Use of hydrophobic moment plot methodology to aid the identification of oblique orientated alpha-helices

A number of alpha-helix forming peptides have been reported which appear to promote membrane fusion and other biological events related to the disruption of a hydrophobic/hydrophilic interface, due to the presence of a hydrophobicity gradient along the helical long axis. When alpha-helices from this class were analysed according to hydrophobic moment plot methodology a linear association was found to exist between the mean hydrophobic moment, , and the corresponding mean hydrophobicity, . This association was described by the least squares regression line: =0.508-0.422 and, here, a methodology to aid the prediction of oblique orientated alpha-helices is presented, based on a 99% prediction band around this regression line. This methodology is intended to provide an initial identification of candidates for further investigation by other techniques such as the molecular hydrophobic potential and laboratory based experimentation, not to assign function.     

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)     

Investigation of hydrophobic moment and hydrophobicity properties for transmembrane <Emphasis Type="Italic">α</Emphasis>-helices

Integral membrane proteins are the primary targets of novel drugs but are largely without solved structures. As a consequence, hydrophobic moment plot methodology is often used to identify putative transmembrane α-helices of integral membrane proteins, based on their local maximum mean hydrophobic moment (<μH>) and the corresponding mean hydrophobicity (<H>). To calculate these properties, the methodology identifies an optimal eleven residue window (L = 11), assuming an amino acid angular frequency, θ, fixed at 100°.     

Modeling alpha-helical transmembrane domains: the calculation and use of substitution tables for lipid-facing residues.

Amino acid substitution tables are calculated for residues in membrane proteins where the side chain is accessible to the lipid. The analysis is based upon the knowledge of the three-dimensional structures of two homologous bacterial photosynthetic reaction centers and alignments of their sequences with the sequences of related proteins. The patterns of residue substitutions show that the lipid-accessible residues are less conserved and have distinctly different substitution patterns from the inaccessible residues in water-soluble proteins. The observed substitutions obtained from sequence alignments of transmembrane regions (identified from, e.g., hydrophobicity analysis) can be compared with the patterns derived from the substitution tables to predict the accessibility of residues to the lipid. A Fourier transform method, similar to that used for the calculation of a hydrophobic moment, is used to detect periodicity in the predicted accessibility that is compatible with the presence of an alpha-helix. If the putative transmembrane region is identified as helical, then the buried and exposed faces can be discriminated. The presence of charged residues on the lipid-exposed face can help to identify the regions that are in contact with the polar environment on the borders of the bilayer, and the construction of a meaningful three-dimensional model is then possible. This method is tested on an alignment of bacteriorhodopsin and two related sequences for which there are structural data at near atomic resolution.     

Residues in the longitudinal, hydrophobic strip-of-helix relate to terminations and crossings of alpha-helices.

An alpha-helix terminates when the virtual extension of its most hydrophobic, longitudinal strip containing Leu, Ile, Val, Phe, and Met lacks those residues. In each of 247 helices a template was fitted to maximize the mean hydrophobicity of positions forming a longitudinal strip-of-helix. The template was then extended into sequences beyond the ends of the helices. Leu, Ile, Val, Phe, and Met occurred in positions in the longitudinal strip-of-helix at an increased frequency (p less than 0.001), but in the first and second positions beyond either end of each true helix, they occurred at the same frequency as for their empirical distribution over all the proteins. Excesses of Asp and Glu were found in the N-terminal loop, and of Arg, His, and Lys in specific positions about the C terminus of helices. The longitudinal hydrophobic strip, the smallest amino acid in that strip, and charged amino acids in that strip, related to rotational and longitudinal orientation of alpha-helices in 15 proteins. Adjacent helices generally crossed through their longitudinal hydrophobic strips. They usually crossed through the smallest residue in the strip. Charged residues, when they occurred in the strips, were excluded from the crossing regions.     

Molecular dissection,tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans

We investigated the possible role of residues at the Ccap position in an alpha-helix on protein stability. A set of 431 protein alpha-helices containing a C'-Gly from the Protein Data Bank (PDB) was analyzed, and the normalized frequencies for finding particular residues at the Ccap position, the average fraction of buried surface area, and the hydrogen bonding patterns of the Ccap residue side-chain were calculated. We found that on average the Ccap position is 70% buried and noted a significant correlation (R=0.8) between the relative burial of this residue and its hydrophobicity as defined by the Gibbs energy of transfer from octanol or cyclohexane to water. Ccap residues with polar side-chains are commonly involved in hydrogen bonding. The hydrogen bonding pattern is such that, the longer side-chains of Glu, Gln, Arg, Lys, His form hydrogen bonds with residues distal (>+/-4) in sequence, while the shorter side-chains of Asp, Asn, Ser, Thr exhibit hydrogen bonds with residues close in sequence (<+/-4), mainly involving backbone atoms. Experimentally we determined the thermodynamic propensities of residues at the Ccap position using the protein ubiquitin as a model system. We observed a large variation in the stability of the ubiquitin variants depending on the nature of the Ccap residue. Furthermore, the measured changes in stability of the ubiquitin variants correlate with the hydrophobicity of the Ccap residue. The experimental results, together with the statistical analysis of protein structures from the PDB, indicate that the key hydrophobic capping interactions between a helical residue (C3 or C4) and a residue outside the helix (C", C3' or C4') are frequently enhanced by the hydrophobic interactions with Ccap residues.     

Protein sequence and structure relationship ARMA spectral analysis: application to membrane proteins.

If it is assumed that the primary sequence determines the three-dimensional folded structure of a protein, then the regular folding patterns, such as alpha-helix, beta-sheet, and other ordered patterns in the three-dimensional structure must correspond to the periodic distribution of the physical properties of the amino acids along the primary sequence. An AutoRegressive Moving Average (ARMA) model method of spectral analysis is applied to analyze protein sequences represented by the hydrophobicity of their amino acids. The results for several membrane proteins of known structures indicate that the periodic distribution of hydrophobicity of the primary sequence is closely related to the regular folding patterns in a protein's three-dimensional structure. We also applied the method to the transmembrane regions of acetylcholine receptor alpha subunit and Shaker potassium channel for which no atomic resolution structure is available. This work is an extension of our analysis of globular proteins by a similar method.     

Secondary structures of rat lipolytic enzymes: circular dichroism studies and relation to hydrophobic moments

To explore the secondary structures of lingual and pancreatic lipases, circular dichroism measurements were performed. Maximum average ellipticities were used to calculate the percentage of alpha-helices, beta-sheets, and random coils. Lingual lipase had an ellipticity of -20235 +/- 140 deg cm2/dmol (mean +/- SE) at 220 nm suggesting 60% alpha-helix, 20% beta-sheet and 20% random coil structure, but the mean ellipticity for pancreatic lipase was -14093 +/- 82 deg cm2/dmol (mean +/- SE) at 210 nm suggesting a 34.8% alpha-helical, 25% beta-sheet and 40% random coil secondary structure. An alpha-helical stretch of residues with a large hydrophobic moment ("globular" alpha-helix by hydrophobic moment plot) from amino acids 382 through 389 at the COOH-terminal end of lingual lipase was noted. This sequence, absent in pancreatic lipase, may account for the avid binding of lingual lipase to fat emulsion particles.     

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.     

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.     

Hydrophobic side-chain size is a determinant of the three-dimensional structure of the p53 oligomerization domain.

The p53 tumor suppressor oligomerization domain, a dimer of two primary dimers, is an independently folding domain whose subunits consist of a beta-strand, a tight turn and an alpha-helix. To evaluate the effect of hydrophobic side-chains on three-dimensional structure, we substituted residues Phe341 and Leu344 in the alpha-helix with other hydrophobic amino acids. Substitutions that resulted in residue 341 having a smaller side-chain than residue 344 switched the stoichiometry of the domain from tetrameric to dimeric. The three-dimensional structure of one such dimer was determined by multidimensional NMR spectroscopy. When compared with the primary dimer of the wild-type p53 oligomerization domain, the mutant dimer showed a switch in alpha-helical packing from anti-parallel to parallel and rotation of the alpha-helices relative to the beta-strands. Hydrophobic side-chain size is therefore an important determinant of a protein fold.     

The amino acid sequence of a gonococcal growth inhibitor from Staphylococcus haemolyticus.

A gonococcal inhibitor produced by Staphylococcus haemolyticus was separated into three components by reverse-phase h.p.l.c. The amino acid composition analysis of each of the three components indicated extensive similarities. N-Terminal sequence analysis of all three components allowed the identification of the first 27-30 residues of each. The complete primary structure of each component was determined from the sequence analysis of trypic peptides and peptides generated by mild acid hydrolysis. Each component is composed of 44 amino acid residues, with evidence suggesting the presence of an N-terminal formylmethionine residue in each. The components I, II and III have respectively 33, 29 and 33 identical amino acid residues in their sequences, which represents 75%, 65.9% and 75% homology. These components contain a high proportion of hydrophobic amino acids, and their hydrophobicity profiles are closely related. Also, each of the three components contains a positively charged residue (lysine) as the third residue, followed by a core of hydrophobic residues. These results suggest that the three components are possible signal sequences of one or more secreted or membrane-associated proteins.     

Properties of integral membrane protein structures: derivation of an implicit membrane potential

Distributions of each amino acid in the trans-membrane domain were calculated as a function of the membrane normal using all currently available alpha-helical membrane protein structures with resolutions better than 4 A. The results were compared with previous sequence- and structure-based analyses. Calculation of the average hydrophobicity along the membrane normal demonstrated that the protein surface in the membrane domain is in fact much more hydrophobic than the protein core. While hydrophobic residues dominate the membrane domain, the interfacial regions of membrane proteins were found to be abundant in the small residues glycine, alanine, and serine, consistent with previous studies on membrane protein packing. Charged residues displayed nonsymmetric distributions with a preference for the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Potentials of mean force along the membrane normal were derived for each amino acid by fitting Gaussian functions to the residue distributions. The individual potentials agree well with experimental and theoretical considerations. The resulting implicit membrane potential was tested on various membrane proteins as well as single trans-membrane alpha-helices. All membrane proteins were found to be at an energy minimum when correctly inserted into the membrane. For alpha-helices both interfacial (i.e. surface bound) and inserted configurations were found to correspond to energy minima. The results demonstrate that the use of trans-membrane amino acid distributions to derive an implicit membrane representation yields meaningful residue potentials.     

Protein engineering to improve the thermostability of glucoamylase from Aspergillus awamori based on molecular dynamics simulations

Twelve mutations were constructed to improve the thermostability of glucoamylase from Aspergillus awamori based on the results of molecular dynamics simulations. The thermal unfolding of the catalytic domain followed a putative hierarchical behavior. In addition, the unfolding of the 13 alpha-helices obeyed the random ordered mechanism, in which the alpha-helices 8, 1 and 11 unfolded more rapidly than the others. The catalytic center was well protected by the (alpha/alpha)(6)-barrel at simulation temperatures up to 600 K, whereas the catalytic base, E400, migrated from its original interior pocket to the surface of the catalytic domain by surmounting the hydrophobic barrier provided by alpha-helices 12 and 13 at 800 K. The disulfide bonds engineered to 'lock' the alpha-helix 11 on the surface of the catalytic domain dramatically increased the thermostability. Substituting G396 and G407 with Ala residues slightly increased the thermostability, whereas their specific activity and catalytic efficiency were reduced. This indicates that the introduced residues with higher hydrophobicity were favorable in the loop between alpha-helices 12 and 13, whereas they partially destroyed the hydrogen bond and salt linkage network in the catalytic center. Alpha-helices 12 and 13 can be stabilized by introducing residues with higher hydrophobicity, except for the H391M mutation.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.     

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.     

Factors influencing the stability of alpha-helices and beta-strands in thermophilic ribonuclease H.

Understanding the influence of structural parameters is crucial to enhance the thermal stability of proteins. In this work, the stability (deltaG) of residues in different secondary structures of Ribonuclease H (RNase H) has been analyzed with 48 amino acid properties. The properties reflecting hydrophobicity show a good correlation with stability. Further, the linear distribution of surrounding hydrophobicity in alpha-helices, obtained from the three dimensional structure of thermophilic RNase H, agrees well with experimental deltaG values. Moreover, the stability parameters correlate better in alpha-helices than those did in beta-strand segments. Multiple regression analysis, incorporating combinations of three properties from among all possible combinations of the 48 properties, increased the correlation coefficient to 0.77.