Transmembrane Segments Of Proteins Research Articles

Lateral gate dynamics of the bacterial translocon using single molecule fluorescence

During translation, transmembrane segments (TMs) of nascent membrane proteins emerging from the ribosome insert into the translocon (SecYEG in bacteria) and reach the phospholipid bilayer through the open lateral gate formed of two helices of SecY. We use single-molecule fluorescence resonance energy transfer (smFRET) to observe lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We show that the lateral gate is highly dynamic with or without the ligands and propose a model-free statistical approach to describe these conformational ensembles.

Dissecting Role of Charged Residue from Transmembrane Domain 5 of Latent Membrane Protein 1 via In Silico Simulations and Wet-Lab Experiments.

Charged residues are frequently found in the transmembrane segments of membrane proteins, which reside in the hydrophobic bilayer environment. Charged residues are critical for the function of membrane protein. However, studies of their role in protein oligomerization are limited. By taking the fifth transmembrane domain (TMD5) of latent membrane protein 1 from the Epstein-Barr virus as a prototype model, in silico simulations and wet-lab experiments were performed to investigate how the charged states affect transmembrane domain oligomerization. Molecular dynamics (MD) simulations showed that the D150-protonated TMD5 trimer was stable, whereas unprotonated D150 created bends in the helices which distort the trimeric structure. D150 was mutated to asparagine to mimic the protonated D150 in TMD5, and the MD simulations of different D150N TMD5 trimers supported that the protonation state of D150 was critical for the trimerization of TMD5. In silico mutations found that D150N TMD5 preferred to interact with TMD5 to form the heterotrimer (1 D150N TMD5:2 protonated TMD5s) rather than the heterotrimer (2 D150N TMD5s:1 protonated TMD5). D150R TMD5 interacted with TMD5 to form the heterotrimer (1 D150R TMD5:2 protonated TMD5). These in silico results imply that D150N TMD5 and D150R TMD5 peptides may be probes for disrupting TMD5 trimerization, which was supported by the dominant-negative ToxR assay in bacterial membranes. In all, this study elucidates the role of charged residues at the membrane milieu in membrane protein oligomerization and provides insight into the development of oligomerization-regulating peptides for modulating transmembrane domain lateral interactions.

Structural Perspectives on Extracellular Recognition and Conformational Changes of Several Type-I Transmembrane Receptors.

Type-I transmembrane proteins represent a large group of 1,412 proteins in humans with a multitude of functions in cells and tissues. They are characterized by an extracellular, or luminal, N-terminus followed by a single transmembrane helix and a cytosolic C-terminus. The domain composition and structures of the extracellular and intercellular segments differ substantially amongst its members. Most of the type-I transmembrane proteins have roles in cell signaling processes, as ligands or receptors, and in cellular adhesion. The extracellular segment often determines specificity and can control signaling and adhesion. Here we focus on recent structural understanding on how the extracellular segments of several diverse type-I transmembrane proteins engage in interactions and can undergo conformational changes for their function. Interactions at the extracellular side by proteins on the same cell or between cells are enhanced by the transmembrane setting. Extracellular conformational domain rearrangement and structural changes within domains alter the properties of the proteins and are used to regulate signaling events. The combination of structural properties and interactions can support the formation of larger-order assemblies on the membrane surface that are important for cellular adhesion and intercellular signaling.

Partial proteolysis improves the identification of the extracellular segments of transmembrane proteins by surface biotinylation

Transmembrane proteins (TMP) play a crucial role in several physiological processes. Despite their importance and diversity, only a few TMP structures have been determined by high-resolution protein structure characterization methods so far. Due to the low number of determined TMP structures, the parallel development of various bioinformatics and experimental methods was necessary for their topological characterization. The combination of these methods is a powerful approach in the determination of TMP topology as in the Constrained Consensus TOPology prediction. To support the prediction, we previously developed a high-throughput topology characterization method based on primary amino group-labelling that is still limited in identifying all TMPs and their extracellular segments on the surface of a particular cell type. In order to generate more topology information, a new step, a partial proteolysis of the cell surface has been introduced to our method. This step results in new primary amino groups in the proteins that can be biotinylated with a membrane-impermeable agent while the cells still remain intact. Pre-digestion also promotes the emergence of modified peptides that are more suitable for MS/MS analysis. The modified sites can be utilized as extracellular constraints in topology predictions and may contribute to the refined topology of these proteins.

Pred-MutHTP: Prediction of disease-causing and neutral mutations in human transmembrane proteins.

Membrane proteins are unique in that segments thereof concurrently reside in vastly different physicochemical environments: the extracellular space, the lipid bilayer, and the cytoplasm. Accordingly, the effects of missense variants disrupting their sequence depend greatly on the characteristics of the environment of the protein segment affected as well as the function it performs. Because membrane proteins have many crucial roles (transport, signal transduction, cell adhesion, etc.), compromising their functionality often leads to diseases including cancers, diabetes mellitus or cystic fibrosis. Here, we report a suite of sequence-based computational methods "Pred-MutHTP" for discriminating between disease-causing and neutral alterations in their sequence. With a data set of 11,846 disease-causing and 9,533 neutral mutations, we obtained an accuracy of 74% and 78% with 10-fold group-wise cross-validation and test set, respectively. The features used in the models include evolutionary information, physiochemical properties, neighboring residue information, and specialized membrane protein attributes incorporating the number of transmembrane segments, substitution matrices specific to membrane proteins as well as residue distributions occurring in specific topological regions. Across 11 disease classes, the method achieved accuracies in the range of 75-85%. The model designed specifically for the transmembrane segments achieved an accuracy of 85% on the test set with a sensitivity and specificity of 86% and 83%, respectively. This renders our method the current state-of-the-art with regard to predicting the effects of variants in the transmembrane protein segments. Pred-MutHTP allows predicting the effect of any variant occurring in a membrane protein-available at https://www.iitm.ac.in/bioinfo/PredMutHTP/.

Probing Conformational States of a Target Protein in Escherichia coli Cells by in vivo Cysteine Cross-linking Coupled with Proteolytic Gel Analysis.

Transporters are dynamic membrane proteins that are essential to the physiology of cells. To function, transporters must cycle between various conformational states, so to understand their mechanistic details, it is critical to characterize how their structure changes during the transport cycle. One approach to studying the dynamics of transporters takes advantage of the chemistry of cysteine by using sulfhydryl-reactive, bi-functional cross-linkers to probe changes in the distance between two specific residues that have been substituted to cysteine. This approach is mostly used to study transporters in vitro, not in their natural cellular environment. Here we describe a protocol based on structure-guided cysteine cross-linking and proteolysis-coupled gel analysis to probe conformational changes of a target transporter in live Escherichia coli cells. Although cross-linking approaches have been used to probe the proximity between transmembrane segments in membrane proteins in vivo, to our knowledge this protocol is the first to be used to interrogate transporter dynamics in cells. The use of this protocol is optimal for proteins with known or modeled structures to guide the replacement of specific residues with cysteines and the selection of cross-linking agents with various spacer arm lengths. This protocol allows for discriminating easily cross-linked and uncross-linked species and does not require the often difficult or unavailable reconstitution of transport activity in an in vitro system. In addition, this protocol could be used to probe the conformation of transporters in cells treated with transport inhibitors in order to better understand their mechanism of action, and potentially dynamic interactions between domains in proteins that are not transporters.

Wavelength-Selective Fluorescence of a Model Transmembrane Peptide: Constrained Dynamics of Interfacial Tryptophan Anchors.

WALPs are prototypical, α-helical transmembrane peptides that represent a consensus sequence for transmembrane segments of integral membrane proteins and serve as excellent models for exploring peptide-lipid interactions and hydrophobic mismatch in membranes. Importantly, the WALP peptides are in direct contact with the lipids. They consist of a central stretch of alternating hydrophobic alanine and leucine residues capped at both ends by tryptophans. In this work, we employ wavelength-selective fluorescence approaches to explore the intrinsic fluorescence of tryptophan residues in WALP23 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Our results show that the four tryptophan residues in WALP23 exhibit an average red edge excitation shift (REES) of 6nm, implying their localization at the membrane interface, characterized by a restricted microenvironment. This result is supported by fluorescence anisotropy and lifetime measurements as a function of wavelength displayed by WALP23 tryptophans in POPC membranes. These results provide a new approach based on intrinsic fluorescence of interfacial tryptophans to address protein-lipid interaction and hydrophobic mismatch.

Revealing the mechanisms of membrane protein export by virulence-associated bacterial secretion systems

Many bacteria export effector proteins fulfilling their function in membranes of a eukaryotic host. These effector membrane proteins appear to contain signals for two incompatible bacterial secretion pathways in the same protein: a specific export signal, as well as transmembrane segments that one would expect to mediate targeting to the bacterial inner membrane. Here, we show that the transmembrane segments of effector proteins of type III and type IV secretion systems indeed integrate in the membrane as required in the eukaryotic host, but that their hydrophobicity in most instances is just below the threshold required for mediating targeting to the bacterial inner membrane. Furthermore, we show that binding of type III secretion chaperones to both the effector’s chaperone-binding domain and adjacent hydrophobic transmembrane segments also prevents erroneous targeting. These results highlight the evolution of a fine discrimination between targeting pathways that is critical for the virulence of many bacterial pathogens.

Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins

Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins.

Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments

Integral membrane proteins are generally under-represented in routine proteomic analyses, mostly because of their relatively low abundance, hydrophobicity and lack of trypsin-cleavage sites. To increase the coverage of membrane proteomes, various strategies have been developed, targeting mostly the extra-membrane segments of membrane proteins. We focused our attention to the rather overlooked hydrophobic transmembrane segments. Such peptides can be isolated after carbonate stripping and protease “shaving” of membranes isolated by simple centrifugation procedure. The treated membranes with embedded hydrophobic peptides can then be solubilized in organic solvents, re-digested with CNBr, delipidated and subjected to LC-MS/MS analysis. We modified the original “hppK” method, and applied it for the analysis of human lymphoma cells. We identified 1224 proteins of which two-thirds were IMPs with 1–16 transmembrane segments. This method allowed us to identify 13 “missing proteins” — proteins with no previous evidence on protein level. Biological significanceIntegral membrane proteins execute numerous essential functions and represent substantial part of eukaryotic proteomes. Our knowledge of their function and expression is, however, limited. Novel approaches extending our knowledge of membrane proteome are therefore highly desired. As we demonstrate here, a non-conventional method which targets rather overlooked hydrophobic transmembrane segments of integral membrane proteins has wide potential to provide the missing information on the membrane proteome. We show that it can deliver identification and potentially also quantification of hundreds of integral membrane proteins including the so called “missing proteins”.

Mycobacterial glycopeptidolipids (GPL) are transported by the MmpL4a membrane protein. Genetic studies have identified critical residues within the transmembrane segments of MmpL proteins postulated to be couple the proton motive force (PMF) to MmpL transport activity. For details, see the article by Bernut et al. on pp. 866–883 of this issue

Mycobacterial glycopeptidolipids (GPL) are transported by the MmpL4a membrane protein. Genetic studies have identified critical residues within the transmembrane segments of MmpL proteins postulated to be couple the proton motive force (PMF) to MmpL transport activity. For details, see the article by Bernut <i>et al</i>. on pp. 866–883 of this issue

Oriented Circular Dichroism: A Method to Characterize Membrane-Active Peptides in Oriented Lipid Bilayers.

The structures of membrane-bound polypeptides are intimately related to their functions and may change dramatically with the lipid environment. Circular dichroism (CD) is a rapid analytical method that requires relatively low amounts of material and no labeling. Conventional CD is routinely used to monitor the secondary structure of peptides and proteins in solution, for example, in the presence of ligands and other binding partners. In the case of membrane-active peptides and transmembrane proteins, these measurements can be applied to, and remain limited to, samples containing detergent micelles or small sonicated lipid vesicles. Such traditional CD analysis reveals only secondary structures. With the help of an oriented circular dichroism (OCD) setup, however, based on the preparation of macroscopically oriented lipid bilayers, it is possible to address the membrane alignment of a peptide in addition to its conformation. This approach has been mostly used for α-helical peptides so far, but other structural elements are conceivable as well. OCD analysis relies on Moffitt's theory, which predicts that the electronic transition dipole moments of the backbone amide bonds in helical polypeptides are polarized either parallel or perpendicular to the helix axis. The interaction of the electric field vector of the circularly polarized light with these transitions results in an OCD spectrum of a membrane-bound α-helical peptide, which exhibits a characteristic line shape and reflects the angle between the helix axis and the bilayer normal. For parallel alignment of a peptide helix with respect to the membrane surface (S-state), the corresponding "fingerprint" CD band around 208 nm will exhibit maximum negative amplitude. If the helix changes its alignment via an obliquely tilted (T-state) to a fully inserted transmembrane orientation (I-state), the ellipticity at 208 nm decreases and the value approaches zero due to the decreased interactions between the field and the transition dipole. Compared to conventional CD, OCD data are not only collected in the biologically relevant environment of a highly hydrated planar lipid bilayer (whose composition can be varied at will), but in addition it provides information about the tilt angle of the polypeptide in the membrane. It is the method of choice for screening numerous different conditions, such as peptide concentration, lipid composition, membrane additives, pH, temperature, and sample hydration. All these factors have been found to affect the peptide alignment in membrane, while having little or no influence on conformation. In many cases, the observed realignment could be related to biological action, such as pore formation by antimicrobial and cell-penetrating peptides, or to binding events of transmembrane segments of integral membrane proteins. Likewise, any lipid-induced conversion from α-helix to β-sheeted conformation is readily picked up by OCD and has been interpreted in terms of protein instability or amyloid-formation.

High-resolution structures of a heterochiral coiled coil

Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein-protein interactions. Coiled-coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled-coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.

Implications of aromatic-aromatic interactions: From protein structures to peptide models.

With increasing structural information on proteins, the opportunity to understand physical forces governing protein folding is also expanding. One of the significant non-covalent forces between the protein side chains is aromatic-aromatic interactions. Aromatic interactions have been widely exploited and thoroughly investigated in the context of folding, stability, molecular recognition, and self-assembly processes. Through this review, we discuss the contribution of aromatic interactions to the activity and stability of thermophilic, mesophilic, and psychrophilic proteins. Being hydrophobic, aromatic amino acids tend to reside in the protein hydrophobic interior or transmembrane segments of proteins. In such positions, it can play a diverse role in soluble and membrane proteins, and in α-helix and β-sheet stabilization. We also highlight here some excellent investigations made using peptide models and several approaches involving aryl-aryl interactions, as an increasingly popular strategy in protein and peptide engineering. A recent survey described the existence of aromatic clusters (trimer, tetramer, pentamer, and higher order assemblies), revealing the self-associating property of aryl groups, even in folded protein structures. The application of this self-assembly of aromatics in the generation of modern bionanomaterials is also discussed.

In silico evaluation of the influence of the translocon on partitioning of membrane segments.

BackgroundThe locations of the TM segments inside the membrane proteins are the consequence of a cascade of several events: the localizing of the nascent chain to the membrane, its insertion through the translocon, and the conformation adopted to reach its stable state inside the lipid bilayer. Even though the hydrophobic h-region of signal peptides and a typical TM segment are both composed of mostly hydrophobic side chains, the translocon has the ability to determine whether a given segment is to be inserted into the membrane. Our goal is to acquire robust biological insights into the influence of the translocon on membrane insertion of helices, obtained from the in silico discrimination between signal peptides and transmembrane segments of bitopic proteins. Therefore, by exploiting this subtle difference, we produce an optimized scale that evaluates the tendency of each amino acid to form sequences destined for membrane insertion by the translocon.ResultsThe learning phase of our approach is conducted on carefully chosen data and easily converges on an optimal solution called the PMIscale (Potential Membrane Insertion scale). Our study leads to two striking results. Firstly, with a very simple sliding-window prediction method, PMIscale enables an efficient discrimination between signal peptides and signal anchors. Secondly, PMIscale is also able to identify TM segments and to localize them within protein sequences.ConclusionsDespite its simplicity, the localization method based on PMIscale nearly attains the highest level of TM topography prediction accuracy as the current state-of-the-art prediction methods. These observations confirm the prominent role of the translocon in the localization of TM segments and suggest several biological hypotheses about the physical properties of the translocon.

Interaction analysis of a ladder-shaped polycyclic ether and model transmembrane peptides in lipid bilayers by using Förster resonance energy transfer and polarized attenuated total reflection infrared spectroscopy

Ladder-shaped polycyclic ethers (LSPs) are predicted to interact with membrane proteins; however, the underlying mechanism has not been satisfactorily elucidated. It has been hypothesized that LSPs possess non-specific affinity to α-helical segments of transmembrane proteins. To verify this hypothesis, we constructed a model LSP interaction system in a lipid bilayer. We prepared 5 types of α-helical peptides and reconstituted them in liposomes. The reconstitution and orientation of these peptides in the liposomes were examined using polarized attenuated total reflection infrared (ATR-IR) spectroscopy and gel filtration. The results revealed that 4 peptides were retained in liposomes, and 3 of them formed stable transmembrane structures. The interaction between the LSP and the peptides was investigated using Förster resonance energy transfer (FRET). In the lipid bilayer, the LSP strongly recognized the peptides that possessed aligned hydrogen donating groups with leucine caps. We propose that this leucine-capped 16-amino acid sequence is a potential LPS binding motif.

Identification of YidC Residues That Define Interactions with the Sec Apparatus

In bacteria, a subset of membrane proteins insert into the membrane via the Sec apparatus with the assistance of the widely conserved essential membrane protein insertase YidC. After threading into the SecYEG translocon, transmembrane segments of nascent proteins are thought to exit the translocon via a lateral gate in SecY, where YidC facilitates their transfer into the lipid bilayer. Interactions between YidC and components of the Sec apparatus are critical to its function. The first periplasmic loop of YidC interacts directly with SecF. We sought to identify the regions or residues of YidC that interact with SecY or with additional components of the Sec apparatus other than SecDF. Using a synthetic lethal screen, we identified residues of YidC that, when mutated, led to dependence on SecDF for viability. Each residue identified is highly conserved among YidC homologs; most lie within transmembrane domains. Overexpression of SecY in the presence of two YidC mutants partially rescued viability in the absence of SecDF, suggesting that the corresponding wild-type YidC residues (G355 and M471) participate in interactions, direct or indirect, with SecY. Staphylococcus aureus YidC complemented depletion of YidC, but not of SecDF, in Escherichia coli. G355 of E. coli YidC is invariant in S. aureus YidC, suggesting that this highly conserved glycine serves a conserved function in interactions with SecY. This study demonstrates that transmembrane residues are critical in YidC interactions with the Sec apparatus and provides guidance on YidC residues of interest for future structure-function analyses.

Membrane Protein TM Segments Are Retained at the Translocon during Integration until the Nascent Chain Cues FRET-Detected Release into Bulk Lipid

Most membrane proteins are integrated cotranslationally into the ER membrane at the translocon, where nonpolar nascent protein transmembrane segments (TMSs) are widely believed to partition directly into the nonpolar membrane interior. However, a FRET approach that monitors the separation between a fluorescent-labeled TMS and fluorescent phospholipids diffusing in the bulk lipid reveals that TMSs do not immediately enter the lipid phase of the membrane. Instead, TMSs are retained at the translocon by protein-protein interactions until their release into bulk lipid is triggered by translation termination or, in some cases, by the arrival of another nascent chain TMS at a translocon. Nascent chain status and structural elements therefore dictate the timing of TMS release into the lipid phase by altering TMS and flanking sequence interactions with translocons, ribosomes, and associated proteins, thereby controlling when successive TMSs assemble in the bilayer and TMS-delineated loops fold.

The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch

Tryptophan (Trp) is abundant in membrane proteins, preferentially residing near the lipid–water interface where it is thought to play a significant anchoring role. Using a total of 3μs of molecular dynamics simulations for a library of hydrophobic WALP-like peptides, a long poly-Leu α-helix, and the methyl-indole analog, we explore the thermodynamics of the Trp movement in membranes that governs the stability and orientation of transmembrane protein segments. We examine the dominant hydrogen-bonding interactions between the Trp and lipid carbonyl and phosphate moieties, cation–π interactions to lipid choline moieties, and elucidate the contributions to the thermodynamics that serve to localize the Trp, by ~4kcal/mol, near the membrane glycerol backbone region. We show a striking similarity between the free energy to move an isolated Trp side chain to that found from a wide range of WALP peptides, suggesting that the location of this side chain is nearly independent of the host transmembrane segment. Our calculations provide quantitative measures that explain Trp's role as a modulator of responses to hydrophobic mismatch, providing a deeper understanding of how lipid composition may control a range of membrane active peptides and proteins.

Anchoring proteins to Escherichia coli cell membranes using hydrophobic anchors derived from a Bacillus subtilis integral membrane protein

Anchored periplasmic expression (APEx) technology aims to express and localize proteins or peptides in the Escherichia coli periplasm. Some reports have suggested that transmembrane segments of integral membrane proteins can be used as membrane anchors in the APEx system. In this study, a series of hydrophobic anchors derived from the first putative transmembrane helix of a Bacillus subtilis integral membrane protein, MrpF, and its truncated forms were investigated for anchored periplasmic expression of alkaline phosphatase (PhoA) in E. coli. Anchoring efficiency of hydrophobic anchors was evaluated by monitoring the expression and activity of anchored PhoA. The length of hydrophobic anchors was found to be critical for anchoring proteins to cell membranes. This study may open new avenues for applying transmembrane segments derived from native membrane proteins as membrane anchors in the APEx system.