Peptides as transmembrane segments: decrypting the determinants for helix-helix interactions in membrane proteins

Although the structural analysis of membrane proteins is advancing, an understanding of the basic principles that underlie their folding and assembly remains limited because of the high insolubility intrinsic to these molecules and concomitant challenges in obtaining crystals. Fortunately, from an experimental standpoint, membrane protein folding can be approximated as the rigid-body docking of pre-formed alpha-helical transmembrane segments one with another to form the final functional protein structure. Peptides derived from the sequences of native alpha-helical transmembrane segments and those that mimic their properties are therefore valuable in the experimental evaluation of protein folding within the membrane. Here we present an overview of the progress made in our laboratory and elsewhere in using peptide models toward defining the sequence requirements and forces stabilizing membrane protein folds.     

Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity

SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of alpha-helical and beta-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of alpha-helical and beta-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the alpha-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion.     

Recognition of transmembrane segments in proteins: review and consistency-based benchmarking of internet servers

Membrane proteins perform a number of crucial functions as transporters, receptors, and components of enzyme complexes. Identification of membrane proteins and prediction of their topology is thus an important part of genome annotation. We present here an overview of transmembrane segments in protein sequences, summarize data from large-scale genome studies, and report results of benchmarking of several popular internet servers.     

Collagenous transmembrane proteins: collagen XVII as a prototype.

Collagenous transmembrane proteins are an emerging group of biologically versatile molecules which function as both cell surface receptors and matrix molecules. The seven group members have interesting structural similarities: they are integral membrane proteins in type II orientation and have one or more collagenous domains in the extracellular C-terminus; interspersed by non-collagenous stretches which confer structural flexibility to the ectodomain. A conserved coiled-coil sequence (linker domain) immediately adjacent to the extracellular face of the cell membrane presumably serves as a nucleus for trimerization and triple-helix folding of each collagen. Intriguingly, the ectodomains of at least some of these molecules are proteolytically shed from the cell surface, releasing a shorter form of the collagen into the extracellular matrix. Collagenous transmembrane proteins are expressed in many different tissues and cells, and are involved in a broad spectrum of biological functions, reaching from epithelial and neural cell adhesion, and epithelial-mesenchymal interactions during morphogenesis to host defense against microbial agents. Several group members are involved in the molecular pathology of genetic and acquired human diseases including epidermolysis bullosa, ectodermal dysplasia, bullous pemphigoid or Alzheimer disease. An extensively investigated member is collagen XVII, a keratinocyte surface protein, which attaches the epidermis to the basement membrane in the skin. In this review, the structure and functions of the currently known collagenous transmembrane proteins are summarized and, as a 'prototype' of the group, collagen XVII and its biology and pathophysiology are delineated.     

Evidence for similar function of transmembrane segments in receptor and membrane-anchored proteins

Transmembrane (TM) regions of receptor proteins should have unique structural and/or chemical characteristics if these regions contain residues functional in TM signal transduction. However, in a survey of the membrane-occurring residues in 37 integral membrane proteins, we found that amino acid compositions of TM regions of receptor proteins (n = 11) could not be distinguished statistically from corresponding regions of membrane-anchored proteins (e.g., recognition molecules) with a functional external domain attached to a single hydrophobic membrane-spanning anchor segment (n = 16). TM regions in both categories of proteins differed from the compositions of TM regions in membrane-transport proteins (n = 10). The analysis implies that TM regions in receptor proteins may function mainly to anchor (and position) receptors in their cellular membranes, and therefore residues in receptors that participate in signal transduction need not be restricted to these regions. In addition to mechanisms involving receptor aggregation, ligand-activated conformational perturbation of a receptor external aqueous domain, resulting in membrane penetration of hydrophobic segment(s) of this domain to produce intramembranous contact with its cytoplasmic domain, is hypothesized as a further possible mode of signal transduction.     

Optimizing synthesis and expression of transmembrane peptides and proteins

Structural studies of full-length membrane proteins have been hindered by their hydrophobicity and low expression in a variety of systems. However, a simplifying aspect of membrane protein folding is that individual transmembrane segments or membrane protein fragments have been observed to represent independent folding domains, and as such, can facilitate the study of packing interactions between TM helices, and the collection of structural information regarding membrane proteins. This review focuses on two categories of techniques--total peptide synthesis and bacterial expression--that can each be optimized for preparation of transmembrane protein segments. First, synthesis of hydrophobic transmembrane peptides that are N- and/or C-tagged with solubilizing residues such as lysine can improve manipulation of the transmembrane core in a variety of biophysical experiments. In this context, we describe general protocol considerations during the synthesis, cleavage, and purification stages of these peptides to identify appropriate parameters that combine to improve yields of hydrophobic peptides. Second, bacterial expression of membrane protein fragments is a useful tool for producing large quantities of hydrophobic protein segments. Targeting protein expression within Escherichia coli can facilitate purification, while attaching the hydrophobic construct to a hydrophilic fusion protein can amplify expression. We show that adapting protein constructs to comply with expression host specifications, in concert with thorough exploration of expression conditions such as the type of media used for expression, temperature, and cell strain, can significantly improve protein yields.     

Synthetic peptides as functional mimics of a viral discontinuous antigenic site.

Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1-VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV.     

Benchmarking of programs that predict the position of transmembrane segments in beta-barrel proteins

We propose a method for comparative analysis of the programs that locate transmembrane segments in proteins. On this basis, we have compiled a sample of beta-barrel protein that allows unequivocal assessment of prediction quality. Upon testing of several Internet servers, B2TMR proves the best, with B2TMPRED, HMM-B2TMR, and PROFtmb following.     

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.     

Composite RNA aptamers as functional mimics of proteins

Individual RNA aptamers are often used to modulate the function of their target proteins, and multi-valent aptamers have been constructed to enhance their activity. To expand the utility of aptamers in manipulating and controlling biological processes, here we advance a general method for the design and construction of composite aptamers. The resulting molecular constructs resemble proteins in that they can form specific interactions with three or more different partners and be readily integrated into existing protein regulatory networks. As the first embodiment of this method, we created a tetra-valent aptamer that simultaneously binds to two molecules of the Drosophila protein B52 and two copies of streptavidin, thus mimicking the function of an antibody in immunochemical assays. We demonstrated that the performance of this ‘aptabody’ rivals that of a monoclonal antibody against B52 in these assays. While this study was performed in vitro and the composite aptamer we made was intended to mimic an existing protein, the same method can be used to accommodate arbitrary combinations of individual aptamers in composite molecular contexts, and these constructs can be delivered into living cells, where they are able to utilize existing cellular infrastructure for their production and processing.     

A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs.     

Synthetic mimics of antimicrobial peptides

Infectious diseases and antibiotic resistance are now considered the most imperative global healthcare problem. In the search for new treatments, host defense, or antimicrobial, peptides have attracted considerable attention due to their various unique properties; however, attempts to develop in vivo therapies have been severely limited. Efforts to develop synthetic mimics of antimicrobial peptides (SMAMPs) have increased significantly in the last decade, and this review will focus primarily on the structural evolution of SMAMPs and their membrane activity. This review will attempt to make a bridge between the design of SMAMPs and the fundamentals of SMAMP-membrane interactions. In discussions regarding the membrane interaction of SMAMPs, close attention will be paid to the lipid composition of the bilayer. Despite many years of study, the exact conformational aspects responsible for the high selectivity of these AMPs and SMAMPs toward bacterial cells over mammalian cells are still not fully understood. The ability to design SMAMPs that are potently antimicrobial, yet nontoxic to mammalian cells has been demonstrated with a variety of molecular scaffolds. Initial animal studies show very good tissue distribution along with more than a 4-log reduction in bacterial counts. The results on SMAMPs are not only extremely promising for novel antibiotics, but also provide an optimistic picture for the greater challenge of general proteomimetics.     

Membrane orientation of carboxyl-terminal half P-glycoprotein: topogenesis of transmembrane segments.

Multiple topological orientations of the carboxyl-terminal half of P-glycoprotein have been observed. One orientation is consistent with the hydropathy-predicted model and contains six transmembrane (TM)-spanning regions. In another orientation, the cytoplasmic-predicted loop between TM8 and TM9 is extracellular and glycosylated. In support of this "alternative" topology, TM8 was previously established to function as a signal-anchor sequence to insert with its amino-terminal end in the cytoplasm and the carboxyl-terminal end in the extracytoplasmic space. However, it is unclear how downstream TM segments fold in the membrane when TM8 functions as a signal-anchor sequence. Here, we created several chimeric Pgp molecules to examine the membrane insertion of TM segments 9 and 10 using a cell-free system. We found that TM9 functions as a stop-transfer sequence when following the signal-anchor sequence, TM8. However, the stop-transfer activity of TM9 depends on the presence of TM10. In the absence of TM10, TM9 partially translocated across the membrane into the endoplasmic reticulum lumen. In contrast, TM9 efficiently stopped the translocation event of the nascent chain in the presence of TM10. Our results suggest that the membrane insertion of TM8 and TM9 establishes the extracellular loop between TM8 and TM9. Formation of this loop apparently involves the interactions between Pgp TM segments, which facilitate proper folding of the Pgp carboxyl-terminal half.     

A modified, dual reporter TOXCAT system for monitoring homodimerization of transmembrane segments of proteins

The TOXCAT assay system developed by Russ and Engelman [TOXCAT: a measure of transmembrane helix association in a biological membrane, Proc. Natl. Acad. Sci. USA 96 (1999) 863-868] provides an in vivo means of selecting for and evaluating the strength of interaction between identical transmembrane alpha-helices. In the course of utilizing TOXCAT to study the architecture of a sodium channel hNa(V)1.5, an apparently strong dimerization of two of its putative transmembrane segments was revealed. Following random mutagenesis of these regions, several amino acids critical for the observed dimerizations were identified. In order to develop a more efficient means of isolating mutations which specifically disrupt dimerization of these transmembrane segments without affecting their membrane-targeting properties, we developed a modification to the original TOXCAT design in which the C-terminal maltose binding protein moiety is replaced by the beta-lactamase. We show that this assay system is capable of simultaneously monitoring the integrity of the chimeric protein, its membrane insertion activity, and the ability of the transmembrane segment under study to dimerize.     

A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm.

We present a novel method that predicts transmembrane domains in proteins using solely information contained in the sequence itself. The PRED-TMR algorithm described, refines a standard hydrophobicity analysis with a detection of potential termini ('edges', starts and ends) of transmembrane regions. This allows one both to discard highly hydrophobic regions not delimited by clear start and end configurations and to confirm putative transmembrane segments not distinguishable by their hydrophobic composition. The accuracy obtained on a test set of 101 non-homologous transmembrane proteins with reliable topologies compares well with that of other popular existing methods. Only a slight decrease in prediction accuracy was observed when the algorithm was applied to all transmembrane proteins of the SwissProt database (release 35). A WWW server running the PRED-TMR algorithm is available at http://o2.db.uoa. gr/PRED-TMR/     

Conformational analysis of bioactive peptides in reverse micelles as mimics of cell membrane environments

Summary Conformational preferences of secretin as a model peptide have been analyzed by CD and IR spectroscopy in reverse micelles of AOT/isooctane/water and compared to those in aqueous TFE, in SDS micelles and in DMPG vesicles. Among the systems examined, reverse micelles and phospholipid vesicles displayed almost identical conformational equilibria. Very high lipid-to-peptide ratios can be obtained in reverse micelles with full retention of optical transparency, even at millimolar peptide concentrations, thus indicating this system to be an interesting mimic of cell membrane environments for spectroscopic analysis of bioactive peptide conformations.Abbreviations TFE trifluoroethanol - SDS sodium dodecyl sulfate - DMPG dimyristoylphosphatidylglycerol - AOT bis(2-ethylhexyl)sulfosuccinate - CMC critical micellar concentration - VIP vasoactive intestinal peptide     

Alpha-hairpin stability and folding of transmembrane segments

Molecular Dynamics (MD) simulations at low dielectric constant have been carried out for peptides matching the double spanning segments of transmembrane proteins. Different folding dynamics have been observed. The peptides folded into the stable helix-turn-helix conformation-alpha-hairpin-with antiparallel-oriented strands or unstable alpha-hairpin conformation that unfolded later into the straight helical structure. The peptide having flexible residues in the TM helices often misfolded into a tangled structure that can be avoided by restricting the flexibility of these residues. General conclusions can be drawn from the observed folding dynamics. The stability and folding of some double spanning transmembrane fragments are self-assembling. The following and/or neighboring peptide chains of the protein may support the stability of the hairpin structure of other fragments. The stability of the TM helices containing flexible residues could be maintained due to contacts with neighboring TM segments.     

A heptad motif of leucine residues found in membrane proteins can drive self-assembly of artificial transmembrane segments

Specific interactions between alpha-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, se- quence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins.