Adaptor proteins are a class of cytoplasmic proteins that bind to phosphorylated residues in receptor tyrosine kinases and trigger signaling cascades that control critically important cellular processes, such as cell survival, growth, differentiation, and motility. Here, we seek to characterize the interaction between epidermal growth factor receptor (EGFR) and the cytoplasmic adaptor protein growth factor receptor-bound protein 2 (Grb2) in a cellular context. To do so, we explore the utility of a highly biologically relevant model system, mammalian cells under reversible osmotic stress, and a recently introduced Förster resonance energy transfer microscopy method, fully quantified spectral imaging. We present a method that allows us to quantify the stoichiometry and the association constant of the EGFR-Grb2 binding interaction in the plasma membrane, in the presence and absence of activating ligand. The method that we introduce can have broad utility in membrane protein research, as it can be applied to different membrane protein-cytoplasmic protein pairs.

•Eadherin is a constitutive dimer under physiological expression conditions.•Ecadherin dimer is stabilized by contacts along the entire length of the protein.•The intracellular domains have a large favorable contribution to dimer stability.•The intracellular domain contribution is independent of soluble proteins or actin.Epithelial cadherin (Ecadherin) is responsible for the intercellular cohesion of epithelial tissues. It forms lateral clusters within adherens cell–cell junctions, but its association state outside these clusters is unknown. Here, we use a quantitative Forster resonance energy transfer (FRET) approach to show that Ecadherin forms constitutive dimers and that these dimers exist independently of the actin cytoskeleton or cytoplasmic proteins. The dimers are stabilized by intermolecular contacts that occur along the entire length of Ecadherin, with the intracellular domains having a surprisingly strong favorable contribution. We further show that Ecadherin mutations and calcium depletion induce structural alterations that propagate from the N terminus all the way to the C terminus, without destabilizing the dimeric state. These findings provide context for the interpretation of Ecadherin adhesion experiments. They also suggest that early events of adherens junction assembly involve interactions between from preformed Ecadherin dimers.Download high-res image (249KB)Download full-size image

pH-triggered membrane-permeabilizing peptides could be exploited in a variety of applications, such as to enable cargo release from endosomes for cellular delivery, or as cancer therapeutics that selectively permeabilize the plasma membranes of malignant cells. Such peptides would be especially useful if they could enable the movement of macromolecules across membranes, a rare property in membrane-permeabilizing peptides. Here we approach this goal by using an orthogonal high-throughput screen of an iterative peptide library to identify peptide sequences that have the following two properties: (i) little synthetic lipid membrane permeabilization at physiological pH 7 at high peptide concentration and (ii) efficient formation of macromolecule-sized defects in synthetic lipid membranes at acidic pH 5 and low peptide concentration. The peptides we selected are remarkably potent macromolecular sized pore-formers at pH 5, while having little or no activity at pH 7, as intended. The action of these peptides likely relies on tight coupling between membrane partitioning, α-helix formation, and electrostatic repulsions between acidic side chains, which collectively drive a sharp pH-triggered transition between inactive and active configurations with apparent pKa values of 5.5–5.8. This work opens new doors to developing applications that utilize peptides with membrane-permeabilizing activities that are triggered by physiologically relevant decreases in pH.

Here we introduce the fully quantified spectral imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling.

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves.Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic single amino acid mutations that cause skeletal and cranial dysplasias, as well as cancer, we also study the effects of these mutations on dimerization. First, we show that the A391E mutation, linked to Crouzon syndrome with acanthosis nigricans and to bladder cancer, significantly enhances FGFR3 dimerization in the absence of ligand and thus induces aberrant receptor interactions. Second, we present results about the effect of three cysteine mutations that cause thanatophoric dysplasia, a lethal phenotype. Such cysteine mutations have been hypothesized previously to cause constitutive dimerization, but we find instead that they have a surprisingly modest effect on dimerization. Most of the studied pathogenic mutations also altered FGFR3 dimer structure, suggesting that both increases in dimerization propensities and changes in dimer structure contribute to the pathological phenotypes. The results acquired with the QI-FRET method further our understanding of the interactions between FGFR3 molecules and RTK molecules in general. Since RTK dimerization regulates RTK signaling, our findings advance our knowledge of RTK activity in health and disease. The utility of the QI-FRET method is not restricted to RTKs, and we thus hope that in the future the QI-FRET method will be applied to other classes of membrane proteins, such as channels and G protein-coupled receptors.

Peptides that self-assemble, at low concentration, into bilayer-spanning pores which allow the passage of macromolecules would be beneficial in multiple areas of biotechnology. However, there are few, if any, natural or designed peptides that have this property. Here we show that the 26-residue peptide “MelP5”, a synthetically evolved gain-of-function variant of the bee venom lytic peptide melittin identified in a high-throughput screen for small molecule leakage, enables the passage of macromolecules across bilayers under conditions where melittin and other pore-forming peptides do not. In surface-supported bilayers, MelP5 forms unusually high conductance, equilibrium pores at peptide:lipid ratios as low as 1:25000. The increase in bilayer conductance due to MelP5 is dramatically higher, per peptide, than the increase due to the parent sequence of melittin or other peptide pore formers. Here we also develop two novel assays for macromolecule leakage from vesicles, and we use them to characterize MelP5 pores in bilayers. We show that MelP5 allows the passage of macromolecules across vesicle membranes at peptide:lipid ratios as low as 1:500, and under conditions where neither osmotic lysis nor gross vesicle destabilization occur. The macromolecule-sized, equilibrium pores formed by MelP5 are unique as neither melittin nor other pore-forming peptides release macromolecules significantly under the same conditions. MelP5 thus appears to belong to a novel functional class of peptide that could form the foundation of multiple potential biotechnological applications.

Plasma membrane derived vesicles are used as a model system for the biochemical and biophysical investigations of membrane proteins and membrane organization. The most widely used vesiculation procedure relies on formaldehyde and dithiothreitol (DTT), but these active chemicals may introduce artifacts in the experimental results. Here we describe a procedure to vesiculate Chinese hamster ovary (CHO) cells, widely used for the expression of recombinant proteins, using a hypertonic vesiculation buffer containing chloride salts and no formaldehyde or DTT. We characterize the size distribution of the produced vesicles. We also show that these vesicles can be used for the biophysical characterization of interactions between membrane proteins.

Here we present a highly efficient protocol for on-the-resin coupling of fluorescent dyes or other functional groups to the N-termini of synthetic peptides prior to cleavage and deprotection. The protocol avoids expensive preactivated dyes and instead employs carboxylated dyes activated by large amounts of coupling reagents. The protocol was used to label peptides with low reactivity such as long hydrophobic peptides and peptides with strong tendencies to form sterically shielding structures or aggregates in solution. In all cases, the yields far exceeded those from commercially available preactivated compounds.

Membrane active peptides exert their biological effects by interacting directly with a cell’s lipid bilayer membrane. These therapeutically promising peptides have demonstrated a variety of activities including antimicrobial, cytolytic, membrane translocating, and cell penetrating activities. Here, we use electrochemical impedance spectroscopy (EIS) on polymer-cushioned supported lipid bilayers constructed on single crystal silicon to study two pairs of closely related membrane active peptides selected from rationally designed, combinatorial libraries to have different activities in lipid bilayers: translocation, permeabilization, or no activity. Using EIS, we observed that binding of a membrane translocating peptide to the lipid bilayer resulted in a small decrease in membrane resistance followed by a recovery back to the original value. The recovery may be directly attributable to peptide translocation. A nontranslocating peptide did not decrease the resistance. The other pair, two membrane permeabilizing peptides, caused an exponential decrease of membrane resistance in a concentration-dependent manner. This permeabilization of the supported bilayer occurs at peptide to lipid ratios as much as 1000-fold lower than that needed to observe effects in vesicle leakage assays and gives new insights into the fundamental peptide–bilayer interactions involved in membrane permeabilization.

Fibroblast growth factors (fgfs) play important roles in embryonic development and in adult life by controlling cell proliferation, differentiation, and migration. There are 18 known fgfs which activate four fibroblast growth factor receptors (FGFRs), with different isoforms due to alternative splicing. The physical basis behind the specificity of the biological responses mediated by different fgf-FGFR pairs is currently unknown. To gain insight into the specificity of FGFR3c, a membrane receptor which is critical for bone development, we studied, analyzed, and compared the activation of FGFR3c over a wide range of fgf1 and fgf2 concentrations. We found that while the strength of fgf2 binding to FGFR3c is lower than the strength of fgf1 binding, the fgf2-bound dimers exhibit higher phosphorylation of the critical tyrosines in the activation loop. As a result, fgf1 and fgf2 elicit a similar FGFR3c response at low, but not at high, concentrations. The results demonstrate the versatility of FGFR3c response to fgf1 and fgf2 and highlight the complexity in fgf signaling.

Thus far, quantitative studies of lateral protein interactions in membranes have been restricted peptides or simplified protein constructs in lipid vesicles or bacterial membranes. Here we show how free energies of membrane protein dimerization can be measured in mammalian plasma membrane-derived vesicles. The measurements, performed in single vesicles, utilize the quantitative imaging FRET (QI-FRET) method. The experiments are described in a step-by-step protocol. The protein characterized is the transmembrane domain of glycophorin A, the most extensively studied membrane protein, known to form homodimers in hydrophobic environments. The results suggest that molecular crowding in cellular membranes has a dramatic effect on the strength of membrane protein interactions.

A robust biomimetic cell membrane platform is critical for mechanistic studies of membrane protein channels. While polymer cushions are believed to facilitate the incorporation of membrane proteins in such a platform, a systematic characterization and optimization of such cushions is rarely performed. Here, we examine the influence of a polymer cushion on the electrical properties of supported 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers produced via a Langmuir−Blodgett deposition/vesicle fusion assembly process on single-crystal silicon. We show that the resistance of DPhPC bilayers is maximized at the calculated crossover concentration of the polymer (5.9 mol % PEG-lipids). Additionally, these bilayers are sufficiently stable to allow impedance analyses to be performed for nearly 3 weeks. These studies reveal the optimal PEG concentration that yields electrically robust bilayers and demonstrate the utility of the platform for future studies of membrane protein channels and membrane active peptides.

The assembly of electrically addressable, planar supported bilayers composed of biologically relevant lipids, such as those used in vesicular systems, will greatly enhance the experimental capabilities in membrane and membrane protein research. Here we assess the electrical properties of bilayers composed of a wide range of physiologically relevant lipids and lipid combinations. We demonstrate that robust, biologically relevant, planar supported bilayers with high resistance composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 25 mol % cholesterol can be constructed with high reproducibility. Furthermore, to enable studies of pore-forming peptides, which are commonly cationic, we demonstrate the construction of bilayers with biologically relevant outer leaflets incorporating up to 10 mol % negatively charged lipids. Unique features of the platform are that (1) the substrate is commercially available, atomically smooth, single-crystal silicon, (2) the polymer cushion allows for the natural incorporation of membrane proteins, and (3) the platform is highly reproducible.

The extracellular domain of a receptor tyrosine kinase has opposing effects on dimerization depending on whether ligand is present.

Here, we employed the Hill equation, used broadly to characterize cooperativity in protein–ligand binding, to describe the dimerization of transmembrane (TM) helices in hydrophobic environments. The Hill analysis of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domain dimerization gives a Hill coefficient of ~1 for lipid bilayers but only ~0.2 for sodium dodecyl sulfate (SDS) micelles. We propose that this finding is indicative of heterogeneity in FGFR3 TM dimer structure and stability in SDS micelles. We further speculate that (1) the Hill equation can be used as a tool to assess the existence of multiple structural states of TM dimers in different hydrophobic environments and (2) the structural heterogeneity, detectable by Hill analysis, may be the underlying reason for the broad peaks and the low resolution NMR studies of peptides in detergents.

Membrane proteins fold, assemble and function within their native fluid lipid environment. Structural studies of fluid lipid bilayers are thus critically important for understanding processes in membranes. Here, we propose a simple approach to visualize the hydrocarbon core using neutron diffraction and deuterated lipids that are commercially available. This method should have broad utility in structural studies of the bilayer response to protein insertion and folding in membranes.

Quantitative measurements of protein interaction strengths, crucial for describing signaling networks and predicting cellular responses to environmental stimuli, are typically performed in dilute buffer solutions. However, protein−protein interactions in cells occur within the context of a crowded system, which is characterized by a high macromolecular concentration. In this paper, we explore the utility of cell-derived vesicles as a model crowded environment for quantitative FRET measurements of protein−protein interactions. We show that the FRET efficiency, and the donor and acceptor concentrations, can be calculated in each vesicle. We also introduce the “quantitative imaging Föster resonance energy transfer” method as a tool that can yield protein interaction strengths within these vesicles.

The energetics of transmembrane (TM) helix dimerization in membranes and the thermodynamic principles behind receptor tyrosine kinase (RTK) TM domain interactions during signal transduction can be studied using Förster resonance energy transfer (FRET). For instance, FRET studies have yielded the stabilities of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domains and two FGFR3 pathogenic mutants, Ala391Glu and Gly380Arg, in the native bilayer environment. To further our understanding of the molecular mechanisms of deregulated FGFR3 signaling underlying different pathologies, we determined the effect of the Gly382Asp FGFR3 mutation, identified in a multiple myeloma cell line, on the energetics of FGFR3 TM domain dimerization. We measured dimerization energetics using a novel FRET acquisition and processing method, termed “emission-excitation FRET (EmEx-FRET),” which improves the precision of thermodynamic measurements of TM helix association. The EmEx-FRET method, verified here by analyzing previously published data for wild-type FGFR3 TM domain, should have broad utility in studies of protein interactions, particularly in cases when the concentrations of fluorophore-tagged molecules cannot be controlled.

The lipid bilayer vesicle is a model of the cellular membrane. Even in this simple system, however, measuring the thermodynamics of membrane protein association is a challenge. Here we discuss Förster resonance energy transfer (FRET) in liposomes as a method to probe the dimerization of transmembrane helices in a membrane environment. Although the measurements are labor intensive, FRET in liposomes can be measured accurately provided that attention is paid to sample homogeneity and sample equilibration. One must also take into account statistical expectations and the FRET that results from random colocalization of donors and acceptors in the bilayer. Without careful attention to these details, misleading results are easy to obtain in membrane FRET experiments. The results that we obtain in model systems are reproducible and depend solely on the concentration of the protein in the bilayer (i.e., on the protein-to-lipid ratio), thereby yielding thermodynamic parameters that are directly relevant to processes in biological membranes.

This review focuses on the methods that are available to study transmembrane (TM) helix dimerization in membrane-like environments (either bacterial membranes or lipid bilayers, as mimics of the eukaryotic cellular membrane), with an emphasis on the utility of surface-supported bilayers in such studies.

•We study the pathogenic mutations: C178S in FGFR1, C342R in FGFR2, and C228R in FGFR3.•The mutations stabilize the full-length FGFR dimers.•The mutations alter the configuration of the transmembrane helices in the dimers.•Aberrant dimerization and structures may both contribute to the pathologies.Missense mutations that introduce or remove cysteine residues in receptor tyrosine kinases are believed to cause pathologies by stabilizing the active receptor tyrosine kinase dimers. However, the magnitude of this stabilizing effect has not been measured for full-length receptors. Here, we characterize the dimer stabilities of three full-length fibroblast growth factor receptor (FGFR) mutants harboring pathogenic cysteine substitutions: the C178S FGFR1 mutant, the C342R FGFR2 mutant, and the C228R FGFR3 mutant. We find that the three mutations stabilize the FGFR dimers. We further see that the mutations alter the configuration of the FGFR transmembrane dimers. Thus, both aberrant dimerization and perturbed dimer structure likely contribute to the pathological phenotypes arising due to these mutations.Download high-res image (117KB)Download full-size image

Using a high throughput screen, we have identified a family of 12-residue long peptides that spontaneously translocate across membranes. These peptides function by a poorly understood mechanism that is very different from that of the well-known, highly cationic cell penetrating peptides such as the tat peptide from HIV. The newly discovered translocating peptides can carry polar cargoes across synthetic bilayers and across cellular membranes quickly and spontaneously without disrupting the membrane. Here we report on the biophysical characterization of a representative translocating peptide from the selected family, TP2, as well as a negative control peptide, ONEG, from the same library. We measured the binding of the two peptides to lipid bilayers, their secondary structure propensities, their dispositions in bilayers by neutron diffraction, and the response of the bilayer to the peptides. Compared to the negative control, TP2 has a greater propensity for membrane partitioning, although it still binds only weakly, and a higher propensity for secondary structure. Perhaps most revealing, TP2 has the ability to penetrate deep into the bilayer without causing significant bilayer perturbations, a property that may help explain its ability to translocate without bilayer permeabilization.

The mechanism of receptor tyrosine kinase (RTK) over-activation due to mutations in their transmembrane (TM) domain is not well understood, and different mechansims have been proposed to contribute to pathogenesis. Here, we address the effect of two such pathogenic mutations (V664E in Neu and A391E in fibroblast growth factor receptor 3 (FGFR3)) on receptor activation in mammalian cells. We develop a quantitative description of receptor activation in terms of free energies of activation, and generate mathematical predictions of active fractions as a function of receptor expression. We test the mathematical predictions by comparing them to Western blot measurements of active fractions of Neu and chimeric Neu_FGFR3 receptors in CHO cells. We show that the predictions describe the experimental data, thus yielding a quantitative measure of receptor over-activation due to the two mutations studied. In CHO cells, the V664E mutation increases the Neu activation propensity by about –1.1 kcal/mol, while the increase due to the A391E mutation is about –0.7 kcal/mol. The two values are similar, and likely represent Glu-mediated stabilization of the active dimeric state. Thus, an increase of the order of ∼ –1 kcal/mol may be sufficient to transform normal signaling processes into pathogenic processes. The results of this study increase our knowledge of the mechanism behind RTK-mediated pathologies, and highlight the potential utility of inhibitors that target the dimerization process. They also suggest the cellular response to stimuli can be understood and predicted based on quantitative knowledge of interaction strengths between proteins involved in signaling.

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as −2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.

•FGFR3 JM domain stabilizes FGFR3 unliganded dimers.•The JM domain contribution to dimer stability is similar to that of a pathogenic mutation.•The contributions of the JM domain and a pathogenic mutation to stability are additive.•The JM domain contribution cancels the repulsive contribution of the EC domain.Receptor tyrosine kinases (RTKs) conduct biochemical signals upon dimerization in the membrane plane. While RTKs are generally known to be activated in response to ligand binding, many of these receptors are capable of forming unliganded dimers that are likely important intermediates in the signaling process. All 58 RTKs consist of an extracellular (EC) domain, a transmembrane (TM) domain, and an intracellular domain that includes a juxtamembrane (JM) sequence and a kinase domain. Here we investigate directly the effect of the JM domain on unliganded dimer stability of FGFR3, a receptor that is critically important for skeletal development. The data suggest that FGFR3 unliganded dimers are stabilized by receptor–receptor contacts that involve the JM domains. The contribution is significant, as it is similar in magnitude to the stabilizing contribution of a pathogenic mutation and the repulsive contribution of the EC domain. Furthermore, we show that the effects of the JM domain and a TM pathogenic mutation on unliganded FGFR3 dimer stability are additive. We observe that the JM-mediated dimer stabilization occurs when the JM domain is linked to FGFR3 TM domain and not simply anchored to the plasma membrane. These results point to a coordinated stabilization of the unliganded dimeric state of FGFR3 by its JM and TM domains via a mechanism that is distinctly different from the case of another well studied receptor, EGFR.Download high-res image (254KB)Download full-size image

The influenza virus matrix protein 2 (M2) assembles into a tetramer in the host membrane during viral uncoating and maturation. It has been used as a model system to understand the relative contributions of protein-lipid and protein-protein interactions to membrane protein structure and association. Here we investigate the effect of lipid chain length on the association of the M2 transmembrane domain into tetramers using Förster resonance energy transfer. We observe that the interactions between the M2 helices are much stronger in 1,2-dilauroyl-sn-glycero-3-phosphocholine than in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Thus, lipid chain length and bilayer thickness not only modulate peptide interactions, but could also be a major determinant of the association of transmembrane helices into functional membrane protein oligomers.

Thanatophoric dysplasia type I (TDI) is a lethal human skeletal growth disorder with a prevalence of 1 in 20,000 to 1 in 50,000 births. TDI is known to arise because of five different mutations, all involving the substitution of an amino acid with a cysteine in fibroblast growth factor receptor 3 (FGFR3). Cysteine mutations in receptor tyrosine kinases (RTKs) have been previously proposed to induce constitutive dimerization in the absence of ligand, leading to receptor overactivation. However, their effect on RTK dimer stability has never been measured experimentally. In this study, we characterize the effect of three TDI mutations, Arg248Cys, Ser249Cys, and Tyr373Cys, on FGFR3 dimerization in mammalian membranes, in the absence of ligand. We demonstrate that the mutations lead to surprisingly modest dimer stabilization and to structural perturbations of the dimers, challenging the current understanding of the molecular interactions that underlie TDI.

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Förster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.

Studies of the assembly of the hexapeptide Acetyl-Trp-Leu5 (AcWL5) into β-sheets in membranes have provided insights into membrane protein folding. Yet, the exact structure of the oligomer in the lipid bilayer is unknown. Here we use neutron diffraction to study the disposition of the peptides in bilayers. We find that pairs of adjacent deuterium-labeled leucines have no well-defined peak or dip in the transmembrane distribution profiles, indicative of heterogeneity in the depth of membrane insertion. At the same time, the monomeric homolog AcWL4 exhibits a homogeneous, well-defined, interfacial location in neutron diffraction experiments. Thus, although the bilayer location of monomeric AcWL4 is determined by hydrophobicity matching or complementarity within the bilayer, the AcWL5 molecules in the oligomer are positioned at different depths within the bilayer because they assemble into a staggered transmembrane β-sheet. The AcWL5 assembly is dominated by protein-protein interactions rather than hydrophobic complementarity. These results have implications for the structure and folding of proteins in their native membrane environment and highlight the importance of the interplay between hydrophobic complementarity and protein-protein interactions in determining the structure of membrane proteins.