Sequence context and modified hydrophobic moment plots help identify 'horizontal' surface helices in transmembrane protein structure prediction

Transmembrane proteins make up at least one-fifth of the genome of most organisms and are critical components of key pathways for cell survival and interactions with the environment. The function of helices found at the membrane surface in transmembrane proteins has not been greatly explored, but it is likely that they play an ancillary role to membrane spanning helices and are analogous to the surface active helices of peripheral membrane proteins, being involved in: lipid association, membrane perturbation, transmembrane signal transduction and regulation, and transmembrane helical bundle formation. Due to the difficulties in obtaining high-resolution structural data for this class of proteins, structure-from-sequence predictive methods continue to be developed as a means to obtain structural models for these largely intractable systems. A simple but effective variant of the hydrophobic moment analysis of amino acid sequences is described here as part of a protocol for distinguishing helical sequences that are parallel to or 'horizontal' at the membrane bilayer/aqueous phase interface from helices that are membrane-embedded or located in extra-membranous domains. This protocol when tested on transmembrane spanning protein amino acid sequences not used in its development, was found to be 84-91% accurate when the results were compared to the partition locations in the corresponding structures determined by X-ray crystallography, and 72% accurate in determining which helices lie horizontal or near horizontal at the lipid interface.     

Influence of proline residues in transmembrane helix packing

Integral membrane proteins often contain proline residues in their alpha-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.     

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.     

Folding amphipathic helices into membranes: amphiphilicity trumps hydrophobicity

High amphiphilicity is a hallmark of interfacial helices in membrane proteins and membrane-active peptides, such as toxins and antimicrobial peptides. Although there is general agreement that amphiphilicity is important for membrane-interface binding, an unanswered question is its importance relative to simple hydrophobicity-driven partitioning. We have examined this fundamental question using measurements of the interfacial partitioning of a family of 17-residue amidated-acetylated peptides into both neutral and anionic lipid vesicles. Composed only of Ala, Leu, and Gln residues, the amino acid sequences of the peptides were varied to change peptide amphiphilicity without changing total hydrophobicity. We found that peptide helicity in water and interface increased linearly with hydrophobic moment, as did the favorable peptide partitioning free energy. This observation provides simple tools for designing amphipathic helical peptides. Finally, our results show that helical amphiphilicity is far more important for interfacial binding than simple hydrophobicity.     

基于小波分析的膜蛋白跨膜区段序列分析和预测

膜蛋白是一类结构独特的蛋白质,在各种细胞中普遍存在,发挥着重要的生理功能。目前仅有少数膜蛋白听结构被实验测出,因此用计算机预测膜蛋白的结构是蛋白质结构预测的主要研究内容之一。膜蛋白一般在膜上形成保守的跨膜螺旋结构,序列特征明显,比较适合用预测的方法确定跨膜螺旋区段的位置。国际上已有一些研究者用人工神经网络方法、多序列比对方法和统计方法进行了预测尝试,取得了一定的成功经验。我们对蛋白质序列数据库中的     

Comprehensive analysis of the numbers,lengths and amino acid compositions of transmembrane helices in prokaryotic,eukaryotic and viral integral membrane proteins of high-resolution structure

We report a comprehensive analysis of the numbers, lengths and amino acid compositions of transmembrane helices in 235 high-resolution structures of integral membrane proteins. The properties of 1551 transmembrane helices in the structures were compared with those obtained by analysis of the same amino acid sequences using topology prediction tools. Explanations for the 81 (5.2%) missing or additional transmembrane helices in the prediction results were identified. Main reasons for missing transmembrane helices were mis-identification of N-terminal signal peptides, breaks in α-helix conformation or charged residues in the middle of transmembrane helices and transmembrane helices with unusual amino acid composition. The main reason for additional transmembrane helices was mis-identification of amphipathic helices, extramembrane helices or hairpin re-entrant loops. Transmembrane helix length had an overall median of 24 residues and an average of 24.9 ± 7.0 residues and the most common length was 23 residues. The overall content of residues in transmembrane helices as a percentage of the full proteins had a median of 56.8% and an average of 55.7 ± 16.0%. Amino acid composition was analysed for the full proteins, transmembrane helices and extramembrane regions. Individual proteins or types of proteins with transmembrane helices containing extremes in contents of individual amino acids or combinations of amino acids with similar physicochemical properties were identified and linked to structure and/or function. In addition to overall median and average values, all results were analysed for proteins originating from different types of organism (prokaryotic, eukaryotic, viral) and for subgroups of receptors, channels, transporters and others.     

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic-aqueous medium

The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C. The parameters extracted indicated all peptides were helical in this membrane mimetic solvent. Using chemical shift indices as the criterion, helicity varied from 64 to 83%. The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer. A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides. Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7. Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues. The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor.     

Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.     

Shorter side chains optimize helix-helix packing

A systematic study of helix-helix packing in a comprehensive database of protein structures revealed that the side chains inside helix-helix interfaces on average are shorter than those in the noninterface parts of the helices. The study follows our earlier study of this effect in transmembrane helices. The results obtained on the entire database of protein structures are consistent with those obtained on the transmembrane helices. The difference in the length of interface and noninterface side chains is small but statistically significant. It indicates that helices, if viewed along their main axis, statistically are not circular, but have a flattened interface. This effect brings the helices closer to each other and creates a tighter structural packing. The results provide an interesting insight into the aspects of protein structure and folding.     

Evaluating tilt angles of membrane-associated helices: comparison of computational and NMR techniques

A computational method to calculate the orientation of membrane-associated alpha-helices with respect to a lipid bilayer has been developed. It is based on a previously derived implicit membrane representation, which was parameterized using the structures of 46 alpha-helical membrane proteins. The method is validated by comparison with an independent data set of six transmembrane and nine antimicrobial peptides of known structure and orientation. The minimum energy orientations of the transmembrane helices were found to be in good agreement with tilt and rotation angles known from solid-state NMR experiments. Analysis of the free-energy landscape found two types of minima for transmembrane peptides: i), Surface-bound configurations with the helix long axis parallel to the membrane, and ii), inserted configurations with the helix spanning the membrane in a perpendicular orientation. In all cases the inserted configuration also contained the global energy minimum. Repeating the calculations with a set of solution NMR structures showed that the membrane model correctly distinguishes native transmembrane from nonnative conformers. All antimicrobial peptides investigated were found to orient parallel and bind to the membrane surface, in agreement with experimental data. In all cases insertion into the membrane entailed a significant free-energy penalty. An analysis of the contributions of the individual residue types confirmed that hydrophobic residues are the main driving force behind membrane protein insertion, whereas polar, charged, and aromatic residues were found to be important for the correct orientation of the helix inside the membrane.     

Calculation of the three-dimensional structure of Saccharomyces cerevisiae cytochrome b inserted in a lipid matrix

Cytochrome b is an integral membrane protein, which forms the core of the ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex. A computer-aided three-dimensional modeling procedure was carried out in four steps. First, the candidate hydrophobic helices were searched for throughout the protein primary sequence by a computer procedure based upon the method of Eisenberg; second, a secondary helical structure was imposed to the transmembrane peptides; third, the helical segments at a lipid-water interface were oriented, and finally the possible interactions between helices with similar properties were investigated. This procedure enabled the identification of nine hydrophobic segments, of which eight are membrane-spanning helices while one has amphipathic properties. Three hydrophilic receptor-binding domains were also identified. Based upon their hydrophobicity profiles, the transmembrane helices could be associated in pairs inside the lipid bilayer. In our folding model proposed for cytochrome b, all mutation sites are not only located on the same side of the membrane but are also in close proximity in the three-dimensional structure. Inhibitor resistance mutational sites which were recently characterized (di Rago, J.-P., and Colson, A.-M. (1988) J. Biol. Chem. 263, 12564-12570) have been located on this model. Moreover, the receptor-binding domains and the mutation sites are close neighbors in the three-dimensional spatial representation.     

Biophysical studies on fragments of the α-factor receptor protein

The receptor for the α-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246–269 (M6) and 273–302 (M7)], two of the interhelical loops [residues 107–125 (E2) and 191–206 (E3)], and to a portion of the carboxyl terminus [residues 350–372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop neptides and the carboxyl terminus peptide were either in a β-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein. © 1994 John Wiley & Sons, Inc.     

Helix packing moments reveal diversity and conservation in membrane protein structure

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.     

A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations.

A structural model of the transmembrane portion of the acetylcholine receptor was developed from sequences of all its subunits by using transfer energy calculations to locate transmembrane alpha-helices and to calculate which helical side chains should be in contact with water inside the channel, with portions of other transmembrane helices, or with lipid hydrocarbon chains. "Knobs-into-holes" side chain packing calculations were used with other factors to stack the transmembrane alpha-helices together. In the model each subunit has the following structures in order along the sequence from the NH2 terminus: a large extracellular domain of undetermined structure, a short apolar alpha-helix that lies on the extracellular lipid surface of the membrane; three apolar transmembrane alpha-helices (I, II, and III), a cytoplasmic domain of undetermined structure, an amphipathic transmembrane alpha-helix (L) that forms the channel lining, a short extracellular alpha-helix, another apolar transmembrane alpha-helix (IV), and a small cytoplasmic domain formed by the COOH-terminal end of the chain. Three concentric layers form the pore. A bundle of five amphipathic L helices forms the channel lining. This bundle is surrounded by a bundle of 10 alternating II and III helices. Helices I and IV cover portions of the outer surface of the bundle formed by helices II and III. Positions of disulfide bridges are predicted and a mechanism for opening and closing conformational changes is proposed that requires tilting transmembrane helices and possibly a thiol-disulfide interchange reaction.     

Interactions involved in the realignment of membrane-associated helices. An investigation using oriented solid-state NMR and attenuated total reflection Fourier transform infrared spectroscopies

A series of histidine-containing peptides (LAH4X6) was designed to investigate the membrane interactions of selected side chains. To this purpose, their pH-dependent transitions from in-plane to transmembrane orientations were investigated by attenuated total reflection Fourier transform infrared and oriented solid-state NMR spectroscopies. Peptides of the same family have previously been shown to exhibit antibiotic and DNA transfection activities. Solution NMR spectroscopy indicates that these peptides form amphipathic helical structures in membrane environments, and the technique was also used to characterize the pK values of all histidines in the presence of detergent micelles. Whereas one face of the amphipathic helix is clearly hydrophobic, the opposite side is flanked by four histidines surrounding six leucine, alanine, glycine, tryptophan, or tyrosine residues, respectively. This diversity in peptide composition causes pronounced shifts in the midpoint pH of the in-plane to transmembrane helical transition, which is completely abolished for the peptides carrying the most hydrophilic amino acid residues. These properties open up a conceptually new approach to study in a quantitative manner the hydrophobic as well as specific interactions of amino acids in membranes. Notably, the resulting scale for whole residue transitions from the bilayer interface to the hydrophobic membrane interior is obtained from extended helical sequences in lipid bilayers.     

Computational study of lipid-destabilizing protein fragments: towards a comprehensive view of tilted peptides

Tilted peptides are short sequence fragments (10-20 residues long) that possess an asymmetric hydrophobicity gradient along their sequence when they are helical. Due to this gradient, they adopt a tilted orientation towards a single lipid/water interface and destabilize the lipids. We have detected those peptides in many different proteins with various functions. While being all tilted-oriented at a single lipid/water interface, no consensus sequence can be evidenced. In order to better understand the relationships between their lipid-destabilizing activity and their properties, we used IMPALA to classify the tilted peptides. This method allows the study of interactions between a peptide and a modeled lipid bilayer using simple restraint functions designed to mimic some of the membrane properties. We predict that tilted peptides have access to a wide conformational space in membranes, in contrast to transmembrane and amphipathic helices. In agreement with previous studies, we suggest that those metastable configurations could lead to the perturbation of the acyl chains organization and could be a general mechanism for lipid destabilization. Our results further suggest that tilted peptides fall into two classes: those from proteins acting on membrane behave differently than destabilizing fragments from interfacial proteins. While the former have equal access to the two layers of the membrane, the latter are confined within a single lipid layer. This could be in relation with the organization of lipid substrate on which the peptides physiologically act.     

Inter-helical hydrogen bond formation during membrane protein integration into the ER membrane

Recent work has shown that efficient di- or trimerization of hydrophobic transmembrane helices in detergent micelles or lipid bilayers can be driven by inter-helix hydrogen bonding involving polar residues such as Asn or Asp. Using in vitro translation in the presence of rough microsomes of a model integral membrane protein, we now show that the formation of so-called helical hairpins, two tightly spaced transmembrane helices connected by a short loop, can likewise be promoted by the introduction of Asn-Asn or Asp-Asp pairs in a long transmembrane hydrophobic segment. These observations suggest that inter-helix hydrogen bonds can form within the context of the Sec61 translocon in the endoplasmic reticulum, implying that hydrophobic segments in a nascent polypeptide chain in transit through the Sec61 channel have immediate access to a non-aqueous subcompartment within the translocon.     

Biophysical studies of signal peptides: Implications for signal sequence functions and the involvement of lipid in protein export

This review discusses efforts to understand the mode of action of signal sequences by biophysical study of synthetic peptides corresponding to these protein localization signals. On the basis of reports from several laboratories, it is now clear that signal peptides may adopt a variety of conformations, depending on their local environment. In membrane-mimetic systems like detergent micelles or lipid vesicles, they have a high tendency to form helices. Ability to take up a helical conformation appears to be required at some point in the function of a signal sequence, since some peptides corresponding to export-defective signal sequences display reduced helical potential. By contrast, functional signal sequences share a high capacity to adopt helices. High affinity for organized lipid assemblies, like monolayers or vesicles, is also a property of functional signal sequences. This correlation suggests a role for direct interaction of signal sequences with the lipids of the cytoplasmic membranein vivo. Supporting this role are studies of the influence of signal peptides on lipid structure, which reveal an ability of these peptides to pertub lipid packing and to alter the phase state of the lipids. Insertion of the signal sequencein vivo could substantially reduce the barrier for translocation of the mature chain. Lastly, synthetic signal peptides have been added to native membranes and found to inhibit translocation of precursor proteins. This approach bridges the biophysical and the biochemical aspects of protein export and promises to shed light on the functional correlates of the properties and interactions observed in model systems.     

Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers

Amphipathic helices in membrane proteins that interact with the hydrophobic/hydrophilic interface of the lipid bilayer have been difficult to structurally characterize. Here, the backbone structure and orientation of an amphipathic helix in the full-length M2 protein from influenza A virus has been characterized. The protein has been studied in hydrated DMPC/DMPG lipid bilayers above the gel to liquid-crystalline phase transition temperature by solid-state NMR spectroscopy. Characteristic PISA (Polar Index Slant Angle) wheels reflecting helical wheels have been observed in uniformly aligned bilayer preparations of both uniformly 15N labeled and amino acid specific labeled M2 samples. Hydrogen/deuterium exchange studies have shown the very slow exchange of some residues in the amphipathic helix and more rapid exchange for the transmembrane helix. These latter results clearly suggest the presence of an aqueous pore. A variation in exchange rate about the transmembrane helical axis provides additional support for this claim and suggests that motions occur about the helical axes in this tetramer to expose the entire backbone to the pore.     

Studies of the minimum hydrophobicity of alpha-helical peptides required to maintain a stable transmembrane association with phospholipid bilayer membranes

The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.