the water-membrane interface with potential hydrogen bonding to residues in receptor loop regions. Although a canonical motif, designated as CCM site, was detected as a specific cholesterol binding site in case of the β2AR, this site was not found to be occupied by cholesterol in other GPCRs possessing the same motif. Cholesterol-receptor interactions can increase the compactness of the receptor structure and are able to enhance the conformational stability towards active or inactive receptor states. Overall, all current data suggest a high plasticity of cholesterol interaction sites in GPCRs.

Structural Determinants of Raft Partitioning for Single-Pass Transmembrane Proteins

Transmembrane proteins (TMPs) comprise ∼30% of the mammalian proteome and mediate nearly all functions of the cell. The subcellular localization, trafficking, signalling, and enzymatic activity of many TMPs is regulated by their association with lipid-driven lateral membrane domains known as lipid rafts. However, the structural determinants of TMP partitioning to raft domains remain almost unknown. We hypothesized that structural features of the transmembrane domain (TMD) of single-pass TMPs would guide raft partitioning and raft-dependent sub-cellular trafficking. To explore TMD-dependent raft partitioning, we isolated plasma membranes as Giant Plasma Membrane Vesicles (GPMVs), a model system that allows direct observation of protein partitioning between raft/non-raft phases by fluorescence microscopy. We generated 96 different fluorescent mutants of a model TMP and quantified their partitioning as a function of TMD features, namely hydrophobic length and accessible surface area. Raft partitioning was related to TMD length, with longer TMDs imparting greater raft association, confirming previously untested predictions. Investigation of 74 mutants with constant TMD length revealed that raft partitioning was also inversely related to the TMD accessible surface area, with larger surface areas preferring the non-raft phase. To explain these results, we present a simple physical model wherein raft partitioning is driven by differences in interfacial energy between the TMD and its surrounding lipid matrix in raft and non-raft phases. This model is in quantitative agreement with experimental observations and provides first predictions of protein-lipid surface tensions differences between coexisting membrane phases. The model could successfully connect predicted raft partitioning with subcellular localization of single-pass TMPs in Eukaryota. These results point the way to a general rule for raft partitioning of TMPs and underline the central role of membrane domains in cellular traffic.

The Biophysics of Living Membranes: Protein Partitioning and Functional Differentiation in Ordered Plasma Membrane Domains

The lipid raft hypothesis posits that ordered, lateral membrane domains are important for the regulation of plasma membrane (PM) structure and function in eukaryotes. Giant Plasma Membrane Vesicles (GPMVs) are isolated plasma membranes that microscopically separate into coexisting ordered and disordered phases, facilitating experimental analysis of the composition and physical properties of ordered raft domains in biological membranes. In these studies, we analyze the biophysical and lipidomic differentiation of the plasma membrane in Mesenchymal Stem Cells (MSC) as the cells undergo differentiation into adipocytes and osteoblasts. During differentiation, dramatic remodeling the PM lipidome, including lipid acyl chain length and unsaturation, results inchanges to membrane order and phase separation. These observations elucidate the compositional determinants of biophysical properties in biological membranes, as well as identify lineage-specific PM features. These results facilitate rational remodeling of membrane phenotypes to direct differentiation, as supplementation with ꙍ-3 docosahexaneoic acid (DHA) promoted the osteoblastic PM phenotype and potentiated osteogenic differentiation. The differences in the physical properties of the coexisting domains lead to preferential protein partitioning between them. We evaluate the structural determinants of raft partitioning of a model transmembrane protein, and find that raft phase partitioning is related to features of the protein's transmembrane domain (TMD), namely the hydrophobic length and surface area of the TMD. Longer TMDs impart greater raft association while TMDs with larger, bulkier amino acid side chains prefer the non-raft phase. We present a simple physical model wherein raft partitioning is driven by phase-dependent differences in interfacial energy between the TMD and its surrounding lipid matrix, and find excellent quantitative agreement with observations that provide the first predictions of protein-lipid surface tension. These results point the way to a general rule for raft partitioning of transmembrane proteins.

How Fast is Your Camera? Timescales for Molecular Motion and their Role in Restraining Molecular Dynamics

How Fast is Your Camera? Timescales for Molecular Motion and their Role in Restraining Molecular Dynamics

Binding of Endogenous Cannabinoid Ligand 2-Arachidonoylglycerol to Cannabinoid Receptor CB2 via the Lipid Matrix

Endogenous cannabinoid ligand, 2-arachidonoylglycerol (2-AG), binds to and activates cannabinoid type-2 receptor CB2, controlling a variety of immune and hematopoietic cellular functions. Due to the high lipophilicity of cannabinoid ligands, there have been extensive discussions that after nonspecific incorporation into membrane, approach of the ligands to the binding pocket of CB2 may take place via lateral diffusion in lipid matrix. While previously reported microsecond-long molecular dynamics simulations showed molecular details in favor of this mechanism, no in-situ experimental evidence for the binding event has yet been made. Here we report on 2H NMR results that show exchange of deuterated 2-AG-d5 between the ligand binding site on CB2 and the surrounding lipid matrix at physiological conditions. Purified, recombinant CB2 was functionally reconstituted into liposomes containing four-fold molar excess of 2-AG-d5, and its deuterium resonances measured over the temperature range of 5-20°C. When the membranes contained CB2, the ligand resonances shifted upfield by 0.5 ppm and broadened significantly. Despite the excess amount of ligand, only one set of time-averaged resonances of the ligand was observed. The result suggests that 2-AG undergoes exchange between receptor-bound and free states on millisecond timescale. The resonance linewidth decreased with decreasing temperature as predicted from reduced exchange rates. The receptor binding was specific since only well-resolved signal of 2-AG in lipid matrix was observed when the binding pocket was blocked with a high affinity synthetic agonist CP-55,940. The experiments contrast to our previous experiments on CP-55,940 binding, where the free state of 2H-labeled CP-55,940-d6 in lipid matrix showed a highly-resolved signal under much slower exchange rate. The present work emphasizes the critical role of lipid matrix to provide a pathway and to regulate approach of lipophilic cannabinoid ligands to the receptor binding pocket.

A Limited 4 Å Radial Displacement of the S4-S5 Linker Is Sufficient for Internal Gate Closing in Kv Channels

Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

Effect of sphingosine on domain morphology in giant vesicles

Sphingosine is a bioactive molecule which is known to participate in the regulation of a number of cellular processes such as apoptosis, cell differentiation, growth, etc. Sphingosine was observed to exhibit different domain morphology depending on the surrounding lipid matrix in biomimetic systems such as giant vesicles. Our current results showed that in a glycerophospholipid matrix sphingosine segregated in gel leaf-like domains whereas cholesterol presence increased its miscibility by melting gel domains in a concentration-dependent manner. Sphingosine and cholesterol did not form merging liquid domains on the micron scale as observed for sphingomyelin and cholesterol. However, we were able to visualize that sphingosine appears as a stabilizer and amplifier of domains in liquid-ordered phase by increasing the temperature of their formation and fraction. These results imply that sphingosine acts as a modulator of the lipid domain formation and thus it could exert its biological role, not only through direct binding to proteins, but also indirectly by influencing their sorting in membranes and modulating the processes of signal transduction.

Equilibrium Water Content in Native Vernix and Its Cellular Component

Vernix caseosa is a naturally occurring substance coating the skin of newborn humans. Structurally, vernix contains fetal corneocytes embedded in a hydrophobic lipid matrix. Despite a relatively high water content approximating 80.7%, vernix exhibits slow water release. In this study, we quantified and contrasted the water release and uptake properties of native vernix and its isolated cellular component over the full range of water activity. Theoretical water sorption models (D'Arcy-Watt, and Frenkel-Halsey-Hill (FHH), and Guggenheim-Anderson-de Boer (GAB)) were fit to the vernix water sorption data. Each of the theoretical models provided a satisfactory description of the equilibrium water content of vernix over the water activity range 0.15-1.0. Vernix corneocytes without the surrounding lipid matrix exhibited markedly increased equilibrium water binding at water activities greater than 0.62 compared to native vernix. Resorption experiments showed full recovery of water content in both native vernix and isolated corneocytes supporting a structured internal domain. These results provide the first quantitative characterization of the water handling properties of native vernix and its cellular component. Such information may prove useful in the design of alternative skin care moisturizing formulations.

Coupling between vesicle shape and lateral distribution of mobile membrane inclusions

Membrane inclusions such as membrane-embedded peptides or proteins exhibit a curvature-dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. This interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. We have studied theoretically the axisymmetric equilibrium shapes of lipid vesicles with mobile inclusions, taking into account that the membrane free energy includes the elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction. Equations describing the shape are derived by minimizing the total free energy at fixed membrane area, enclosed volume, and number of inclusions and are then solved numerically. It is shown that vesicle shape may assume a symmetry that differs from that of the vesicle with no inclusions. If the inclusion-membrane interaction exceeds a certain value, there is no axisymmetric solution of the equations with a continuous and derivable lateral density of inclusions over the whole area of the vesicle. When approaching the critical vesicle shape, the shapes obtained differ qualitatively from those described by the area difference elasticity model of the elastic properties of lipid membranes. In general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in a way such that the lateral distribution of inclusions becomes inhomogeneous.

Structure of bacteriorhodopsin at 1.55 Å resolution

The atomic structure of the light-driven ion pump bacteriorhodopsin and the surrounding lipid matrix was determined by X-ray diffraction of crystals grown in cubic lipid phase. In the extracellular region, an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base and the proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a π-bulge that causes a non-proline? kink. The bulge is stabilized by hydrogen-bonding of the main-chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The results indicate extensive involvement of bound water molecules in both the structure and the function of this seven-helical membrane protein. A bilayer of 18 tightly bound lipid chains forms an annulus around the protein in the crystal. Contacts between the trimers in the membrane plane are mediated almost exclusively by lipids.

A theoretical model for the association of amphiphilic transmembrane peptides in lipid bilayers

A theoretical model is proposed for the association of trans-bilayer peptides in lipid bilayers. The model is based on a lattice model for the pure lipid bilayer, which accounts accurately for the most important conformational states of the lipids and their mutual interactions and statistics. Within the lattice formulation the bilayer is formed by two independent monolayers, each represented by a triangular lattice, on which sites the lipid chains are arrayed. The peptides are represented by regular objects, with no internal flexibility, and with a projected area on the bilayer plane corresponding to a hexagon with seven lattice sites. In addition, it is assumed that each peptide surface at the interface with the lipid chains is partially hydrophilic, and therefore interacts with the surrounding lipid matrix via selective anisotropic forces. The peptides would therefore assemble in order to shield their hydrophilic residues from the hydrophobic surroundings. The model describes the self-association of peptides in lipid bilayers via lateral and rotational diffusion, anisotropic lipid-peptide interactions, and peptide-peptide interactions involving the peptide hydrophilic regions. The intent of this model study is to analyse the conditions under which the association of trans-bilayer and partially hydrophilic peptides (or their dispersion in the lipid matrix) is lipid-mediated, and to what extent it is induced by direct interactions between the hydrophilic regions of the peptides. The model properties are calculated by a Monte Carlo computer simulation technique within the canonical ensemble. The results from the model study indicate that direct interactions between the hydrophilic regions of the peptides are necessary to induce peptide association in the lipid bilayer in the fluid phase. Furthermore, peptides within each aggregate are oriented in such a way as to shield their hydrophilic regions from the hydrophobic environment. The average number of peptides present in the aggregates formed depends on the degree of mismatch between the peptide hydrophobic length and the lipid bilayer hydrophobic thickness: The lower the degree of mismatch is the higher this number is.

Membrane‐linked digestion in fish

Membrane‐linked digestion (MLD) processes were first described in 1959. These processes are closely linked to specific histological structures that can be seen in the brush border area of animals from several different classes. The brush border found in different types of fish permits the digestion process that takes place in the intestine to be increased by a factor of approximately 40. This specific digestive structure contains many enzymes that are found in the microvilli proper, such as alkaline phosphatase, maltase, saccharase and several dipeptidases, and enzymes that are adsorbed to the microvilli, such as α‐amylase, carboxypeptidase, and several lipases. There is a strong correlation between the specific food intake and the activity of the MLD. In predatory fish, proteinases predominate; in bentho‐or planktonophages, carbohydrases are the predominant enzymes. Seasonal changes in enzymatic activities have been detected, especially in fish of boreal regions. Adaptive changes to environmental temperature are described; these are the end result of basic structural changes and changes in the surrounding lipid matrix. The energy of activation of different enzymes changes at different temperatures and is a good indicator of adaptation to environmental conditions. The process of MLD plays a central role in all digestive systems.

Formation of non-bilayer structures induced by M13 coat protein depends on the conformation of the protein

A comparison is made of the interaction of the coat protein of bacteriophage M13 in a predominant α-helix conformation and in a predominant β-sheet conformation. To perform a systematic study of the interaction between the protein in these two different forms and the surrounding lipid matrix. NMR spectra of 2H-nuclei of specific labelled phospholipid systems are measured. In addition 31P-NMR is employed to provide information about the morphological structure adopted by the reconstituted lipid/protein systems. From the 2H-NMR studies on specific headgroup and chain deuterium labelled phospholipids it is found that the protein in the predominant β-sheet conformation causes of fraction of lipids to be trapped. By combining the results from the headgroup and acyl chains of the phospholipids, it is concluded that the trapped lipids are arranged in a non-bilayer structure, probably caused by a misfitting of the hydrophobic core of the protein and the membrane bilayer. The protein in the predominant α-helix conformation perfectly fits in the lipid bilayer and has only minor influences on the surrounding lipid matrix. A new model is proposed to explain the presence of the trapped lipids in the lipid/protein systems.