A helix propensity scale based on experimental studies of peptides and proteins

The average globular protein contains 30% alpha-helix, the most common type of secondary structure. Some amino acids occur more frequently in alpha-helices than others; this tendency is known as helix propensity. Here we derive a helix propensity scale for solvent-exposed residues in the middle positions of alpha-helices. The scale is based on measurements of helix propensity in 11 systems, including both proteins and peptides. Alanine has the highest helix propensity, and, excluding proline, glycine has the lowest, approximately 1 kcal/mol less favorable than alanine. Based on our analysis, the helix propensities of the amino acids are as follows (kcal/mol): Ala = 0, Leu = 0.21, Arg = 0.21, Met = 0.24, Lys = 0.26, Gln = 0.39, Glu = 0.40, Ile = 0.41, Trp = 0.49, Ser = 0.50, Tyr = 0. 53, Phe = 0.54, Val = 0.61, His = 0.61, Asn = 0.65, Thr = 0.66, Cys = 0.68, Asp = 0.69, and Gly = 1.     

Helix propensities are identical in proteins and peptides

Our understanding of the factors stabilizing alpha-helical structure has been greatly enhanced by the study of model alpha-helical peptides. However, the relationship of these results to the folding of helices in intact proteins is not well characterized. Helix propensities measured in model peptides are not in good agreement with those from proteins. In order to address these questions, we have measured helix propensities in the alpha-helix of ribonuclease T1 and a helical peptide of identical sequence. We have previously demonstrated excellent agreement between peptide and protein for the nonpolar amino acids [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2833-2837]. Most other amino acids also show good agreement, although certain polar amino acids are exceptions. Helix propensities measured in the ribonuclease T1 peptide/protein are compared with those measured in other systems. Reasonable agreement is found between most systems; however, our propensities differ substantially from those measured in several model peptide systems. Alanine-based peptides overestimate the propensity differences by a factor of 2, and host/guest experiments underestimate them by a factor of 2-3.     

Helix stabilizing factors and stabilization of thermophilic proteins: an X-ray based study

We have compared the X-ray structures of 13 thermophilic proteins with their mesophilic homologues, in order to bring out differences in the stability of helices. The energy terms of a helix-coil transition algorithm were used to evaluate helix stability. Helices of thermophilic proteins are more stable than the mesophilic homologues in 69% of cases. This is due mainly to intrinsic helical propensities of amino acids, whereas minor effects are linked to main chain H-bonds, side chain-side chain interactions, capping motifs and charge-dipole effects. Furthermore, the frequency of 10 helix stabilizing factors recognized by appropriate sequence patterns was evaluated. The only factor occurring significantly in the thermostable proteins was the lack of beta branched residues. Other factors do not show a definite trend, although their occurrence in proteins is believed to be important for stability. This is discussed in the light of protein engineering applications.     

Long-term results of total penile reconstruction with a prefabricated lateral arm free flap

Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the alpha-helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.     

C--H...O hydrogen bond involving proline residues in alpha-helices

Despite proline being assumed to be a helix-breaker, a large number of alpha-helices are found to contain Pro in globular as well as membrane proteins. Proline has no free NH group and therefore cannot form the conventional intra-helical NH.O=C hydrogen bond. An analysis of known protein structures has shown that the Cdelta protons are involved in C--H...O hydrogen bonds, usually two, with the carbonyl groups in the preceding turn of the helix (four and three residues away). These interactions satisfy the hydrogen bond forming potential of the carbonyl groups, which would otherwise, in the case of membrane-bound helices, be unfavorably exposed to hydrophobic surroundings. Depending on the type (based on the location of the carbonyl group, usually three, four or five residues preceding Pro) of C--H...O interactions, the kink in the helix may be of different magnitude. The puckering (UP or DOWN) of the pyrrolidine ring of Pro residues is controlled by the type of the C--H...O bond present, and the form that provides a better hydrogen bond geometry is preferred.     

De novo design, synthesis, and characterization of a pore-forming small globular protein and its insertion into lipid bilayers

The question of how to design a water-soluble globular protein remains. We report here the synthesis of a native-like and pore-forming small globular protein (SGP, 69 amino acid residues). The protein was designed to have four helices: a Trp-containing short hydrophobic helix in the middle surrounded by three Tyr-containing long basic amphiphilic helices. Size-exclusion chromatography and CD measurements indicated that in buffer solution SGP is monomeric with a 50% helical structure. SGP did not completely denature even at high temperature (90 degrees C) and at relatively high Gu x HCl concentration so that the denaturant concentration at the midpoint of the transition is 5 M. Dye binding studies and fluorescence energy transfer experiments showed that SGP possesses a hydrophobic binding site and its Trp of the central helix is present at a relatively hydrophobic region and accepts the energy from Tyr(s) in other amphiphilic helices, indicating that SGP takes a stable globular-like structure in aqueous solution. From the depth-dependent fluorescent studies using egg PC liposomes containing n-doxyl fatty acids and brominated phospholipid as quenchers, it was found that the hydrophobic central alpha-helix is able to enter spontaneously into the lipid bilayers and the Trp in the central alpha-helix is located at about the middle of the alkyl chain in the outer layer of the phospholipid bilayer. The peptide is also able to increase the membrane permeability with two modes of current (basal current and single ion channel) in planar phospholipid bilayers, indicating the spontaneous insertion of the protein into the lipid bilayer (basal current) and then the formation of a uniform size of channel pore (14 pS). SGP is useful as a basic and starting model to find good amino acid sequences that fold to a desired protein structure and to search translocation mechanisms from aqueous solution into lipid bilayers.     

Statistical analysis of predicted transmembrane alpha-helices

Statistical analyses were undertaken for putative transmembrane alpha-helices obtained from a database representing the subset of membrane proteins available in Swiss-Prot. The average length of a transmembrane alpha-helix was found to be 22-21 amino acids with a large variation around the mean. The transfer free energy from water to oil of a transmembrane alpha-helix in bitopic proteins, -48 kcal/mol, is higher than that in polytopic proteins, -39 kcal/mol, and is nearly identical to that obtained by assuming a random distribution of solely hydrophobic amino acids in the alpha-helix. The amino acid composition of hydrophobic residues is similar in bitopic and polytopic proteins. In contrast, the more polar the amino acids are, the less likely they are to be found in bitopic proteins compared to polytopic ones. This most likely reflects the ability of alpha-helical bundles to shield the polarity of residues from the hydrophobic bilayer. One half of all amino acids were distributed nonrandomly in both bitopic and polytopic proteins. A preference was found for tyrosine and tryptophan residues to be at the ends of transmembrane alpha-helices. Correlated distribution analysis of amino acid pairs indicated that most amino acids are independently distributed in each helix. Exceptions are cysteine, tyrosine, and tryptophan which appear to cluster closely to one another and glycines which are preferentially found on the same side of alpha-helices.     

Stereochemical punctuation marks in protein structures: glycine and proline containing helix stop signals

An analysis on the nature of alpha-helix stop signals has been carried out, using a dataset of 1057 helices identified from 250 high resolution (相似文献   

Long charge-rich alpha-helices in systemic autoantigens

Autoimmune diseases are characterized by the presence of antibodies and T-cells targeting restricted sets of host proteins. This phenomenon may be due in part to greater non-specific immunogenicity for these proteins compared to others which are not autoantigenic. We used computer-based methods to analyze the sequenced human autoantigens for distinctive patterns of potential immunologic importance. Sequences longer than 27 residues predicted by these methods to form coiled-coil alpha-helices with a probability greater than 0.9 were detected in 40 of 109 (36.7%) of the known human autoantigens. These include many predominantly systemic disease-specific autoantigens not previously known to contain this structure. In comparison, 8.7% of human proteins in the Swissprot data base, and 1.1% of the proteins in the Brookhaven data base were found to contain such segments. These predicted coiled-coil alpha-helices are distinguished from most globular protein helices by greater length, higher charge content, and a heptad repeat multivalency. Coiled-coil segments correlate in part with known autoantibody epitopes and may contribute to autoantigenic potential. Systemic autoantigens generally are either basic or contain extended, multivalent, charge-rich segments such as coiled-coils.     

Comparison between the phi distribution of the amino acids in the protein database and NMR data indicates that amino acids have various phi propensities in the random coil conformation

It has been indicated that amino acids have various intrinsic phi and psi propensities, as demonstrated from the comparison between experimental secondary structure propensities and their relative statistical distribution in the protein database for the appropriate region of the Ramachandran plot. However, this does not eliminate the possibility that these experimental propensities are the result of context effects due to the secondary structure environment of the mutated position. To demonstrate that there are at least real intrinsic phi propensities, independent of context effects, we have used two different nuclear magnetic resonance parameters related to the phi dihedral angle (J3 alpha HN coupling constants and the chemical shift of the C alpha H proton), determined in random-coil tetra- and pentapeptides, and/or in proteins. Comparison of the experimentally determined values for these parameters with the theoretical ones determined from the analysis by different empirical and theoretical equations of the phi dihedral angle statistical distribution of the amino acids in the protein database, supports the idea that each amino acid has, at least, different phi intrinsic propensities. Consideration of all conformations, or only coil conformations, in the protein database produces similar results. The reasonable correlation between these experimental and theoretical data and the hydrogen-exchange data in random-coil peptides suggests that maximisation of hydrophobic surface-buried and hydrogen-bond formation with the solvent could be responsible for these different random-coil conformational preferences. Analysis of the intrinsic propensities for beta-strand, alpha-helix and polyproline II dihedral angles of the 20 amino acids in coil conformations, indicates that the side-chain of the amino acids is mainly determining the relative preferences for the phi angle.     

Slow alpha helix formation during folding of a membrane protein

Very little is known about the folding of proteins within biological membranes. A "two-stage" model has been proposed on thermodynamic grounds for the folding of alpha helical, integral membrane proteins, the first stage of which involves formation of transmembrane alpha helices that are proposed to behave as autonomous folding domains. Here, we investigate alpha helix formation in bacteriorhodopsin and present a time-resolved circular dichroism study of the slow in vitro folding of this protein. We show that, although some of the protein's alpha helices form early, a significant part of the protein's secondary structure appears to form late in the folding process. Over 30 amino acids, equivalent to at least one of bacteriorhodopsin's seven transmembrane segments, slowly fold from disordered structures to alpha helices with an apparent rate constant of about 0.012 s-1 at pH 6 or 0.0077 s-1 at pH 8. This is a rate-limiting step in protein folding, which is dependent on the pH and the composition of the lipid bilayer.     

Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening.     

Solution structure of recombinant human interleukin-6

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.     

Design, expression, and initial characterization of MB1, a de novo protein enriched in essential amino acids

Using recently emerging protein folding principles we have designed a protein enriched in the essential amino acids methionine, threonine, lysine and leucine. Our preliminary study of consensus residues (based on charge, hydrophobicity and volume) of natural alpha-helical bundle proteins indicated that the residues M, T, K, and L could be inserted in an alpha-helical bundle structure. We therefore attempted to create a stable de novo protein, highly enriched in these essential amino acids, that would adopt the alpha-helical bundle fold. The design process was an iterative one. The consensus residues (based on the properties profile) for bundle helices were found considering the four helices taken together, helices I to IV individually, or only their N- and C-termini. Using these data, the helices in our de novo protein were designed by inserting the residues M, T, K and L as often as possible at positions where their volume, hydrophobicity and charge match the consensus found in natural bundle helices. Short sequences of strong turn formers were used to join the helices and adjust the predicted p1 to 7.7, while a number of local and global factors were used to refine our design. Further, the sequence was checked to eliminate various known protease targets in E. coli. The sequence of our de novo protein, MB1, is: MAT-EDMTDMMTTLFKTMQLLTK-SEPTA-MDEATKTATTMKNHLQNLMQK-TKNKE DMTDMATTYFKTMQLLTK-TEPSA-MDEATKTATTMKNHLQNLMQK-GVA+ ++ , where dashes separate long helices from short, turn forming linkers. A gene coding for this protein was assembled from synthetic oligonucleotides, then fused to the maltose binding protein gene under the control of a tac promoter. The fusion protein was expressed in E. coli, purified and cleaved to yield maltose binding protein and our de novo protein, MB1. MB1 was found to be helical, to have the expected molecular weight (11 kDa) and the expected content (57%) of the essential amino acids M, T, K and L.     

Capping interactions in isolated alpha helices: position-dependent substitution effects and structure of a serine-capped peptide helix

The influence of an amino acid on the stability of alpha-helical structure depends on the position of the residue in the helix with respect to the ends. Short alpha helices in proteins are stabilized both by H-bonding of the main-chain NH and CO groups and by capping interactions between side chains and unfulfilled peptide groups at the N and C termini. Peptide models based on consensus position-dependent helix sequences allow one to model capping effects in isolated helices and to establish a base line for these interactions in proteins. We report here an extended series of substitutions in the cap positions of our peptide models and the solution structure of peptide S3, with serine at the N-cap position defined as the N-terminal residue with partly helix and partly coil conformation. The resulting model, determined by 2D 1H NMR, is consistent with a structure at the N-cap involving H-bonding between the serine gamma oxygen and the peptide NH of the glutamic acid residue three amino acids toward the C terminus. A bifurcated H-bond of Ser O gamma with the NH of Asp5 is possible also, since this group is within interacting distance. This provides direct evidence that specific side-chain interactions with the main chain stabilize isolated alpha-helical structure.     

Helix-stabilizing nonpolar interactions between tyrosine and leucine in aqueous and TFE solutions: 2D-1H NMR and CD studies in alanine-lysine peptides

Interactions between side chains spaced (i,i + 3) and (i,i + 4) may explain the context dependence of helix propensities observed in different systems. Nonpolar residues with these spacings occur frequently in protein helices and stabilize isolated peptide helices. Here (i,i + 3) and (i,i + 4) nonpolar interactions between Tyr and Leu in different solution conditions are studied in detail in alanine-based peptides using 2D 1H NMR and CD spectroscopy. Helix contents analyzed using current models for helix-coil transitions yield interaction energies which demonstrate significant helix stabilization in aqueous 1 M NaCl solutions by Tyr-Leu or Leu-Tyr pairs when spaced (i,i + 4) and, to a smaller extent, when spaced (i,i + 3), comparable to those estimated for other residue pairs. The interactions persist in solutions containing TFE, a helix-stabilizing solvent believed to diminish hydrophobic interactions, but not in helix-destabilizing 6 M urea. 1H NMR resonances for all peptides and solution conditions except in 6 M urea were completely assigned. NMR data indicate that the N-terminal residues are more helical and that the N-acetyl group participates in helix formation. The two (i,i + 4) spaced pairs show the same pattern of NOE cross-peaks between the Tyr and Leu side chains, as do the two (i,i + 3) pairs in 1 M NaCl as well in TFE solutions, and correspond well with that expected for the specific Tyr-Leu pair with side-chain contacts in protein helices.     

Uncoupling hydrophobicity and helicity in transmembrane segments. Alpha-helical propensities of the amino acids in non-polar environments

Although the chains of amino acids in proteins that span the membrane are demonstrably helical and hydrophobic, little attention has been paid toward addressing the range of helical propensities of individual amino acids in the non-polar environment of membranes. Because it is inappropriate to apply soluble protein-based structure prediction algorithms to membrane proteins, we have used de novo designed peptides (KKAAAXAAAAAXAAWAAXAAAKKKK-amide, where X indicates one of the 20 commonly occurring amino acids) that mimic a protein membrane-spanning domain to determine the alpha-helical proclivity of each residue in the isotropic non-polar environment of n-butanol. Peptide helicities measured by circular dichroism spectroscopy were found to range from theta222 = -17,000 degrees (Pro) to -38,800 degrees (Ile) in n-butanol. The relative helicity of each amino acid is shown to be well correlated with its occurrence frequency in natural transmembrane segments, indicating that the helical propensity of individual residues in concert with their hydrophobicity may be a key determinant of the conformations of protein segments in membranes.     

The structure of Fis mutant Pro61Ala illustrates that the kink within the long alpha-helix is not due to the presence of the proline residue

The influence of proline on bending of the alpha-helix was investigated by replacement of the proline residue located in the middle of the long alpha-helix of the Fis protein with alanine, serine, or leucine. Each of the three substitutions folded into a stable protein with the same or higher melting points than the wild-type, but only Pro61Ala was functionally active in stimulating Hin-mediated DNA inversion. Pro61Ala formed crystals that were isomorphous with the wild-type protein allowing the structure to be determined at 1.9-A resolution by x-ray diffraction methods. The structure of the Pro61Ala mutant is almost identical to the wild-type protein, consistent with its near wild-type activity. One of the alpha-helices, the B-helix, is kinked in the wild-type Fis protein by 20 degrees which was previously assumed to be caused solely by the presence of proline 61 in the center of the helix. However, the B-helix is still kinked by 16 degrees when proline 61 is replaced by alanine. Local peptide backbone movement around residue 57 adjusts the geometry of the helix to accommodate the new main chain hydrogen bond between the -CO group in Glu57 and the -NH group in Ala61. Thus, the kink of the alpha-helix in Pro61Ala does not require the presence of proline.     

Role of main-chain electrostatics, hydrophobic effect and side-chain conformational entropy in determining the secondary structure of proteins

The physiochemical bases of amino acid preferences for alpha-helical, beta-strand, and other main-chain conformational states in proteins is controversial. Hydrophobic effect, side-chain conformational entropy, steric factors, and main-chain electrostatic interactions have all been advanced as the dominant physical factors which determine these preferences. Many attempts to resolve the controversy have focused on small model systems. The disadvantage of such systems is that the amino acids in small molecules are largely exposed to the solvent. In proteins, however, the amino acids are in contact with the solvent to a different degree, causing a large variability of strengths of all interactions. The estimates of mean strengths of interactions in the actual protein environment are therefore essential to resolve the controversy. In this work the experimental protein structures are used to estimate the mean strengths of various interactions in proteins. The free energy contributions of the interactions are implemented into the Lifson-Roig theory to calculate the helix and strand free energy profiles. From the profiles the secondary structures of proteins and peptides are predicted using simple rules. The role of hydrophobic effect, side-chain conformational entropy, and main-chain electrostatic interactions in determining the secondary structure of proteins is assessed from the abilities of different models, describing stability of secondary structures, to correctly predict alpha-helices, beta-strands and coil in 130 proteins. The three-state accuracy of the model, which contains only the free energy terms due to the main-chain electrostatics with 40 coefficients, is 68.7%. This accuracy is approaching to the accuracy of currently the best secondary structure prediction algorithm based on neural networks (72%); however, many thousands of parameters have to be optimized during the training of the neural networks to reach this level of accuracy. The correlation coefficient between the calculated and the experimental helix contents of 37 alanine based peptides is 0.91. If the hydrophobic and the side-chain conformational entropy terms are included into the helix-coil transition parameters, the accuracy of the algorithm does not improve significantly. However, if the main-chain electrostatic interactions are excluded from the helix-coil and strand-coil transition parameters, the accuracy of the algorithm reaches only 59.5%. These results support the dominant role of the short-range main-chain electrostatics in determining the secondary structure of proteins and peptides. The role of the hydrophobic effect and the side-chain conformational entropy is small.