Association In Membranes Research Articles

A thermodynamic analysis of CLC transporter dimerization in lipid bilayers

The CLC-ec1 chloride/proton antiporter is a membrane-embedded homodimer with subunits that can dissociate and associate, but the thermodynamic driving forces favor the assembled dimer at biological densities. Yet, the physical reasons for this stability are confounding as dimerization occurs via the burial of hydrophobic interfaces away from the lipid solvent. For binding of nonpolar surfaces in aqueous solution, the driving force is often attributed to the hydrophobic effect, but this should not apply in the membrane since there is very little water. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG°. To ensure that the reaction reached equilibrium at different temperatures, we utilized a Förster resonance energy transfer assay to report on relaxation kinetics of subunit exchange as a function of temperature. Equilibration times were then applied to measure CLC-ec1 dimerization isotherms at different temperatures using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in Escherichia coli-like membranes exhibits a nonlinear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects such as the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the nonbilayer defect required to solvate the monomeric state is one source of the observed change in heat capacity and indicates the existence of a generalizable driving force for protein association in membranes.

Building a molecular model for ion channel and transporter stability in membranes through a partnership of experiments and computation.

Ion channels and transporters are the molecular gatekeepers of biology, governing the passage of atoms and molecules in and out of cells. They provide essential nutrients for metabolism, eliminate waste, enable cell-to-cell communication and store the potential energy that fuels life. Yet, despite their overwhelming importance, we still lack a physical and molecular understanding of why membrane transport proteins form stable structures that enable their specific functions within the oil-filled environment of the lipid bilayer. Our lack of understanding here is well rationalized by the inherent challenge of measuring equilibrium reactions of membrane protein assembly in membranes, compounded by the complexity of the reaction solvent as a structured lipid bilayer. However, through a combination of novel experimental single-molecule microscopy approaches coupled with coarse-grained and all-atom computational modeling, we can now measure equilibrium constants of protein association in membranes and dissect out the important molecular contributions from the protein and surrounding membrane. Here, I present our results on two systems where reversible binding within membranes can be quantitatively assessed - the homodimeric CLC-ec1 chloride/proton antiporter and the dual-topology homodimeric Fluc fluoride ion channel. By combining experimental and computational studies, we identify a driving force for protein association that arises from state-dependent perturbation of the membrane structure and a mechanism for tuning dimerization stability by altering solvation energetics through preferential solvation. These results provide generalizable mechanisms that drive membrane protein assembly in membranes that are expected to apply to all ion channels and transporters.

On Computing Equilibrium Binding Constants for Protein-Protein Association in Membranes.

Protein association in lipid membranes is fundamental to membrane protein function and of great biomedical relevance. All-atom and coarse-grained models have been extensively used to understand the protein-protein interactions in the membrane and to compute equilibrium association constants. However, slow translational and rotational diffusion of protein in membrane presents challenges to the effective sampling of conformations defining the ensembles of free and bound states contributing to the association equilibrium and the free energy of dimerization. We revisit the homodimerization equilibrium of the TM region of glycophorin A. Conformational sampling is performed using umbrella sampling along previously proposed one-dimensional collective variables and compared with sampling over a two-dimensional collective variable space using the MARTINI v2.2 force field. We demonstrate that the one-dimensional collective variables suffer from restricted sampling of the native homodimer conformations leading to a biased free energy landscape. Conversely, simulations along the two-dimensional collective variable effectively characterize the thermodynamically relevant native and non-native interactions contributing to the association equilibrium. These results demonstrate the challenges associated with accurately characterizing binding equilibria when multiple poses contribute to the bound state ensemble.

Temperature Dependency of CLC-ec1 Dimerization in Lipid Bilayers

Why do greasy membrane proteins form stable complexes via greasy binding interfaces in the similarly greasy lipid solvent? To answer this question, we must quantify the thermodynamic factors that drive membrane protein association in membranes. Previously, we found that the CLC-ec1 Cl-/H+ antiporter participates in an equilibrium dimerization reaction in 2:1 POPE:POPG lipid bilayers. The dimer assembles via a large membrane embedded interface lined by non-polar residues, and the complex is stable, exhibiting a free energy of dimerization of −11 kcal/mole relative to the subunit/lipid standard state. Thus, CLC-ec1 provides an ideal model system for studying this question. In the current study, we carried out a van't Hoff analysis of the CLC-ec1 (WT) dimerization equilibrium in lipid bilayers. To verify that we are studying the equilibrated system, we examined subunit-exchange kinetics between WT-Cy3, and WT-Cy5 EPL (E. coli polar lipid) proteoliposomes, fused to form multi-lamellar membranes. Mixing of subunits led to formation of heterodimers (WT-Cy3/WT-Cy5) and the resulting increase in bulk Förster Resonance Energy Transfer (FRET) was monitored as a function of time, and temperature (22-56°C). Additionally, temperature jump type experiments conducted for the co-labelled WT showed changes in FRET, with both fused and co-labelled samples converging to the same FRET- plateaus. We found that rate of subunit-exchange increases with temperature, and that FRET- plateau decreases above 44oC indicating a decrease in oligomerization, with the chloride transport function remaining unchanged across the same temperature range. To measure the full dimerization binding isotherm, we equilibrated WT-Cy5 in EPL membranes at various temperatures and carried out subunit capture single-molecule photobleaching analysis for measurement of Keq(T). Finally, a van't Hoff plot of ln(Keq) vs. 1/T is presented, allowing a thermodynamic dissection of enthalpy, entropy, and heat capacity changes associated with CLC dimerization in membranes.

Multisite reversible association in membranes and solutions: From non-Markovian to Markovian kinetics.

The role of diffusion on the kinetics of reversible association to a macromolecule with two inequivalent sites is studied. Previously, we found that, in the simplest possible description, it is not sufficient to just renormalize the rate constants of chemical kinetics, but one must introduce direct transitions between the bound states in the kinetic scheme. The physical reason for this is that a molecule that just dissociated from one site can directly rebind to the other rather than diffuse away into the bulk. Such a simple description is not valid in two dimensions because reactants can never diffuse away into the bulk. In this work, we consider a variety of more sophisticated implementations of our recent general theory that are valid in both two and three dimensions. We compare the predicted time dependence of the concentrations for a wide range of parameters and establish the range of validity of various levels of the general theory.

The Biology of Gangliosides.

Gangliosides comprise a varied family of glycosphingolipid structures bearing one or more sialic acid residues. They are found in all mammalian tissues but are most abundant in the brain, where they represent the quantitatively major class of sialoglycans. As prominent molecular determinants on cell surfaces, they function as molecular-recognition partners for diverse glycan-binding proteins ranging from bacterial toxins to endogenous cell-cell adhesion molecules. Gangliosides also regulate the activity of plasma membrane proteins, including protein tyrosine kinases, by lateral association in the same membranes in which they reside. Their roles in molecular recognition and membrane protein regulation implicate gangliosides in human physiology and pathology, including infectious diseases, diabetes, cancer, and neurodegeneration. The varied structures and biosynthetic pathways of gangliosides are presented here, along with representative examples of their biological functions in health and disease.

A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers.

The thermodynamic reasons why membrane proteins form stable complexes inside the hydrophobic lipid bilayer remain poorly understood. This is largely because of a lack of membrane-protein systems amenable for equilibrium studies and a limited number of methods for measuring these reactions. Recently, we reported the equilibrium dimerization of the CLC-ec1 Cl-/H+ transporter in lipid bilayers (Chadda et al. 2016. eLife https://doi.org/10.7554/eLife.17438), which provided a new type of model system for studying protein association in membranes. The measurement was conducted using the subunit-capture approach, involving passive dilution of the protein in large multilamellar vesicles, followed by single-molecule photobleaching analysis of the Poisson distribution describing protein encapsulation into extruded liposomes. To estimate the fraction of dimers (FDimer ) as a function of protein density, the photobleaching distributions for the nonreactive, ideal monomer and dimer species must be known so that random co-capture probabilities can be accounted for. Previously, this was done by simulating the Poisson process of protein reconstitution into a known size distribution of liposomes composed of Escherichia coli polar lipids (EPLs). In the present study, we investigate the dependency of FDimer and ΔG° on the modeling through a comparison of different liposome size distributions (EPL versus 2:1 POPE/POPG). The results show that the estimated FDimer values are comparable, except at higher densities when liposomes become saturated with protein. We then develop empirical controls to directly measure the photobleaching distributions of the nonreactive monomer (CLC-ec1 I201W/I422W) and ideal dimer (WT CLC-ec1 cross-linked by glutaraldehyde or CLC-ec1 R230C/L249C cross-linked by a disulfide bond). The measured equilibrium constants do not depend on the correction method used, indicating the robustness of the subunit-capture approach. This strategy therefore presents a model-free way to quantify protein dimerization in lipid bilayers, offering a simplified strategy in the ongoing effort to characterize equilibrium membrane-protein reactions in membranes.

Measuring the Free Energy of ClC-ec1 Dimerization in Membranes using Single Molecule Photobleaching Analysis

Why do greasy membrane proteins interact with their greasy protein partners instead of the similarly greasy lipid bilayer? This is a question at the root of membrane protein oligomerization and folding in membranes. Recently, we turned to the homodimeric ClC-ec1 Cl-/H+ antiporter, in order to develop a new model system that can be used to investigate this question. The ClC-ec1 dimerization interface is large (1200 A2) and lined by ∼20 non-polar residues, forming an interaction surface exhibiting high shape complementarity. Previous studies showed that ClC-ec1 is folded and functional in both monomeric and dimeric states. Using a single-molecule microscopy approach, we measured the photobleaching distributions across a wide range of subunit/lipid dilutions, with the lowest representing a sub-biological condition of 1 subunit per 40 E. coli membranes. The data shows that wild-type ClC-ec1 follows a monomer to dimer reaction that is reversible and fits to an equilibrium isotherm, allowing for determination of the dimerization free energy in 2:1 POPE/POPG lipid bilayers (Chadda et al., eLife 2016). Next, two approaches were used to investigate how residues at the interaction interface influence the free energy of dimerization. First, bulky TRP residues were introduced resulting in a destabilization of the dimer by 2 kcal/mole per TRP. Second, subtractive substitutions to ALA were constructed introducing cavities at the interface. While most of the ALA substitutions appear to have no effect, L194A destabilizes the dimer by 2 kcal/mole in lipid bilayers while maintaining both fold and function. These studies present ClC-ec1 as a robust model system for probing the physical forces driving protein association in membranes.

Synthesis of Hydrogen-Bonded Pore-Switchable Cylindrical Vesicles via Visible-Light-Mediated RAFT Room-Temperature Aqueous Dispersion Polymerization

Analogous to cellulose, polymers whose monomer units possess both hydrogen donators and acceptors are generally insoluble in ambient water because of hydrogen bonding (HB). Herein we present stimuli-responsive long aqueous cylindrical vesicles (nanotubes) synthesized directly using HB-driven polymerization-induced self-assembly (PISA) under visible-light-mediated RAFT aqueous dispersion polymerization at 25 °C. The PISA undergoes an unprecedented film/silk-to-ribbon-to-vesicle transition and films/silks/ribbons formed at low DPs (∼25-85) of core-forming block in free-flowing aqueous solution. Pore-switchable nanotubes are synthesized by electrostatic repulsive perturbation of the HB association in anisotropic vesicular membranes via inserting minor ionized monomer units into the core-forming block. These nanotubes are synthesized at >35% solids, and tubular membranes are more sensitive than spherical counterparts in response to aqueous surroundings. This facile, robust, and general strategy paves a new avenue toward scale-up production of advanced intelligent nanomaterials.

The dimerization equilibrium of a ClC Cl(-)/H(+) antiporter in lipid bilayers.

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states - monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.

Adaptable Lipid Matrix Promotes Protein-Protein Association in Membranes.

The cell membrane is "stuffed" with proteins, whose transmembrane (TM) helical domains spontaneously associate to form functionally active complexes. For a number of membrane receptors, a modulation of TM domains' oligomerization has been shown to contribute to the development of severe pathological states, thus calling for detailed studies of the atomistic aspects of the process. Despite considerable progress achieved so far, several crucial questions still remain: How do the helices recognize each other in the membrane? What is the driving force of their association? Here, we assess the dimerization free energy of TM helices along with a careful consideration of the interplay between the structure and dynamics of protein and lipids using atomistic molecular dynamics simulations in the hydrated lipid bilayer for three different model systems - TM fragments of glycophorin A, polyalanine and polyleucine peptides. We observe that the membrane driven association of TM helices exhibits a prominent entropic character, which depends on the peptide sequence. Thus, a single TM peptide of a given composition induces strong and characteristic perturbations in the hydrophobic core of the bilayer, which may facilitate the initial "communication" between TM helices even at the distances of 20-30 Å. Upon tight helix-helix association, the immobilized lipids accommodate near the peripheral surfaces of the dimer, thus disturbing the packing of the surrounding. The dimerization free energy of the modeled peptides corresponds to the strength of their interactions with lipids inside the membrane being the lowest for glycophorin A and similarly higher for both homopolymers. We propose that the ability to accommodate lipid tails determines the dimerization strength of TM peptides and that the lipid matrix directly governs their association.

Counting Membrane Proteins in Liposomes for Single-Molecule Microscopy

We can learn a lot by studying membrane proteins using single-molecule techniques. For example, electrical recordings of single ion channels have provided a wealth of information about key functional states. To obtain structural information, fluorescence microscopy can be used to monitor single-molecule Forster resonance energy transfer revealing dynamic conformational details. Experiments of this sort are being done but usually examine the protein in detergent micelles rather than a lipid environment. However, for many questions, studying the protein in lipid membranes is essential. While it is possible to carry out microscopy on proteins in supported membranes, formation of the bilayer can be complicated and often limits the lipid conditions. Another method is to simply examine the protein in liposomes, the benefits being that it is quick, easy and reconstitution is robust under different lipid compositions. In order to prepare this approach for single-molecule studies, we must establish the conditions in which we have a single protein molecule per liposome. Reconstitution statistics follows a Poisson distribution, which can be measured as a function of protein density and liposome size. Here we measure the size distributions of liposomes formed with E. Coli polar lipids and those containing mixtures of phosphatidyl-ethanolamine (PE), -glycerol (PG) and -choline (PC), by cryo-electron microscopy and fluorescence intensity of Alexa-488 labelled PE by total internal reflection fluorescence microscopy. The protein occupancy is measured as a function of protein density by photo-bleaching analysis of the Cl-/H+ transporter CLC-ec1 labelled with Cyanine-5. The results provide a foundation for future single-molecule studies of membrane proteins, such as the identification of conformational changes as well as the determination of protein stoichiometry with respect to studying the thermodynamics of protein association in membranes.

Self-Association of Transmembrane Domains of ErbB2 Receptors in Cholesterol-Containing Membranes

The transmembrane domain of ErbB2 receptors presents two separate GxxxG motifs that are proposed to be connected to stability and activity of the dimer. Recently developed parallel Monte Carlo methods are employed to study the association of ErbB2 TM domains in cholesterol-containing membranes with coarse-grained models that retain a level of amino-acid specificity. Extensive sampling along separation between the two helices shows that GxxxG motifs play a critical role during the recognition stage. In pure phospholipid bilayers association occurs by contacts formed at the C-terminus promoted by the presence of phenylalanine residues. Helices subsequently rotate to eventually form a stable dimer favored by lipid entropic contributions. In contrast, at intermediate cholesterol concentrations a different pathway is followed that involves dimers with a weaker interface towards the N-terminus. However, at high cholesterol content, a switch towards the C-terminus is observed with an overall non-monotonic change of the dimerization affinity. This conformational switch modulated by cholesterol has important implications on the thermodynamic, structural and kinetic characteristics of helix-helix association in lipid membranes.[1] Janosi L. and Doxastakis M., “Accelerating flat-histogram methods for potential of mean force calculations”, J. Chem. Phys., 131, 054105 (2009)[2] Janosi L., Prakash A. and Doxastakis M., “Lipid-Modulated sequence-specific association of Glycophorin A in membranes”, Biophys. J., 99, 284-292 (2010)[3] Prakash A., Janosi L. and Doxastakis M., “Self-association of models of transmembrane domains of ErbB receptors in a lipid bilayer”, Biophys. J., 99, 3657-3665 (2010)[4] Prakash A., Janosi L. and Doxastakis M., “GxxxG motifs, phenylalanine and cholesterol guide the self-association of transmembrane domains of ErbB2 receptors”, Biophys. J., in press

GxxxG Motifs, Phenylalanine, and Cholesterol Guide the Self-Association of Transmembrane Domains of ErbB2 Receptors

GxxxG motifs are common in transmembrane domains of membrane proteins and are often introduced to artificial peptides to inhibit or promote association to stable structures. The transmembrane domain of ErbB2 presents two separate such motifs that are proposed to be connected to stability and activity of the dimer. Using molecular simulations, we show that these sequences play a critical role during the recognition stage, forming transient complexes that lead to stable dimers. In pure phospholipid bilayers association occurs by contacts formed at the C-terminus promoted by the presence of phenylalanine residues. Helices subsequently rotate to eventually pack at short separations favored by lipid entropic contributions. In contrast, at intermediate cholesterol concentrations, a different pathway is followed that involves dimers with a weaker interface toward the N-terminus. However, at high cholesterol content, a switch toward the C-terminus is observed with an overall nonmonotonic change of the dimerization affinity. This conformational switch modulated by cholesterol has important implications on the thermodynamic, structural, and kinetic characteristics of helix-helix association in lipid membranes.

Phase transitions and spatially ordered counterion association in ionic-lipid membranes: A statistical model

We propose a statistical model to account for the gel-fluid anomalous phase transitions in charged bilayer- or lamellae-forming ionic lipids. The model Hamiltonian comprises effective attractive interactions to describe neutral-lipid membranes as well as the effect of electrostatic repulsions of the discrete ionic charges on the lipid headgroups. The latter can be counterion dissociated (charged) or counterion associated (neutral), while the lipid acyl chains may be in gel (low-temperature or high-lateral-pressure) or fluid (high-temperature or low-lateral-pressure) states. The system is modeled as a lattice gas with two distinct particle types--each one associated, respectively, with the polar-headgroup and the acyl-chain states--which can be mapped onto an Ashkin-Teller model with the inclusion of cubic terms. The model displays a rich thermodynamic behavior in terms of the chemical potential of counterions (related to added salt concentration) and lateral pressure. In particular, we show the existence of semidissociated thermodynamic phases related to the onset of charge order in the system. This type of order stems from spatially ordered counterion association to the lipid headgroups, in which charged and neutral lipids alternate in a checkerboard-like order. Within the mean-field approximation, we predict that the acyl-chain order-disorder transition is discontinuous, with the first-order line ending at a critical point, as in the neutral case. Moreover, the charge order gives rise to continuous transitions, with the associated second-order lines joining the aforementioned first-order line at critical end points. We explore the thermodynamic behavior of some physical quantities, like the specific heat at constant lateral pressure and the degree of ionization, associated with the fraction of charged lipid headgroups.

MHC Class II and CD9 in Human Eosinophils Localize to Detergent-Resistant Membrane Microdomains

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.

Eye lens membrane junctional microdomains: a comparison between healthy and pathological cases

The eye lens is a transparent tissue constituted of tightly packed fiber cells. To maintain homeostasis and transparency of the lens, the circulation of water, ions and metabolites is required. Junctional microdomains connect the lens cells and ensure both tight cell-to-cell adhesion and intercellular flow of fluids through a microcirculation system. Here, we overview membrane morphology and tissue functional requirements of the mammalian lens. Atomic force microscopy (AFM) has opened up the possibility of visualizing the junctional microdomains at unprecedented submolecular resolution, revealing the supramolecular assembly of lens-specific aquaporin-0 (AQP0) and connexins (Cx). We compare the membrane protein assembly in healthy lenses with senile and diabetes-II cataract cases and novel data of the lens membranes from a congenital cataract. In the healthy case, AQP0s form characteristic square arrays confined by connexons. In the cases of senile and diabetes-II cataract patients, connexons were degraded, leading to malformation of AQP0 arrays and breakdown of the microcirculation system. In the congenital cataract, connexons are present, indicating probable non-membranous grounds for lens opacification. Further, we discuss the energetic aspects of the membrane organization in junctional microdomains. The AFM hence becomes a biomedical nano-imaging tool for the analysis of single-membrane protein supramolecular association in healthy and pathological membranes.

Lipid-Modulated Sequence-Specific Association of Glycophorin A in Membranes

Protein association in lipid membranes is a complex process with thermodynamics directed by a multitude of different factors. Amino-acid sequence is a molecular parameter that affects dimerization as shown by limited directed mutations along the transmembrane domains. Membrane-mediated interactions are also important although details of such contributions remain largely unclear. In this study, we probe directly the free energy of association of Glycophorin A by means of extensive parallel Monte Carlo simulations with recently developed methods and a model that accounts for sequence-specificity while representing lipid membranes faithfully. We find that lipid-induced interactions are significant both at short and intermediate separations. The ability of molecules to tilt in a specific hydrophobic environment extends their accessible interfaces, leading to intermittent contacts during protein recognition. The dimer with the lowest free energy is largely determined by the favorable lipid-induced attractive interactions at the closest distance. Finally, the coarse-grained model employed herein, together with the extensive sampling performed, provides estimates of the free energy of association that are in excellent agreement with existing data.

Computer simulations and modeling-assisted ToxR screening in deciphering 3D structures of transmembrane α-helical dimers: ephrin receptor A1

Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide–peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix–helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active ‘peptide interceptors’ specifically targeting membrane domains of proteins.

Tracking membrane protein association in model membranes.

Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue.We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well.After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 Å, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a stoichiometry for the complex that exhibits a strong pH dependance: from 2 to 6 MexA per OprM trimer when the pH decreases from 7.5 to 5.5.Our technique allows to study membrane protein associations in a membrane environment. It provides some challenging information about complexes such as geometry and stoichiometry.