Strong Correlation Between Statistical Transmembrane Tendency and Experimental Hydrophobicity Scales for Identification of Transmembrane Helices

Direct physical chemistry measurements of the hydrophobicity of amino acids or their derivatives have often been used to estimate the propensity of amino acids to participate in transmembrane helices. In this short note, it is found that there is a very high degree of correlation (r = 0.944–0.965) between an average physical chemistry hydrophobicity scale (an average of scales derived, e.g., from the solubility of amino acid derivatives in organic solvents versus water or their binding to hydrophobic particles) and the statistically based transmembrane tendency scale (derived from the relative abundance of residues in known transmembrane and soluble protein sequences (Zhao and London, Protein Sci 15:1987–2001, 2006)). This correlation indicates that, other than hydrophobicity, amino acid properties/interactions that promote or inhibit transmembrane helix formation in a specific membrane protein largely cancel out when averaged over all transmembrane sequences. In other words, other than hydrophobicity, there are no properties of a specific amino acid residue within a hydrophobic segment that have a strong systematic effect upon transmembrane helix formation independent of the remainder of the sequence in that hydrophobic segment. However, proline is an exception to this rule.     

Protein Structure Classification Based on Conserved Hydrophobic Residues

Protein folding is frequently guided by local residue interactions that form clusters in the protein core. The interactions between residue clusters serve as potential nucleation sites in the folding process. Evidence postulates that the residue interactions are governed by the hydrophobic propensities that the residues possess. An array of hydrophobicity scales has been developed to determine the hydrophobic propensities of residues under different environmental conditions. In this work, we propose a graph-theory-based data mining framework to extract and isolate protein structural features that sustain invariance in evolutionary-related proteins, through the integrated analysis of five well-known hydrophobicity scales over the 3D structure of proteins. We hypothesize that proteins of the same homology contain conserved hydrophobic residues and exhibit analogous residue interaction patterns in the folded state. The results obtained demonstrate that discriminatory residue interaction patterns shared among proteins of the same family can be employed for both the structural and the functional annotation of proteins. We obtained on the average 90 percent accuracy in protein classification with a significantly small feature vector compared to previous results in the area. This work presents an elaborate study, as well as validation evidence, to illustrate the efficacy of the method and the correctness of results reported.     

Hydrophobicity of transmembrane proteins: spatially profiling the distribution

A hallmark of soluble globular protein tertiary structure is a hydrophobic core and a protein exterior populated predominantly by hydrophilic residues. Recent hydrophobic moment profiling of the spatial distribution of 30 globular proteins of diverse size and structure had revealed features of this distribution that were comparable. Analogous profiling of the hydrophobicity distribution of the alpha-helical buried bundles of several transmembrane proteins, as the lipid/protein interface is approached from within the bilayer, reveals spatial hydrophobicity profiles that contrast with those obtained for the soluble proteins. The calculations, which enable relative changes of hydrophobicity to be simply identified over the entire spatial extent of the multimer within the lipid bilayer, show the accumulated zero-order moments of the bundles to be mainly inverted with respect to that found for the soluble proteins. This indicates a statistical increase in the average residue hydrophobic content as the lipid bilayer is approached. This result differs from that of a relatively recent calculation and qualitatively agrees with earlier calculations involving lipid exposed and buried residues of the alpha-helices of transmembrane proteins. Spatial profiling, over the entire spatial extent of the multimer with scaled values of residue hydrophobicity, provides information that is not available from calculations using lipid exposure alone.     

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.     

Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins

Protein segments that form amphipathic alpha-helices in their native state have periodic variation in the hydrophobicity values of the residues along the segment, with a 3.6 residue per cycle period characteristic of the alpha-helix. The assignment of hydrophobicity values to amino acids (hydrophobicity scale) affects the display of periodicity. Thirty-eight published hydrophobicity scales are compared for their ability to identify the characteristic period of alpha-helices, and an optimum scale for this purpose is computed using a new eigenvector method. Two of the published scales are also characterized by eigenvectors. We compare the usual method for detecting periodicity based on the discrete Fourier transform with a method based on a least-squares fit of a harmonic sequence to a sequence of hydrophobicity values. The two become equivalent for very long sequences, but, for shorter sequences with lengths commonly found in alpha-helices, the least-squares procedure gives a more reliable estimate of the period. The analog to the usual Fourier transform power spectrum is the "least-squares power spectrum", the sum of squares accounted for in fitting a sinusoid of given frequency to a sequence of hydrophobicity values. The sum of the spectra of the alpha-helices in our data base peaks at 97.5 degrees, and approximately 50% of the helices can account for this peak. Thus, approximately 50% of the alpha-helices appear to be amphipathic, and, of those that are, the dominant frequency at 97.5 degrees rather than 100 degrees indicates that the helix is slightly more open than previously thought, with the number of residues per turn closer to 3.7 than 3.6. The extra openness is examined in crystallographic data, and is shown to be associated with the C terminus of the helix. The alpha amphipathic index, the key quantity in our analysis, measures the fraction of the total spectral area that is under the 97.5 degrees peak, and is a characteristic of hydrophobicity scales that is consistent for different sets of helices. Our optimized scale maximizes the amphipathic index and has a correlation of 0.85 or higher with nine previously published scales. The most surprising feature of the optimized scale is that arginine tends to behave as if it were hydrophobic; i.e. in the crystallographic data base it has a tendency to be on the hydrophobic face of teh amphipathic helix. Although the scale is optimal only for predicting alpha-amphipathicity, it also ranks high in identifying beta-amphipathicity and in distinguishing interior from exterior residues in a protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cysteine function governs its conservation and degeneration and restricts its utilization on protein surfaces

Cysteine (Cys) is an enigmatic amino acid residue. Although one of the least abundant, it often occurs in the functional sites of proteins. Whereas free Cys is a polar amino acid, Cys in proteins is often buried, and its classification on the hydrophobicity scale is ambiguous. We hypothesized that the deviation of Cys residues from the properties of a free amino acid is due to their reactivity and addressed this possibility by examining Cys in large protein structure data sets. Compared to other amino acids, Cys was characterized by the most extreme conservation pattern, with the majority of Cys being either highly conserved or poorly conserved. In addition, clustering of Cys with another Cys residue was associated with high conservation, whereas exposure of Cys on protein surfaces was associated with low conservation. Moreover, although clustered Cys behaved as polar residues, isolated Cys was the most buried residue of all, in disagreement with known chemical properties of Cys. Thus, the anomalous hydrophobic behavior and conservation pattern of Cys can be explained by elimination of isolated Cys from protein surfaces during evolution and by clustering of other Cys residues. These findings indicate that Cys abundance is governed by Cys function in proteins rather than by the sheer chemical-physical properties of free amino acids, and suggest that a high tendency of Cys to be functionally active can considerably limit its abundance on protein surfaces.     

An examination of the conservation of surface patch polarity for proteins

MOTIVATION: The solubility of a protein is crucial for its function and is therefore an evolutionary constraint. As the solubility of a protein is related to the distribution of polar and hydrophobic residues on its solvent accessible surface, such a constraint should provide a valuable insight into the evolution of protein surfaces. We examine how the surfaces of proteins have evolved by considering how the average hydrophobicities of patches of surface residues vary across homologous proteins. We derive distributions for the average hydrophobicity/philicity of surface patches at a residue-based level-which we refer to as the residue hydrophobic density. This is computed for a set of 28 monomeric proteins and their homologues. The resulting distributions are compared with a set of randomized sequences, with the same residue content. RESULTS: We find that the patches, involving typically more than 10 residues, maintain a more hydrophilic surface than one would expect from a random substitution model, indicating a cooperative behaviour for these surfaces residues in terms of this single variable. SUPPLEMENTARY INFORMATION: Additional plots for all of the proteins examined in this paper can be found at: http://www.ebi.ac.uk/~shanahan/PCon/index.html     

The hydrophobic moment and its use in the classification of amphiphilic structures (review)

Amphiphilic alpha-helices play a major role in membrane dependent processes and are manifested in the primary structure of a protein by the periodic appearance of hydrophobic residues. Based on these periodic sequences, the hydrophobic moment was introduced, , which essentially treats the hydrophobicity of amino acid residues as a two-dimensional vector sum and provides a measure of amphiphilicity within regular repeat structures. To identify putative amphiphilic alpha-helix forming sequences, hydrophobic moment analysis assumes an amino acid residue periodicity of 100 and scans protein primary structures to find the 11-residue window with maximal . Taken with the window's mean hydrophobicity, , hydrophobic moment plot analysis uses the coordinate pair, [, ] to classify alpha-helices as either surface active, globular or transmembrane. More recently, this latter analysis has been extended to recognize candidate oblique orientated alpha-helices. Here, the hydrophobic moment is reviewed and data to query the logic of using a fixed window length and a fixed residue angular periodicity in hydrophobic moment analysis are provided. In addition, problems associated with the use of such analysis to predict alpha-helix structure/function relationships are considered.     

The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum

Signal sequences for insertion of proteins into the endoplasmic reticulum induce translocation of either the C- or the N-terminal sequence across the membrane. The end that is translocated is primarily determined by the flanking charges and the hydrophobic domain of the signal. To characterize the hydrophobic contribution to topogenesis, we have challenged the translocation machinery in vivo in transfected COS cells with model proteins differing exclusively in the apolar segment of the signal. Homo-oligomers of hydrophobic amino acids as different in size and shape as Val(19), Trp(19), and Tyr(22) generated functional signal sequences with similar topologies in the membrane. The longer a homo-oligomeric sequence of a given residue, the more N-terminal translocation was obtained. To determine the topogenic contribution of all uncharged amino acids in the context of a hydrophobic signal sequence, two residues in a generic oligoleucine signal were exchanged for all uncharged amino acids. The resulting scale resembles a hydrophobicity scale with the more hydrophobic residues promoting N-terminal translocation. In addition, the helix breakers glycine and proline showed a position-dependent effect, which raises the possibility of a conformational contribution to topogenesis.     

A two-component nucleation model of protein hydrophobicity

A fundamental characteristic of soluble globular protein structure is a hydrophobic core and protein exterior comprised predominantly of hydrophilic residues. This distribution of amino acid residue hydrophobicity, from protein interior to exterior, has recently been profiled with the use of hydrophobic moments. The calculations enable comparison of the radial hydrophobicity distribution of different proteins and had revealed two features common to 30 proteins of diverse size and structure. One, a global feature, is the overall shape of the second-order ellipsoidal hydrophobic moment. The second, a specific feature, is a quasi-invariant hydrophobic-ratio of distances. Both features are dependent upon the rates of increase, from protein interior to exterior, of the accumulated numbers of hydrophobic and hydrophilic amino acid residues. These rates can be simulated simply with a two-component nucleation model of protein hydrophobicity. The model provides insight into the origin of the shape of the observed hydrophobic moment profiles and of the observed range of hydrophobic ratios. Consistent with observation, it is shown that a relatively wide range of hydrophobic and hydrophilic rates of increase yield a relatively narrow range of hydrophobic ratios. Furthermore, the model identifies one factor, the decrease in residue density with increasing distance from the protein interior, that is critical in providing the range of values that is comparable with the observed range.     

Characterization of ligand-binding properties of the human BMP type II receptor extracellular domain

ActR-IIA, ActR-IIB, and BMPR-II are low-affinity type II receptors that bind bone morphogenetic proteins (BMPs) in the same overall manner. The binding of BMPs by ActR-IIs has been analyzed structurally and functionally, but no detailed analysis of BMPR-II has been reported. The objective of this study was to determine ligand-binding epitopes and specificity determinants in two regions, the hydrophobic patch and the A-loop of the BMPR-II extracellular domain (ECD). A series of alanine-substituted variants was generated using a recently published X-ray structure of the unliganded form of the ovine BMPR-II ECD as a guide. These variants were characterized using one-dimensional NMR and functional activity assays with BMP-2, BMP-7 and GDF-5 as ligands. The results showed that alanine substitutions of conserved residues W85 and Y113 within the hydrophobic patch of the ECD differentially perturbed BMP ligand binding without disrupting receptor folding, suggesting that they are critical determinants for ligand binding and ligand specificity. Our results further revealed that the nonconserved residue L69 in the hydrophobic patch contributes to ligand-binding activity and specificity. Mutations of several residues within the A-loop resulted in minimal effects on the binding of the different BMP ligands. Overall, these observations identify several amino acid residues that play different roles in BMPR-II and ActR-II and thereby enable BMPR-II and ActR-IIs to bind different subclasses of BMP ligands.     

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.     

Development of an isotope dilution mass spectrometry assay for HbA1c based on enzyme-cleaved peptide analysis

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R(2) = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.     

Signal sequences containing multiple aromatic residues.

Using homopolymeric units of either phenylalanine or tryptophan to replace the natural core segment of the Escherichia coli alkaline phosphatase signal peptide, the hydrophobicity requirements for protein export and processing were further explored. The mutant signal peptide containing polyphenylalanine functioned at least as efficiently as the wild-type, while the signal incorporating polytryptophan was dysfunctional. The transport properties of these mutants confirm our work with sequences rich in aliphatic residues; namely that a high mean hydrophobicity per residue is critical for complete and rapid precursor processing and for translocation of the protein. The efficient transport properties of the polyphenylalanine-containing signal peptide demonstrate that neither the bulky, aromatic nature of phenylalanine nor the unusually high hydrophobicity of this mutant peptide adversely alters function. This study also suggests that the low occurrence of phenylalanine in natural signal sequences is not of functional consequence but probably reflects the low number of DNA codons for this residue. The polytryptophan-containing precursor was membrane inserted but not translocated. This type of transport defect suggests that this is a weakly hydrophobic signal peptide, consistent with hydropathy scales, which indicate that tryptophan is comparable to alanine. This application of polymeric sequences provides a function-based assay for the evaluation of amino acid hydrophobicity.     

Sequence determinants regulating fibrillation of human alpha-synuclein

α-Synuclein is a neural protein that comprises the fibrillar core of Lewy bodies, a histologically defining lesion of Parkinson’s disease. To investigate the role of each specific residue of the α-synuclein molecule in fibril formation, amino acid substitutions were introduced throughout the molecule. Incorporation of proline, especially in the region spanning residues 37-89, drastically retarded fibril formation. Substitutions with polar residues showed that the hydrophobicity of the central hydrophobic region is also important in fibrillation regulation. In the N-terminal repeated region, increasing the number of negative charges interfered with fibrillation. In contrast, single amino acid substitutions in the C-terminal acidic region of α-synuclein had only minimal effects on fibrillation. More than 20 different single amino acid substitutions that were sufficient to prevent fibrillation of α-synuclein were obtained, and most of them were impaired in both nucleation and fibril elongation. Identification of sequence determinants regulating fibrillation of amyloidogenic proteins may provide valuable information for designing peptide analog drugs to prevent protein amyloidosis.     

Underlying Hydrophobic Sequence Periodicity of Protein Tertiary Structure

Abstract

Hydropathy plots or window averages over local stretches of the sequence of residue hydrophobicity have revealed patterns related to various protein tertiary structural features. This has enabled identification of regions of the sequence that are at the surface or within the interior of globular soluble proteins, regions located within the lipid bilayer of transmembrane proteins, portions of the sequence that characterize repeating motifs, as well as motifs that usefully characterize different protein structural families. This, therefore, provides one example of the generally expressed maxim that “sequence determines structure”. On the other hand, a number of previous investigations have shown the rapidly varying values of residue hydrophobicity along the sequence to be distributed approximately randomly. So one might question just how much of the sequence actually determines structure. It is, therefore, of interest to extract that part of this rapidly varying distribution of residue hydrophobicity that is responsible for the longer wavelength variations that correlate with protein tertiary structural features and to determine their prevalence within the entire distribution. This is accomplished by a finite Fourier analysis of the sequence of residue hydrophobicity and of a new measure of residue distance from the protein interior. Calculations are performed on a number of globins, immunoglobulins, cuprodoxins, and papain-like structures. The spectral power of the Fourier amplitudes of the frequencies extracted, whose inverse transforms underlie the windowed values of residue hydrophobicity is shown to be a small fraction of the total power of the hydrophobicity distribution and thereby consistent with a distribution that might appear to be predominantly random. The wide range of sequence identity between proteins having the same fold, all exhibiting similar small fractions of power amplitude that correlate with the longer wavelength inside-to- outside excursions of the amino acid residues, supports the general contention that close sequence identity is an expression of a close evolutionary relationship rather than an expression of structural similarity. Practical implications of the present analysis for protein structure prediction and engineering are also described.     

How do side chains orient globally in protein structures?

An angle Omega is defined to serve as a metric for global side-chain orientations, which reflects the orientation of the side chain relative to the radial vector from the center of the protein to an amino acid. The side-chain orientations of buried residues exhibit characteristically different orientations than do exposed residues, in both monomeric and dimeric structures. Overall, buried side chains point mostly inward, whereas surface side chains tend to point outward from the surface. This difference in behavior also correlates well with the residue hydrophobicity; so a global side-chain orientation can be viewed as a direct structural manifestation of hydrophobicity. When various solvent-accessible layers are considered, the behavior is relatively continuous between centrally located and exposed residues. In the case of interfacial residues between subunits, there are statistically significant differences between exposed residues and interface residues for ALA, ARG, ASN, ASP, GLU, HIS, LYS, THR, VAL, MET, PRO, and overall the interface residues have an increased tendency to point inward. Presumably, these substantial differences in orientations of side chains may be a manifestation of hydrophobic forces.     

A role for surface hydrophobicity in protein-protein recognition.

The role of hydrophobicity as a determinant of protein-protein interactions is examined. Surfaces of apo-protein targets comprising 9 classes of enzymes, 7 antibody fragments, hirudin, growth hormone, and retinol-binding protein, and their associated ligands with available X-ray structures for their complexed forms, are scanned to determine clusters of surface-accessible amino acids. Clusters of surface residues are ranked on the basis of the hydrophobicity of their constituent amino acids. The results indicate that the location of the co-crystallized ligand is commonly found to correspond with one of the strongest hydrophobic clusters on the surface of the target molecule. In 25 of 38 cases, the correspondence is exact, with the position of the most hydrophobic cluster coinciding with more than one-third of the surface buried by the bound ligand. The remaining 13 cases demonstrate this correspondence within the top 6 hydrophobic clusters. These results suggest that surface hydrophobicity can be used to identify regions of a protein''s surface most likely to interact with a binding ligand. This fast and simple procedure may be useful for identifying small sets of well-defined loci for possible ligand attachment.