Structure-based statistical analysis of transmembrane helices

Recent advances in determination of the high-resolution structure of membrane proteins now enable analysis of the main features of amino acids in transmembrane (TM) segments in comparison with amino acids in water-soluble helices. In this work, we conducted a large-scale analysis of the prevalent locations of amino acids by using a data set of 170 structures of integral membrane proteins obtained from the MPtopo database and 930 structures of water-soluble helical proteins obtained from the protein data bank. Large hydrophobic amino acids (Leu, Val, Ile, and Phe) plus Gly were clearly prevalent in TM helices whereas polar amino acids (Glu, Lys, Asp, Arg, and Gln) were less frequent in this type of helix. The distribution of amino acids along TM helices was also examined. As expected, hydrophobic and slightly polar amino acids are commonly found in the hydrophobic core of the membrane whereas aromatic (Trp and Tyr), Pro, and the hydrophilic amino acids (Asn, His, and Gln) occur more frequently in the interface regions. Charged amino acids are also statistically prevalent outside the hydrophobic core of the membrane, and whereas acidic amino acids are frequently found at both cytoplasmic and extra-cytoplasmic interfaces, basic amino acids cluster at the cytoplasmic interface. These results strongly support the experimentally demonstrated biased distribution of positively charged amino acids (that is, the so-called the positive-inside rule) with structural data.     

TM Finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales

Based on the principle of dual prediction by segment hydrophobicity and nonpolar phase helicity, in concert with imposed threshold values of these two parameters, we developed the automated prediction program TM Finder that can successfully locate most transmembrane (TM) segments in proteins. The program uses the results of experiments on a series of host-guest TM segment mimic peptides of prototypic sequence KK AAAXAAAAAXAAWAAXAAAKKKK-amide (where X = each of the 20 commonly occurring amino acids) through which an HPLC-derived hydropathy scale, a hydrophobicity threshold for spontaneous membrane insertion, and a nonpolar phase helical propensity scale were determined. Using these scales, the optimized prediction algorithm of TM Finder defines TM segments by first searching for competent core segments using the combination of hydrophobicity and helicity scales, and then performs a gap-joining operation, which minimizes prediction bias caused by local hydrophilic residues and/or the choice of window size. In addition, the hydrophobicity threshold requirement enables TM Finder to distinguish reliably between membrane proteins and globular proteins, thereby adding an important dimension to the program. A full web version of the TM Finder program can be accessed at http://www.bioinformatics-canada.org/TM/.     

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.     

Empirical lipid propensities of amino acid residues in multispan alpha helical membrane proteins

Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (TransMembrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of high-resolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 A. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis.     

UMARS: Un-MAppable Reads Solution

Background

Few high-resolution structures of integral membranes proteins are available, as crystallization of such proteins needs yet to overcome too many technical limitations. Nevertheless, prediction of their transmembrane (TM) structure by bioinformatics tools provides interesting insights on the topology of these proteins.

Methods

We describe here how to extract new information from the analysis of hydrophobicity variations or hydrophobic pulses (HPulses) in the sequence of integral membrane proteins using the Hydrophobic Pulse Predictor, a new tool we developed for this purpose. To analyze the primary sequence of 70 integral membrane proteins we defined two levels of analysis: G1-HPulses for sliding windows of n = 2 to 6 and G2-HPulses for sliding windows of n = 12 to 16.

Results

The G2-HPulse analysis of 541 transmembrane helices allowed the definition of the new concept of transmembrane unit (TMU) that groups together transmembrane helices and segments with potential adjacent structures. In addition, the G1-HPulse analysis identified helix irregularities that corresponded to kinks, partial helices or unannotated structural events. These irregularities could represent key dynamic elements that are alternatively activated depending on the channel status as illustrated by the crystal structures of the lactose permease in different conformations.

Conclusions

Our results open a new way in the understanding of transmembrane secondary structures: hydrophobicity through hydrophobic pulses strongly impacts on such embedded structures and is not confined to define the transmembrane status of amino acids.     

Transmembrane helices before, during, and after insertion

The transmembrane (TM) helix is the fundamental structural unit of helix-bundle membrane proteins. Recent biophysical studies provide new insights into the interactions of TM helices with each other and with membrane lipid bilayers. The biological process of helix insertion is carried out by translocon complexes acting in concert with ribosomes. An electron cryo-microscopic reconstruction of these complexes reveals their architecture in new detail, and shows that the complex is constructed from four SecY/Sec61 heterotrimers and two TRAP complexes. A disulfide bridge study shows that elongating polypeptide chains pass through the pore previously identified in the X-ray structure of an archaeal SecY heterotrimer. The fundamental code used by the translocon to select polypeptide segments for insertion as TM helices has been broken. A detailed analysis of the TM amino acid distributions of helix-bundle membrane proteins of known structure recapitulates this code.     

Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.     

Prediction of protein helices with a derivative of the strip-of-helix hydrophobicity algorithm

The strip-of-helix hydrophobicity algorithm was devised to identify protein sequences which, when coiled as alpha or 3(10) helices, had one axial, hydrophobic strip and otherwise variably hydrophilic residues. The strip-of-helix hydrophobicity algorithm also ranked such sequences according to an index, the mean hydrophobicity of amino acids in the axial strip. This algorithm well predicted T cell-presented fragments of antigenic proteins. A derivative of this algorithm (the structural helices algorithm (SHA] was tested for the prediction of helices in crystallographically defined proteins. For the SHA, eight amino acid sequences, 2 cycles plus one amino acid in an alpha helix, with strip-of-helix hydrophobicity indices greater than 2.5, were selected with overlapping segments joined. These selections were terminated according to simple "capping rules," which took into account the roles of N-terminal Asn or Pro and C-terminal Gly in the stability of helices. In analyses of 35 crystallographically defined proteins with known alpha and 3(10) helices, the predictions with the SHA overlapped (had overlap indices x greater than or equal to 0.5) with 34% of known helices, touched (had overlap indices 0.5 greater than x greater than 0) or overlapped with 66% of known helices, or were neighboring (came within 6 residues) or touched or overlapped with 82% of known helices. At each level of judging the quality of prediction, the SHA was usually less sensitive (correct predictions/total number of known helices) and more efficient (correct predictions/total number of predictions) than the Chou-Fasman and Garnier-Robson methods. It was simpler in design and calculation. The chemical mechanisms underlying these algorithms appear to apply both to protein folding and to selection of T cell-presented antigenic sequences.     

Exploring the sequence patterns in the alpha-helices of proteins

This paper reports an extensive sequence analysis of the alpha-helices of proteins. alpha-Helices were extracted from the Protein Data Bank (PDB) and were divided into groups according to their sizes. It was found that some amino acids had differential propensity values for adopting helical conformation in short, medium and long alpha-helices. Pro and Trp had a significantly higher propensity for helical conformation in short helices than in medium and long helices. Trp was the strongest helix conformer in short helices. Sequence patterns favoring helical conformation were derived from a neighbor-dependent sequence analysis of proteins, which calculated the effect of neighboring amino acid type on the propensity of residues for adopting a particular secondary structure in proteins. This method produced an enhanced statistical significance scale that allowed us to explore the positional preference of amino acids for alpha-helical conformations. It was shown that the amino acid pair preference for alpha-helix had a unique pattern and this pattern was not always predictable by assuming proportional contributions from the individual propensity values of the amino acids. Our analysis also yielded a series of amino acid dyads that showed preference for alpha-helix conformation. The data presented in this study, along with our previous study on loop sequences of proteins, should prove useful for developing potential 'codes' for recognizing sequence patterns that are favorable for specific secondary structural elements in proteins.     

Characteristics affecting expression and solubilization of yeast membrane proteins

Biochemical and structural analysis of membrane proteins often critically depends on the ability to overexpress and solubilize them. To identify properties of eukaryotic membrane proteins that may be predictive of successful overexpression, we analyzed expression levels of the genomic complement of over 1000 predicted membrane proteins in a recently completed Saccharomyces cerevisiae protein expression library. We detected statistically significant positive and negative correlations between high membrane protein expression and protein properties such as size, overall hydrophobicity, number of transmembrane helices, and amino acid composition of transmembrane segments. Although expression levels of membrane and soluble proteins exhibited similar negative correlations with overall hydrophobicity, high-level membrane protein expression was positively correlated with the hydrophobicity of predicted transmembrane segments. To further characterize yeast membrane proteins as potential targets for structure determination, we tested the solubility of 122 of the highest expressed yeast membrane proteins in six commonly used detergents. Almost all the proteins tested could be solubilized using a small number of detergents. Solubility in some detergents depended on protein size, number of transmembrane segments, and hydrophobicity of predicted transmembrane segments. These results suggest that bioinformatic approaches may be capable of identifying membrane proteins that are most amenable to overexpression and detergent solubilization for structural and biochemical analyses. Bioinformatic approaches could also be used in the redesign of proteins that are not intrinsically well-adapted to such studies.     

TIM23-mediated insertion of transmembrane α-helices into the mitochondrial inner membrane

While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.     

The position of the Gly-xxx-Gly motif in transmembrane segments modulates dimer affinity.

Although the intrinsic low solubility of membrane proteins presents challenges to their high-resolution structure determination, insight into the amino acid sequence features and forces that stabilize their folds has been provided through study of sequence-dependent helix-helix interactions between single transmembrane (TM) helices. While the stability of helix-helix partnerships mediated by the Gly-xxx-Gly (GG4) motif is known to be generally modulated by distal interfacial residues, it has not been established whether the position of this motif, with respect to the ends of a given TM segment, affects dimer affinity. Here we examine the relationship between motif position and affinity in the homodimers of 2 single-spanning membrane protein TM sequences: glycophorin A (GpA) and bacteriophage M13 coat protein (MCP). Using the TOXCAT assay for dimer affinity on a series of GpA and MCP TM segments that have been modified with either 4 Leu residues at each end or with 8 Leu residues at the N-terminal end, we show that in each protein, centrally located GG4 motifs are capable of stronger helix-helix interactions than those proximal to TM helix ends, even when surrounding interfacial residues are maintained. The relative importance of GG4 motifs in stabilizing helix-helix interactions therefore must be considered not only in its specific residue context but also in terms of the location of the interactive surface relative to the N and C termini of alpha-helical TM segments.     

Hydrophobicity and prediction of the secondary structure of membrane proteins and peptides.

Reliability of the hydropathy method to predict the formation of membrane-spanning alpha-helices by integral membrane proteins and peptides whose structure is known from X-ray crystallography is analysed. It is shown that Kyte-Doolittle hydropathy plots do not predict accurately 22 transmembrane alpha-helices in the reaction centres (RC) of the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides (R-26). The accuracy of prediction for these proteins was improved using an optimised Kyte-Doolittle hydrophobicity scale. However, this hydrophobicity scale did not improve the predictions for the alphabeta-peptides of the B800-850 (LH2) complexes of the photosynthetic bacteria Rhodopseudomonas acidophila and Rhodospirillum molischianum, which were excluded from the optimisation procedure. The best and worst predictions of membrane-spanning alpha-helices for the RC proteins and LH2 peptides, respectively, were obtained with a propensity scale (PRC) calculated from the amino acid sequences and X-ray data for the RC proteins. A propensity scale (PLH) obtained using the amino acid sequences and X-ray data for the alphabeta-peptides of the LH2 complexes did not give an acceptable prediction of the transmembrane segments in the LH2 peptides; moreover, it markedly contradicted the PRC scale. Amino acids have been concluded to have no significant preference to localisation in transmembrane segments. Therefore, the predictive ability of the hydropathy methodology appears to be limited: the number of transmembrane segments can be correctly calculated for the best case only, and the lengths and positions of membrane-spanning alpha-helices in a protein amino acid sequence can not be predicted exactly.     

Intra-Helical Salt Bridge Contribution to Membrane Protein Insertion

Salt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. It is possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied yet. In this work, we show that potentially salt-bridge forming pairs are statistically over-represented in TM-helices. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and whole cell systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute ～0.5 kcal/mol to the apparent free energy of membrane insertion (ΔG_app). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.     

Energetics, stability, and prediction of transmembrane helices

We show that the peptide backbone of an alpha-helix places a severe thermodynamic constraint on transmembrane (TM) stability. Neglect of this constraint by commonly used hydrophobicity scales underlies the notorious uncertainty of TM helix prediction by sliding-window hydropathy plots of membrane protein (MP) amino acid sequences. We find that an experiment-based whole-residue hydropathy scale (WW scale), which includes the backbone constraint, identifies TM helices of membrane proteins with an accuracy greater than 99 %. Furthermore, it correctly predicts the minimum hydrophobicity required for stable single-helix TM insertion observed in Escherichia coli. In order to improve membrane protein topology prediction further, we introduce the augmented WW (aWW) scale, which accounts for the energetics of salt-bridge formation. An important issue for genomic analysis is the ability of the hydropathy plot method to distinguish membrane from soluble proteins. We find that the method falsely predicts 17 to 43 % of a set of soluble proteins to be MPs, depending upon the hydropathy scale used.     

Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix

α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N(cyt)-C(lum) or N(lum)-C(cyt)) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the "missing hydrophobicity" in polytopic membrane proteins.     

Analysis and prediction of helix-helix interactions in membrane channels and transporters

Membrane proteins span a large variety of different functions such as cell-surface receptors, redox proteins, ion channels, and transporters. Proteins with functional pores show different characteristics of helix-helix packing as other helical membrane proteins. We found that the helix-helix contacts of 13 nonhomologous high-resolution structures of membrane channels and transporters are mainly accomplished by weakly polar amino acids (G > S > T > F) that preferably create contacts every fourth residue, typical for right-handed helix crossings. There is a strong correlation between the now available biological hydrophobicity scale and the propensities of the weakly polar and hydrophobic residues to be buried at helix-helix interfaces or to be exposed to the lipids in membrane channels and transporters. The polar residues, however, make no major contribution towards the packing of their transmembrane helices, and are therefore subsumed to be primarily exposed to the polar milieu during the folding process. The contact formation of membrane channels and transporters is therefore ruled by the solubility of the residues, which we suppose to be the driving force for the assembly of their transmembrane helices. By contrast, in 14 nonhomologous high-resolution structures of other membrane protein coils, also large and polar amino acids (D > S > M > Q) create characteristic contacts every 3.5th residues, which is a signature for left-handed helix crossings. Accordingly, it seems that dependent on the function, different concepts of folding and stabilization are realized for helical membrane proteins. Using a sequence-based matrix prediction method these differences are exploited to improve the prediction of buried and exposed residues of transmembrane helices significantly. When the sequence motifs typical for membrane channels and transporters were applied for the prediction of helix-helix contacts the quality of prediction rises by 16% to an average value of 76%, compared to the same approach when only single amino acid positions are taken into account.     

Evolution and adaptation of single-pass transmembrane proteins

A comparative analysis of 6039 single-pass (bitopic) membrane proteins from six evolutionarily distant organisms was performed based on data from the Membranome database. The observed repertoire of bitopic proteins is significantly enlarged in eukaryotic cells and especially in multicellular organisms due to the diversification of enzymes, emergence of proteins involved in vesicular trafficking, and expansion of receptors, structural, and adhesion proteins. The majority of bitopic proteins in multicellular organisms are located in the plasma membrane (PM) and involved in cell communication. Bitopic proteins from different membranes significantly diverge in terms of their biological functions, size, topology, domain architecture, physical properties of transmembrane (TM) helices and propensity to form homodimers. Most proteins from eukaryotic PM and endoplasmic reticulum (ER) have the N-out topology. The predicted lengths of TM helices and hydrophobic thicknesses, stabilities and hydrophobicities of TM α-helices are the highest for proteins from eukaryotic PM, intermediate for proteins from prokaryotic cells, ER and Golgi apparatus, and lowest for proteins from mitochondria, chloroplasts, and peroxisomes. Tyr and Phe residues accumulate at the cytoplasmic leaflet of PM and at the outer leaflet of membranes of bacteria, Golgi apparatus, and nucleus. The propensity for dimerization increases from unicellular to multicellular eukaryotes, from enzymes to receptors, and from intracellular membrane proteins to PM proteins. More than half of PM proteins form homodimers with a 2:1 ratio of right-handed to left-handed helix packing arrangements. The inverse ratio (1:2) was observed for dimers from the ER, Golgi and vesicles.     

Membrane profile-based probabilistic method for predicting transmembrane segments via multiple protein sequence alignment

Prediction of transmembrane (TM) segments of amino acid sequences of membrane proteins is a well-known and very important problem. The accuracy of its solution can be improved for approaches that do not use a homology search in an additional data bank. There is a lack of tested data in this area of research, because information on the structure of membrane proteins is scarce. In this work we created a test sample of structural alignments for membrane proteins. The TM segments of these proteins were mapped according to aligned 3D structures resolved for these proteins. A method for predicting TM segments in an alignment was developed on the basis of the forward-backward algorithm from the HMM theory. This method allows a user not only to predict TM segments, but also to create a probabilistic membrane profile, which can be employed in multiple alignment procedures taking the secondary structure of proteins into account. The method was implemented in a computer program available at http://bioinf.fbb.msu.ru/fwdbck/. It provides better results than the MEMSAT method, which is nearly the only tool predicting TM segments in multiple alignments, without a homology search.     

Why is the biological hydrophobicity scale more accurate than earlier experimental hydrophobicity scales?

The recognition of transmembrane helices by the translocon is primarily guided by the average hydrophobicity of the potential transmembrane helix. However, the exact hydrophobicity of each amino acid can be identified in several different ways. The free energy of transfer for amino acid analogues between a hydrophobic media, for example, octanol and water can be measured or obtained from simulations, the hydrophobicity can also be estimated by statistical properties from known transmembrane segments and finally the contribution of each amino acid type for the probability of translocon recognition has recently been measured directly. Although these scales correlate quite well, there are clear differences between them and it is not well understood which scale represents neither the biology best nor what the differences are. Here, we try to provide some answers to this by studying the ability of different scales to recognize transmembrane helices and predict the topology of transmembrane proteins. From this analysis it is clear that the biological hydrophobicity scale as well scales created from statistical analysis of membrane helices perform better than earlier experimental scales that are mainly based on measurements of amino acid analogs and not directly on transmembrane helix recognition. Using these results we identified the properties of the scales that perform better than other scales. We find, for instance, that the better performing scales consider proline more hydrophilic. This shows that transmembrane recognition is not only governed by pure hydrophobicity but also by the helix preferences for amino acids, as proline is a strong helix breaker. Proteins 2014; 82:2190–2198. © 2014 Wiley Periodicals, Inc.