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By analyzing transmembrane (TM) helices in known structures, we find that some polar amino acids are more frequent at the N terminus than at the C terminus. We propose the asymmetry occurs because most polar amino acids are better able to snorkel their polar atoms away from the membrane core at the N terminus than at the C terminus. Two findings lead us to this proposition: (1) side-chain conformations are influenced strongly by the N or C-terminal position of the amino acid in the bilayer, and (2) the favored snorkeling direction of an amino acid correlates well with its N to C-terminal composition bias. Our results suggest that TM helix predictions should incorporate an N to C-terminal composition bias, that rotamer preferences of TM side-chains are position-dependent, and that the ability to snorkel influences the evolutionary selection of amino acids for the helix N and C termini. [Copyright &y& Elsevier]

An inherent problem in transmembrane protein topology prediction and signal peptide prediction is the high similarity between the hydrophobic regions of a transmembrane helix and that of a signal peptide, leading to cross-reaction between the two types of predictions. To improve predictions further, it is therefore important to make a predictor that aims to discriminate between the two classes. In addition, topology information can be gained when successfully predicting a signal peptide leading a transmembrane protein since it dictates that the N terminus of the mature protein must be on the non-cytoplasmic side of the membrane. Here, we present Phobius, a combined transmembrane protein topology and signal peptide predictor. The predictor is based on a hidden Markov model (HMM) that models the different sequence regions of a signal peptide and the different regions of a transmembrane protein in a series of interconnected states. Training was done on a newly assembled and curated dataset. Compared to TMHMM and SignalP, errors coming from cross-prediction between transmembrane segments and signal peptides were reduced substantially by Phobius. False classifications of signal peptides were reduced from 26.1% to 3.9% and false classifications of transmembrane helices were reduced from 19.0% to 7.7%. Phobius was applied to the proteomes of Homo sapiens and Escherichia coli. Here we also noted a drastic reduction of false classifications compared to TMHMM/SignalP, suggesting that Phobius is well suited for whole-genome annotation of signal peptides and transmembrane regions. The method is available at http://phobius.cgb.ki.se/ as well as at http://phobius.binf.ku.dk/ [Copyright &y& Elsevier]

The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum, was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in β-dodecylmaltoside and purified by streptactin affinity chromatography. Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution. Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane. Reconstitution with E. coli polar lipids produced 2D crystals with unit cell parameters of 182 A˚×154 A˚, γ=90° exhibiting p22121 symmetry. Electron cryo-microscopy yielded a projection map at 7.5 A˚. The unit cell contains four non-crystallographic trimers of BetP. Within each monomer, ten to 12 density peaks characteristic of transmembrane α-helices surround low-density regions that define potential transport pathways. Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation. The projection map of BetP shows no clear resemblance to other secondary transporters of known structure. [Copyright &y& Elsevier]

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences. [Copyright &y& Elsevier]

De novo folding simulations of the major pVIII coat protein from filamentous fd bacteriophage, using a newly developed implicit membrane generalized Born model and replica-exchange molecular dynamics, are presented and discussed. The quality of the predicted structures, judged by comparison of the root-mean-square deviations of a room temperature ensemble of conformations from the replica-exchange simulations and experimental structures from both solid-state NMR in lipid bilayers and solution-phase NMR on the protein in micelles, was quite good, reinforcing the general quality of the folding simulations. The transmembrane helical segment of the protein was well defined in comparison with experiment and the amphipathic helical fragment remained at the membrane/aqueous phase boundary while undergoing significant conformational flexibility due to the loop connecting the two helical segments of the protein. Additional comparisons of computed solid-state NMR properties, the 15N chemical shift and 15N–1H dipolar coupling constants, showed semi-quantitative agreement with the corresponding measurements. These findings suggest an emerging potential for the de novo investigation of integral membrane peptides and proteins and a mechanism to assist experimental approaches to the characterization and structure determination of these important systems. [Copyright &y& Elsevier]

Integral membrane proteins often contain proline residues in their α-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In addition, both glycosylation mapping in biological membranes and molecular dynamics showed that the presence of a proline residue at the lipid/water interface has as an effect the extension of the helical end. Thus, helix packing can be an important factor that determines appearance of proline in TM helices. Membrane proteins might accumulate proline residues at the two ends of their TM segments in order to modulate the exposition of key amino acid residues at the interface for molecular recognition events while allowing stable association and native folding. [Copyright &y& Elsevier]

The Bacillus subtilis chemoreceptor McpB is a dimer of identical subunits containing two transmembrane (TM) segments (TM1, residues 17–34: TM2, residues 280–302) in each monomer with a 2-fold axis of symmetry. To study the organization of the TM domains, the wild-type receptor was mutated systematically at the membrane bilayer/extracytoplasmic interface with 15 single cysteine (Cys) substitutions in each of the two TM domains. Each single Cys substitution was capable of complementing a null allele in vivo, suggesting that no significant perturbation of the native tertiary or quaternary structure of the chemoreceptor was introduced by the mutations. On the basis of patterns of disulfide crosslinking between subunits of the dimeric receptor, an α-helical interface was identified between TM1 and TM1′ (containing residues 32, 36, 39, and 43) and between TM2 and TM2′ (containing residues 276, 277, 280, 283 and 286). Pairs of cysteine substitutions (positions 34/280 and 38/273) in TM1 and TM2 were used to further elucidate specific contacts within a monomer subunit, enabling a model to be constructed defining the organization of the TM domain. Crosslinking of residues that were 150–180° removed from position 32 (positions 37, 41, and 44) suggested that the receptors may be organized as an array of trimers of dimers in vivo. All crosslinking was unaffected by deletion of cheB and cheR (loss of receptor demethylation/methylation enzymes) or by deletion of cheW and cheV (loss of proteins that couple receptors with the autophosphorylating kinase). These findings indicate that the organization of the transmembrane region and the stability of the quaternary complex of receptors are independent of covalent modifications of the cytoplasmic domain and conformations in the cytoplasmic domain induced by the coupling proteins. [Copyright &y& Elsevier]

Polar residues are capable of mediating the association of membrane-embedded helices through the formation of side-chain/side-chain inter-helical hydrogen bonds. However, the extent to which native van der Waals packing of the residues surrounding the polar locus can enhance, or interfere with, the interaction of polar residues has not yet been studied. We examined the propensities of four polar residues (aspartic acid, asparagine, glutamic acid, and glutamine) to promote self-association of transmembrane (TM) domains in several biologically derived sequence environments, including (i) four naturally occurring TM domains that contain a Glu or Gln residue (Tnf5/CD40 ligand, C79a/Ig-α, C79b/Ig-β, and Fut3/α-fucosyltransferase); and (ii) variants of bacteriophage M13 major coat protein TM segment with Asp and Asn at interfacial and non-interfacial positions. Self-association was quantified by the TOXCAT assay, which measures TM helix self-oligomerization in the Escherichia coli inner membrane. While an appropriately placed polar residue was found in several cases to significantly stabilize TM helix–helix interactions through the formation of an interhelical hydrogen bond, in other cases the strongly polar residues did not enhance the association of the two helices. Overall, these results suggest that an innate structural mechanism may operate to control non-specific association of membrane-embedded polar residues. [Copyright &y& Elsevier]

The twin-arginine translocation (Tat) system transports folded proteins across bacterial plasma membranes and the chloroplast thylakoid membrane. Here, we investigate the composition and structural organization of three different purified Tat complexes from Escherichia coli, Salmonella typhimurium and Agrobacterium tumefaciens. First, we demonstrate the functional activity of these Tat systems in vivo, since expression of the tatABC operons from S. typhimurium or A. tumefaciens in an E. coli tat null mutant strain resulted in efficient Tat-dependent export of an E. coli cofactor-containing substrate, TMAO reductase. The three isolated, affinity-tagged Tat complexes comprised TatA, TatB and TatC in each case, demonstrating a strong interaction between these three subunits. Single-particle electron microscopy studies of all three complexes revealed approximately oval-shaped, asymmetric particles with maximal dimensions up to 13 nm. A common feature is a number of stain-excluding densities surrounding more or less central pools of stain, suggesting protein-lined pores or cavities. The characteristics of size variation among the particles suggest a modular form of assembly and/or the recruitment of varying numbers of TatBC/TatA units. Despite low levels of sequence homology, the combined data indicate structural and functional conservation in the Tat systems of these three bacterial species. [Copyright &y& Elsevier]

We describe an effective procedure for modeling the structures of simple transmembrane helix homo-oligomers. The method differs from many previous approaches in that the only structural constraint we use to help select the correct model is the oligomerization state of the protein. The method involves the following steps: (1) perform 100–250 independent Monte Carlo energy minimizations of helix pairs to produce a large collection of well-packed structures; (2) filter the minimized structures to find those that are consistent with the expected symmetry of the oligomer; (3) cluster the structures that pass the symmetry filter; and (4) select a representative of the most populous cluster as the final prediction. We applied the method to the transmembrane helices of five proteins and compare our results to the available experimental data. Our predictions of glycophorin A, neu, the M2 channel and phospholamban resulted in a single model for each protein that agreed with the experimental results. In the case of erbB-2, however, we obtained three structurally distinct clusters of approximately equal sizes, so it was not possible to identify a clearly favored structure. This may reflect a real heterogeneity of packing modes for erbB-2, which is known to interact with different receptor subunits. Our method should be useful for obtaining structural models of transmembrane domains, improving our understanding of structure/function relationships for particular membrane proteins. [Copyright &y& Elsevier]

We produced the human leukotriene B4 (LTB4) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% α-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB4 with Ka=7.8(±0.2)×108 M−1 and a stoichiometric ratio of 0.98(±0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB4. We report evidence that both partners, LTB4 and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB4 conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix. [Copyright &y& Elsevier]

Higher-order interactions are important for protein folding and assembly. We introduce the concept of interhelical three-body interactions as derived from Delaunay triangulation and alpha shapes of protein structures. In addition to glycophorin A, where triplets are strongly correlated with protein stability, we found that tight interhelical triplet interactions exist extensively in other membrane proteins, where many types of triplets occur far more frequently than in soluble proteins. We developed a probabilistic model for estimating the value of membrane helical interaction triplet (MHIT) propensity. Because the number of known structures of membrane proteins is limited, we developed a bootstrap method for determining the 95% confidence intervals of estimated MHIT values. We identified triplets that have high propensity for interhelical interactions and are unique to membrane proteins, e.g. AGF, AGG, GLL, GFF and others. A significant fraction (32%) of triplet types contains triplets that may be involved in interhelical hydrogen bond interactions, suggesting the prevalent and important roles of H-bond in the assembly of TM helices. There are several well-defined spatial conformations for triplet interactions on helices with similar parallel or antiparallel orientations and with similar right-handed or left-handed crossing angles. Often, they contain small residues and correspond to the regions of the closest contact between helices. Sequence motifs such as GG4 and AG4 can be part of the three-body interactions that have similar conformations, which in turn can be part of a higher-order cooperative four residue spatial motif observed in helical pairs from different proteins. In many cases, spatial motifs such as serine zipper and polar clamp are part of triplet interactions. On the basis of the analysis of the archaeal rhodopsin family of proteins, tightly packed triplet interactions can be achieved with several different choices of amino acid residues. [Copyright &y& Elsevier]

Voltage-sensitive K+ channels (Kv) serve numerous important roles, e.g. in the control of neuron excitability and the patterns of synaptic activity. Here, we use electron microscopy (EM) and single particle analysis to obtain the first, complete structure of Kv1 channels, purified from rat brain, which contain four transmembrane channel-forming α-subunits and four cytoplasmically-associated β-subunits. The 18 A˚ resolution structure reveals an asymmetric, dumb-bell-shaped complex with 4-fold symmetry, a length of 140 A˚ and variable width. By fitting published X-ray data for recombinant components to our EM map, the modulatory (β)4 was assigned to the innermost 105 A˚ end, the N-terminal (T1)4 domain of the α-subunit to the central 50 A˚ moiety and the pore-containing portion to the 125 A˚ membrane part. At this resolution, the selectivity filter could not be localised. Direct contact of the membrane component with the central (T1)4 domain occurs only via peripheral connectors, permitting communication between the channel and β-subunits for coupling of responses to changes in excitability and metabolic status of neurons. [Copyright &y& Elsevier]

The mechanism of membrane insertion and folding of a β-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded β-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles. We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA. Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer. When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively. The formation of β-sheet secondary structure and closure of the β-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer. OmpA, and presumably other β-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of α-helical bundle membrane proteins. These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins. [Copyright &y& Elsevier]

Proline residues in the transmembrane (TM) α-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM α-helices. We have examined 199 transmembrane α-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM α-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments. [Copyright &y& Elsevier]

The Na,K-ATPase, or sodium pump, is a ubiquitously expressed membrane-bound enzyme that controls the transmembrane (TM) gradients of sodium and potassium ions in animal cells. The enzyme comprises two subunits, α and β, and in the kidney, is also associated with a small single-spanning membrane protein, the γ subunit. This 65 amino acid residues protein has been linked to a form of dominant renal hypomagnesaemia resulting from substitution of a highly conserved glycine residue (Gly41) to arginine residue. In order to characterize the quaternary structure of the γ subunit, and effects of the G41R mutation thereupon, we synthesized a series of peptides (wild-type and mutant) that span the γ subunit TM region. Using circular dichroism spectroscopy, we show that the 32-amino acid residue peptides are random coils in aqueous buffer but spontaneously adopt an α-helical conformation in the presence of detergent micelles (sodium dodecyl sulfate, SDS, and perfluorooctanoate, PFO). Furthermore, fluorescence resonance energy transfer experiments, combined with polyacrylamide gel electrophoresis, demonstrate that while γ-TM does not self-associate in SDS, it forms oligomers in PFO, a detergent that tolerates relatively weak associations between membrane proteins. Importantly, oligomerization of γ-TM is abrogated in a peptide that contains either the disease-causing mutation G41R, or the more conservative mutation G41L. On the other hand, a peptide that contains a Gly-to-Arg substitution on a different face of the helix, at position 35, retains its ability to oligomerize. Our results provide evidence for a link between renal hypomagnesaemia and γ subunit oligomerization. [Copyright &y& Elsevier]

Pairs of helices in transmembrane (TM) proteins are often tightly packed. We present a scoring function and a computational methodology for predicting the tertiary fold of a pair of α-helices such that its chances of being tightly packed are maximized. Since the number of TM protein structures solved to date is small, it seems unlikely that a reliable scoring function derived statistically from the known set of TM protein structures will be available in the near future. We therefore constructed a scoring function based on the qualitative insights gained in the past two decades from the solved structures of TM and soluble proteins. In brief, we reward the formation of contacts between small amino acid residues such as Gly, Cys, and Ser, that are known to promote dimerization of helices, and penalize the burial of large amino acid residues such as Arg and Trp. As a case study, we show that our method predicts the native structure of the TM homodimer glycophorin A (GpA) to be, in essence, at the global score optimum. In addition, by correlating our results with empirical point mutations on this homodimer, we demonstrate that our method can be a helpful adjunct to mutation analysis. We present a data set of canonical α-helices from the solved structures of TM proteins and provide a set of programs for analyzing it (http://ashtoret.tau.ac.il/~sarel). From this data set we derived 11 helix pairs, and conducted searches around their native states as a further test of our method. Approximately 73% of our predictions showed a reasonable fit (RMS deviation <2 A˚) with the native structures compared to the success rate of 8% expected by chance. The search method we employ is less effective for helix pairs that are connected via short loops (<20 amino acid residues), indicating that short loops may play an important role in determining the conformation of α-helices in TM proteins. [Copyright &y& Elsevier]

Proline residues in the helical segments of soluble and transmembrane proteins have received special attention from both a structural and functional perspective. A feature of these helices is the structural distortion termed “proline-kink”, which has been associated with the presence of the proline residue. However, a recent report on the yeast heat-shock transcription factor of Kluyveromyces lactis (HSF_KL) suggests that these proline-associated deformations can be achieved in the absence of proline residues, thus raising the question of the mechanisms responsible for the structural mimicry of proline-related features. In this study, the specific interactions responsible for the distortion were characterized by comparative analysis of the atomic details of the packing interactions that surround the evolutionarily conserved proline-kink in the α2 helix of HSF_KL and a set of 39 structurally related proteins that lacked the distortion. The mechanistic details inferred from this analysis were confirmed with molecular dynamics simulations. The study shows that the packing interactions between the α2 and α1 helices in HSF_KL are responsible for the stabilization of the conserved kink, whether a proline residue that divides the helix into segments is present or not. The proline-kink can facilitate the formation of tertiary packing interactions that would otherwise not be possible. However, it is the ability to establish differential packing interactions for the helix segments, rather than the structural properties of the proline-kink itself, that emerges as the key factor for the characteristic distortion. [Copyright &y& Elsevier]