Turns in transmembrane helices: determination of the minimal length of a "helical hairpin" and derivation of a fine-grained turn propensity scale.

We have recently reported a first experimental turn propensity scale for transmembrane helices. This scale was derived from measurements of how efficiently a given residue placed in the middle of a 40 residue poly(Leu) stretch induces the formation of a "helical hairpin" with two rather than one transmembrane segment. We have now extended these studies, and have determined the minimum length of a poly(Leu) stretch compatible with the formation of a helical hairpin. We have also derived a more fine-grained turn propensity scale by (i) introducing each of the 20 amino acid residues into the middle of the shortest poly(Leu) stretch compatible with helical hairpin formation, and (ii) introducing pairs of residues in the middle of the 40 residue poly(Leu) stretch. The new turn propensities are consistent with the amino acid frequencies found in short hairpin loops in membrane proteins of known 3D structure.     

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.     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.     

Highly restricted distributions of hydrophobic and charged amino acids in longitudinal quadrants of alpha-helices

Helix formation in folding proteins is stabilized by binding of recurrent hydrophobic side chains in one longitudinal quadrant against the locally most hydrophobic region of the protein. To test this hypothesis, we fitted sequences of 247 alpha-helices of 55 proteins to the circular (infinite) template (symbol; see text) to maximize the strip-of-helix hydrophobicity index (the mean hydrophobicity of residues in (symbol; see text) positions). These template-predicted configurations closely matched crystallographic structures in 87% of four- or five-turn helices compared. We determined the longitudinal quadrant distributions of amino acids in the template-fitted, sheet projections of alpha-helices with respect to the best longitudinal, hydrophobic strip on each helix and to the N and C termini, interiors, and entire helices. Amino acids Leu, Ile, Val, and Phe were concentrated in one longitudinal quadrant (p less than 0.001). Lys, Arg, Asp, and Glu were not in the quadrant of Leu, Ile, Val, and Phe (p less than 0.001). Significant quadrant distributions for other amino acids and for termini of the helices were also found.     

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.     

Comparison of helix interactions in membrane and soluble alpha-bundle proteins

Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble alpha-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with respect to membrane protein structure prediction and membrane protein stability.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

A structural dissection of amino acid substitutions in helical transmembrane proteins

The evolution of protein folds is under strong constraints from their surrounding environment. Although folding in water‐soluble proteins is driven primarily by hydrophobic forces, the nature of the forces that determine the folding and stability of transmembrane proteins are still not fully understood. Furthermore, the chemically heterogeneous lipid bilayer has a non‐uniform effect on protein structure. In this article, we attempt to get an insight into the nature of this effect by examining the impact of various types of local structure environment on amino acid substitution, based on alignments of high‐resolution structures of polytopic helical transmembrane proteins combined with sequences of close homologs. Compared to globular proteins, burying amino acid sidechains, especially hydrophilic ones, led to a lower increase in conservation in both the lipid‐water interface region and the hydrocarbon core region. This observation is due to surface residues in HTM proteins especially in the HC region being relatively highly conserved, suggesting higher evolutionary constraints from their specific interactions with the surrounding lipid molecules. Polar and small residues, particularly Pro and Gly, show a noticeable increase in conservation as they are positioned more towards the centre of the membrane, which is consistent with their recognized key roles in structural stability. In addition, the examination of hydrogen bonds in the membrane environment identified some exposed hydrophilic residues being better conserved when not hydrogen‐bonded to other residues, supporting the importance of lipid‐protein sidechain interactions. The conclusions presented in this study highlight the distinct features of substitution matrices that take into account the membrane environment, and their potential role in improving sequence‐structure alignments of transmembrane proteins. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Amphiphilicity index of polar amino acids as an aid in the characterization of amino acid preference at membrane-water interfaces

MOTIVATION: An amphiphilicity index of amino acid residues was developed for improving the method of transmembrane helix prediction. RESULTS: The transfer energy of a hydrocarbon stem group beyond the gamma-carbon was calculated from the accessible surface area, and used to index the amphiphilicity of the residue. Non-zero amphiphilicity index values were obtained for lysine, arginine, histidine, glutamic acid, glutamine, tyrosine and tryptophan. Those residues were found to be abundant in the end regions of transmembrane helices, indicating their preference for the membrane-water interface. The moving average of the amphiphilicity index actually showed significant peaks in the end regions of most transmembrane helices. A dispersion diagram of average amphiphilicity index versus average hydrophobicity index was devised to facilitate discrimination of transmembrane helices. AVAILABILITY: The amphiphilicity index has been incorporated into a system, SOSUI, for the discrimination of membrane proteins and the prdiction of tranmembrane helical regions (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html).     

Are membrane proteins "inside-out" proteins?

One of the central paradigms of structural biology is that membrane proteins are "inside-out" proteins, in that they have a core of polar residues surrounded by apolar residues. This is the reverse of the characteristics found in water-soluble proteins. We have decided to test this paradigm, now that sufficient numbers of transmembrane alpha-helical structures are accessible to statistical analysis. We have analyzed the correlation between accessibility and hydrophobicity of both individual residues and complete helices. Our analyses reveal that hydrophobicity of residues in a transmembrane helical bundle does not correlate with any preferred location and that the hydrophilic vector of a helix is a poor indicator of the solvent exposed face of a helix. Neither polar nor hydrophobic residues show any bias for the exterior or the interior of a transmembrane domain. As a control, analysis of water-soluble helical bundles performed in a similar manner has yielded clear correlations between hydrophobicity and accessibility. We therefore conclude that, based on the data set used, membrane proteins as "inside-out" proteins is an unfounded notion, suggesting that packing of alpha-helices in membranes is better understood by maximization of van der Waal's forces, rather than by a general segregation of hydrophobicities driven by lipid exclusion.     

Amino-acid solvation structure in transmembrane helices from molecular dynamics simulations

Understanding the solvation of amino acids in biomembranes is an important step to better explain membrane protein folding. Several experimental studies have shown that polar residues are both common and important in transmembrane segments, which means they have to be solvated in the hydrophobic membrane, at least until helices have aggregated to form integral proteins. In this work, we have used computer simulations to unravel these interactions on the atomic level, and classify intramembrane solvation properties of amino acids. Simulations have been performed for systematic mutations in poly-Leu helices, including not only each amino acid type, but also every z-position in a model helix. Interestingly, many polar or charged residues do not desolvate completely, but rather retain hydration by snorkeling or pulling in water/headgroups--even to the extent where many of them exist in a microscopic polar environment, with hydration levels corresponding well to experimental hydrophobicity scales. This suggests that even for polar/charged residues a large part of solvation cost is due to entropy, not enthalpy loss. Both hydration level and hydrogen bonding exhibit clear position-dependence. Basic side chains cause much less membrane distortion than acidic, since they are able to form hydrogen bonds with carbonyl groups instead of water or headgroups. This preference is supported by sequence statistics, where basic residues have increased relative occurrence at carbonyl z-coordinates. Snorkeling effects and N-/C-terminal orientation bias are directly observed, which significantly reduces the effective thickness of the hydrophobic core. Aromatic side chains intercalate efficiently with lipid chains (improving Trp/Tyr anchoring to the interface) and Ser/Thr residues are stabilized by hydroxyl groups sharing hydrogen bonds to backbone oxygens.     

Local hydrophobicity stabilizes secondary structures in proteins

The probability of occurrence of helix and β-sheet residues in 47 globular proteins was determined as a function of local hydrophobicity, which was defined by the sum of the Nozaki-Tanford transfer free energies at two nearest-neighbors on both sides of the amino acid sequence. In general, hydrophilic amino acids favor neither helix nor β-sheet formations when neighbor residues are also hydrophilic but favor helix formation at higher local hydrophobicity. On the other hand, some hydrophobic amino acids such as Met, Leu, and Ile favor helix formation when neighbor residues are hydrophilic. None of the hydrophobic amino acids favor β-sheet formation with hydrophilic neighbors, but most of them strongly favor β-sheet formation at high local hydrophobicity. When the average of 20 amino acids is taken, both helix and β-sheet residue probabilities are higher at higher local hydrophobicity, although the increase is steeper for β-sheets. Therefore, β-sheet formation is more influenced by local hydrophobicity than helix formation. Generally, helices are nearer the surface and tend to have hydrophilic and hydrophobic faces at opposite sides. The tendency of alternating regions of hydrophilic and hydrophobic residues in a helical sequence was revealed by calculating the correlation of the Nozaki-Tanford values. Such amphipathic helices may be important in protein–protein and protein–lipid interactions and in forming hydrophilic channels in the membrane. The choice of 30 nonhomologous proteins as the data set did not alter the above results.     

Alpha-periodicity analysis of small multidrug resistance (SMR) efflux transporters.

Proteins in the small multidrug resistance (SMR) family of transport proteins are about 110 amino acids in length and are predicted to have four transmembrane helices. This family is divided into a two groups, one of which we have referred to as small multidrug pumps (Smp) and confer resistance to a wide variety of quaternary ammonium compounds through a proton-drug efflux antiport mechanism. Members of the second group within this family have, as yet, not had their substrate profile characterized and are referred to as Sug proteins. Alpha-periodicity analysis was conducted on a set of six homologous proteins of the SMR family consisting of three established Smp and three Sug proteins. Several amino acid properties were used in the analysis including hydropathy, variability, and a substitution matrix for lipid exposed amino acids. The scanning window was varied between 8 and 14 residues and the alpha-periodicity was calculated from the peaks in the Fourier transform power spectra in the region between 3.0 and 4.3 residues/turn. This analysis adds to the hydropathy analysis to give a more confident prediction of which residues are within the lipid bilayer for each of the four transmembrane helices. Information was also obtained that allowed for the identification of zones within each transmembrane helix that face the interior of the helical bundle on one side and are lipid exposed on the other face.     

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.     

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.     

Deuterium/hydrogen exchange factors measured by solution nuclear magnetic resonance spectroscopy as indicators of the structure and topology of membrane proteins

Deuterium/hydrogen exchange factors (chi) were measured for the backbone amide sites of the membrane-bound forms of the 50-residue fd coat protein and the 23-residue magainin2 peptide in lipid micelles by solution nuclear magnetic resonance spectroscopy. By combining kinetic and thermodynamic effects, deuterium/hydrogen exchange factors overcome the principal limitations encountered in the measurements of kinetic protection factors and thermodynamic fractionation factors for membrane proteins. The magnitudes of the exchange factors can be correlated with the structure and topology of membrane-associated polypeptides. In fd coat protein, residues in the transmembrane helix have exchange factors that are substantially smaller than those in the amphipathic surface helix or the loop connecting the two helices. For the amphipathic helical peptide, magainin2, the exchange factors of residues exposed to the solvent are appreciably larger than those that face the hydrocarbon portion of membrane bilayers. These examples demonstrate that deuterium/hydrogen exchange factors can be measured by solution NMR spectroscopy and used to identify residues in transmembrane helices as well as to determine the polarity of amphipathic helices in membrane proteins.     

Defining the structural basis for assembly of a transmembrane cytochrome

To define the structural basis for cofactor binding to membrane proteins, we introduce a manageable model system, which allows us, for the first time, to study the influence of individual transmembrane helices and of single amino acid residues on the assembly of a transmembrane cytochrome. In vivo as well as in vitro analyses indicate central roles of single amino acid residues for either interaction of the transmembrane helices or for binding of the cofactor. The results clearly show that interaction of the PsbF transmembrane helix is independent from binding of the heme cofactor. On the other hand, binding of the cofactor highly depends on helix-helix interactions. By site-directed mutagenesis critical amino acid residues were identified, which are involved in the assembly of a functional transmembrane cytochrome. Especially, a highly conserved glycine residue is critical for interaction of the transmembrane helices and assembly of the cytochrome. Based on the two-stage-model of alpha-helical membrane protein folding, the presented results clearly indicate a third stage of membrane protein folding, in which a cofactor binds to a pre-assembled transmembrane protein.     

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.