Critical role of the N-loop and beta1-strand hydrophobic clusters of RANTES-derived peptides in anti-HIV activity

HIV initiates its infectious cycle by docking to CD4 and a chemokine receptor, most commonly CCR5. RANTES, a natural CCR5 ligand, is a potent inhibitor of HIV-1. Despite the lack of structural information on the RANTES-CCR5 complex, determinants of HIV blockade were previously identified within the RANTES N-loop and beta1-strand regions. A prototype N-loop/beta1-strand peptide, named R11-29, contains two terminal hydrophobic stretches separated by a central hydrophilic region. Here, the role of the terminal hydrophobic clusters was investigated by means of amino acid substitutions or deletions. Most hydrophobic residues in these clusters were shown to be fundamental for the anti-HIV activity. However, increasing the hydrophobicity of the two clusters using non-natural amino acids did not significantly improve the potency of the peptides. These results may provide instrumental knowledge for the rational design of RANTES-derivative molecules with increased anti-HIV activity.     

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.     

Mutation matrices and physical-chemical properties: Correlations and implications

To investigate how the properties of individual amino acids result in proteins with particular structures and functions, we have examined the correlations between previously derived structure-dependent mutation rates and changes in various physical-chemical properties of the amino acids such as volume, charge, α-helical and β-sheet propensity, and hydrophobicity. In most cases we found the ΔG of transfer from octanol to water to be the best model for evolutionary constraints, in contrast to the much weaker correlation with the ΔG of transfer from cyclohexane to water, a property found to be highly correlated to changes in stability in site-directed mutagenesis studies. This suggests that natural evolution may follow different rules than those suggested by results obtained in the laboratory. A high degree of conservation of a surface residue's relative hydrophobicity was also observed, a fact that cannot be explained by constraints on protein stability but that may reflect the consequences of the reverse-hydrophobic effect. Local propensity, especially α-helical propensity, is rather poorly conserved during evolution, indicating that non-local interactions dominate protein structure formation. We found that changes in volume were important in specific cases, most significantly in transitions among the hydrophobic residues in buried locations. To demonstrate how these techniques could be used to understand particular protein families, we derived and analyzed mutation matrices for the hypervariable and framework regions of antibody light chain V regions. We found a surprisingly high conservation of hydrophobicity in the hypervariable region, possibly indicating an important role for hydrophobicity in antigen recognition. Proteins 27:336–344, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of hydrophobicity in the alpha and beta chemokine families and its relevance to dimerization.

The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, alpha and beta, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25-70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 alpha and 9 beta chemokine sequences, based on the coordinates of the prototypic alpha and beta chemokines, interleukin-8 (IL-8), and human macrophage inflammatory protein-1 beta (hMIP-1 beta), respectively, is presented. The monomers of the alpha and beta chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the alpha and beta chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the alpha and beta chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL-8 and hMIP-1 beta are preserved for all the alpha and beta chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the alpha and beta chemokine subfamilies.     

A turn propensity scale for transmembrane helices.

Using a model protein with a 40 residue hydrophobic transmembrane segment, we have measured the ability of all the 20 naturally occurring amino acids to form a tight turn when placed in the middle of the hydrophobic segment. Turn propensities in a transmembrane helix are found to be markedly different from those of globular proteins, and in most cases correlate closely with the hydrophobicity of the residue. The turn propensity scale may be used to improve current methods for membrane protein topology prediction.     

Hydrophobicity Density Profiles to Predict Thermal Stability Enhancement in Proteins

A hydrophobicity density is defined for a protein through its hydrophobicity tensor (similar to the inertia tensor), by using the Eisenberg hydrophobicity scale of the hydrophobic amino acids of a protein. This allows calculation of the radii of the corresponding hydrophobic ellipsoid of a protein and thus subsequently of its hydrophobic density. A hydrophobicity density profile is then obtained by simulating point mutations of each amino acid of a protein either to a high hydrophobicity value or to zero hydrophobicity. It is found that an increase in the hydrophobic density of the protein correlates with an increase of its mid-point transition temperature. From this profile it is possible to determine the amino acids or domain stretches in a protein that are most amenable to mutation in order to increase the thermal stability. The model is tested to predict the thermostabilisation effects of two mutations in a β-glucanase: M29G and M29F. This model is compared with other hydrophobicity-related profiles described by other authors.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Surface hydrophobicity of hemoglobins A, F and S determined by gel filtration column chromatography in high-phosphate buffer

We found that hemoglobins A, F and S could be separated on TSK-GEL-SW columns by differences in surface hydrophobicity when eluted with 1.8 M phosphate buffer, pH 7.4. The elution pattern of the oxy- and deoxy-forms of hemoglobins A, S and F from a TSK-GEL-SW-type gel filtration column is useful for measuring surface hydrophobicity. The elution volumes of oxyhemoglobins F, A and S on the TSK-GEL-SW column in 1.8 M potassium phosphate buffer, pH 7.4, related linearly to the log of their solubility; the higher the surface hydrophobicity, the lower the solubility. There was no linear relationship between the solubilities and the elution volumes of these hemoglobins in the deoxy-form; deoxy-Hb S was far from the lines formed by deoxy-Hb A and deoxy-Hb F. These data suggest that the solubility of oxyhemoglobins is related to simple hydrophobic interactions caused by the total surface hydrophobicity, but the extremely low solubility of deoxy-Hb S must be the result of a stereospecific strong hydrophobic interaction between amino acids at the contact regions of deoxy-Hb S molecules.     

Effect of site-directed point mutations on protein misfolding: A simulation study

A Monte Carlo simulation based sequence design method is proposed to investigate the role of site-directed point mutations in protein misfolding. Site-directed point mutations are incorporated in the designed sequences of selected proteins. While most mutated sequences correctly fold to their native conformation, some of them stabilize in other nonnative conformations and thus misfold/unfold. The results suggest that a critical number of hydrophobic amino acid residues must be present in the core of the correctly folded proteins, whereas proteins misfold/unfold if this number of hydrophobic residues falls below the critical limit. A protein can accommodate only a particular number of hydrophobic residues at the surface, provided a large number of hydrophilic residues are present at the surface and critical hydrophobicity of the core is preserved. Some surface sites are observed to be equally sensitive toward site-directed point mutations as the core sites. Point mutations with highly polar and charged amino acids increases the misfold/unfold propensity of proteins. Substitution of natural amino acids at sites with different number of nonbonded contacts suggests that both amino acid identity and its respective site-specificity determine the stability of a protein. A clash-match method is developed to calculate the number of matching and clashing interactions in the mutated protein sequences. While misfolded/unfolded sequences have a higher number of clashing and a lower number of matching interactions, the correctly folded sequences have a lower number of clashing and a higher number of matching interactions. These results are valid for different SCOP classes of proteins.     

Activity of amino acids as a function of their hydrophobicity in the process of the autolysis of theSaccharomyces cerevisiae yeast

The effect of amino acids on the process of autolysis of baker’s yeast was studied. It is shown that the addition of amino acids inhibits the increase in concentration of amino nitrogen during the process of autolysis. The effect of the inhibition is connected with the hydrophobicity of amino acids, the relationship being of the symbasis nature; it is especially obvious at high concentrations of the latter. The deviation from the symbasis as a result of the effect of low concentrations is the most typical for the highly hydrophobic amino acids (tyrosine, phenylalanine, isoleucine, tryptophan), which can be explained by their solubilization in lipid components of the cell. Hydrophilic glutamic acid suppresses both protease activity and nuclease activity of endoenzymes, which can be explained by its membranotrophic effect.     

Peptide binding specificity of the chaperone calreticulin

Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composition. A large set of available synthetic peptides (n=127) was tested for binding to calreticulin and the results analysed by multivariate data analysis. The parameter that correlated best with binding was hydrophobicity while beta-turn potential disfavoured binding. Only hydrophobic peptides longer than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has a peptide-binding specificity for hydrophobic sequences and delineate the fine specificity of calreticulin for hydrophobic amino acid residues.     

Differences in hydrophobic properties for human alpha 2-macroglobulin and pregnancy zone protein as studied by affinity phase partitioning

Human alpha 2-macroglobulin and pregnancy zone protein are related with regard to primary structure, physicochemical properties, and quarternary structure. Both proteins undergo conformational changes when they form complexes with proteinases or react with primary amines. The surface properties of the native, chymotrypsin-treated and methylamine-treated forms of alpha 2-macroglobulin and pregnancy zone protein were studied by partitioning in aqueous two-phase systems composed of 7.5% dextran T70 and 5% poly(ethylene glycol) 8000. All proteins and their derivatives had a high potential for hydrophobic interaction as analyzed in terms of affinity for poly(ethylene glycol) esters of fatty acids included in the phase systems. Treatment of alpha 2-macroglobulin with methylamine or chymotrypsin increased the surface hydrophobicity significantly compared to that of the native protein. No difference in hydrophobic interaction was found for native and methylamine-treated pregnancy zone protein, but the chymotrypsin-treated protein showed a marked increase in binding to the hydrophobic ligand. The changes in surface hydrophobicity parallel changes in receptor binding properties of the derivatized forms of alpha 2-macroglobulin and could be a signal for binding to cell-surface receptors, followed by internalization.     

The ProteoMiner in the proteomic arena: a non-depleting tool for discovering low-abundance species

Combinatorial ligand libraries, composed by millions of hexapeptides, are here reviewed in terms of their ability of capturing the low-abundance proteome. First, the physico-chemical properties of such libraries are dealt with, especially in regard to the proper length of the bait. The capturing ability of single amino acids has been assessed demonstrating that there exist a protein adsorption capability dichotomy, by which 8 amino acids (Arg, Lys, His, Phe, Tyr, Trp, Val and Leu) are classified as interacting with a large number of proteins with all the remaining amino acids with limited capturing capabilities. The highest performance in capturing the largest possible population of proteins is offered by the three hydrophobic, aromatic amino acids, i.e. Phe, Tyr and Trp, suggesting that hydrophobic motifs are those responsible for the strongest, and most frequently occurring, interactions. By exploring baits ranging from single, individual amino acids, to di-, tri-, tetra- penta- and hexapeptides, it was demonstrated that the 6-mer baits are the ones with the most promising length for capturing the largest possible population of proteins and that probably longer lengths would hardly be needed. Some examples are given on the ability to explore the low-abundance proteome in two systems, notably chicken egg white and yolk. In both cases, by using the peptide library methodology, it is possible to detect at least twice as many protein species as compared to the best results obtained so far with the most advanced proteomics studies using highly sophisticated mass spectrometry tools.     

Conformational Ensembles of Antibodies Determine Their Hydrophobicity

A major challenge in the development of antibody biotherapeutics is their tendency to aggregate. One root cause for aggregation is exposure of hydrophobic surface regions to the solvent. Many current techniques predict the relative aggregation propensity of antibodies via precalculated scales for the hydrophobicity or aggregation propensity of single amino acids. However, those scales cannot describe the nonadditive effects of a residue’s surrounding on its hydrophobicity. Therefore, they are inherently limited in their ability to describe the impact of subtle differences in molecular structure on the overall hydrophobicity. Here, we introduce a physics-based approach to describe hydrophobicity in terms of the hydration free energy using grid inhomogeneous solvation theory (GIST). We apply this method to assess the effects of starting structures, conformational sampling, and protonation states on the hydrophobicity of antibodies. Our results reveal that high-quality starting structures, i.e., crystal structures, are crucial for the prediction of hydrophobicity and that conformational sampling can compensate errors introduced by the starting structure. On the other hand, sampling of protonation states only leads to good results when combined with high-quality structures, whereas it can even be detrimental otherwise. We conclude by pointing out that a single static homology model may not be adequate for predicting hydrophobicity.     

Hydrophobic clusters in protein structures

Globular proteins fold such that the hydrophobic groups are packed inside forming hydrophobic clusters, and the hydrophilic groups are present on the surface. In this article we analyze clusters of hydrophobic groups of atoms in 781 protein structures selected from the PDB. Our analysis showed that every structure consists of two types of clusters: at least one large cluster that forms the hydrophobic core and probably dictates the protein fold; and numerous smaller clusters, which might be involved in the stabilization of the fold. We also analyzed the preference of the hydrophobic groups in each of the amino acids toward forming hydrophobic clusters. We find that hydrophobic groups from the hydrophilic amino acids also contribute toward cluster formation. Proteins 2008. © 2008 Wiley‐Liss, Inc.     

Influence of carbon source and surface hydrophobicity on the aggregation of the yeast Kluyveromyces bulgaricus

Aggregation of the yeast Kluyveromyces bulgaricus is mediated by the galactose-specific lectin KbCWL1. This lectin contains hydrophobic amino acids and its activity is calcium dependent. A specific fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid in the free acid form (ANS; Sigma Chemical Co., St. Louis, Missouri), was used to study the hydrophobic areas on the cellular surface of K. bulgaricus. Changes in surface hydrophobicity during the growth and aggregation of yeast cells were studied. Surface hydrophobicity increased during growth and depended on the amount of yeast cells in the culture medium. During growth, the size of the hydrophobic areas on the cell surface was measured using ANS and was found to increase with the percentage of flocculating yeasts. Our results strongly suggest that the hydrophobic areas of the cell walls of yeast cells are involved in the aggregation of K. bulgaricus.     

The pH-dependence of the peptidase activity of aminoacylase.

Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveals that only dipeptides with uncharged N-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the N-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent. Hydrophobic, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors. Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the N-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.     

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.     

Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450

The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively. This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase. We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s. The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form. Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms. This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s. This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.     

A fast method for the quantitative estimation of the distribution of hydrophobic and hydrophilic segments in alpha-helices of membrane proteins

The work presents a fast quantitative approach for estimating the orientations of hydrophilic and hydrophobic regions in the helical wheels of membrane-spanning alpha-helices of transmembrane proteins. The common hydropathy analysis provides an estimate of the integral hydrophobicity in a moving window which scans an amino acid sequence. The new parameter, orientation hydrophobicity, is based on the estimate of hydrophobicity of the angular segment that scans the helical wheel of a given amino acid sequence. The corresponding procedure involves the treatment of transmembrane helices as cylinders with equal surface elements for each amino acid residue. The orientation hydrophobicity, P(phi), phi = 0-360 degrees, of a helical cylinder is given as a sum of hydrophobicities of individual amino acids which are taken as the S-shaped functions of the angle between the centre of amino acid surface element and the centre of the segment. Non-zero contribution to P(phi) comes only from the amino acids belonging to the angular segment for a given angle phi. The size of the angular segment is related to the size of the channel pore. The amplitudes of amino acid S-functions are calibrated in the way that their maximum values (reached when the amino acid is completely exposed into the pore) are equal to the corresponding hydropathy index in the selected scale (here taken as Goldman-Engelman-Steitz hydropathy scale). The given procedure is applied in the studies of three ionic channels with well characterized three-dimensional structures where the channel pore is formed by a bundle of alpha-helices: cholera toxin B, nicotinic acetylcholine homopentameric alpha7 receptor, and phospholamban. The estimated maximum of hydrophilic properties at the helical wheels are in a good agreement with the spatial orientations of alpha-helices in the corresponding channel pores.