Alpha-helix stability in proteins. II. Factors that influence stability at an internal position.

The solvent-exposed residue Ala32 in the second alpha-helix of barnase was replaced by all other naturally occurring amino acids and the concomitant effects on the protein stability were determined. The results are assumed to reflect both the distinct conformational preferences of the different amino acids and also possible intrahelical interactions. The conformational preferences may be fully rationalized by invoking only a few physical principles. The results agree well with recently experimentally determined rank-order of helix-forming tendencies determined on a model peptide. There is very weak correlation between the results and the experimental host-guest values. There is a weak correlation between our results and the statistical helix propensities and a slightly better correlation with the positional-dependent statistical parameters of J. S. Richardson, and D. C. Richardson.     

A survey of amino acid side-chain interactions in 21 proteins

Based on the atomic co-ordinate data for 21 representative proteins, the frequencies of long-range interactions between side-chain groups and 15 different types of side-chain atoms have been determined. The observed frequencies are compared to the results expected for random association in order to define a scale of relative affinities. Thirty-five residue-atom pairs exhibit frequencies of interaction that differ by at least 50% from the expected values. The amino acids tend to fall into three classes: non-polar, neutral and polar amino acids. The data are regrouped in a different way to determine the average affinity of each amino acid side-chain group for all other types of side-chain groups. Fourteen side-chain pairs have at least 50% fewer interactions than expected, while 21 side-chain pairs have at least 50% more interactions than expected. Unusual patterns of association are discussed and compared with current ideas about the organization of protein structure.     

The Glu 2- ... Arg 10+ side-chain interaction in the C-peptide helix of ribonuclease A

Previous studies have identified Lys 1, Glu 2, and His 12 as the charged residues responsible for the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A). Here we examine whether the helix-stabilizing behavior of Glu 2- results from a Glu 2- ... Arg 10+ interaction, which is known to be present in the crystal structure of ribonuclease A. The general approach is to measure the helix content of C-peptide analogs as a function of three variables: pH (titration of ionizing groups), amino acid identity (substitution test), and NaCl concentration (ion screening test). In order to interpret the results of residue replacement, several factors in addition to the putative Glu 2- ... Arg 10+ interaction have been studied: intrinsic helix-forming tendencies of amino acids; interactions of charged residues with the alpha-helix macrodipole; and helix-lengthening effects. The results provide strong evidence that the Glu 2- ... Arg 10+ interaction is linked to helix formation and contributes to the stability of the isolated C-peptide helix. NMR evidence supports these conclusions and suggests that this interaction also acts as the N-terminal helix stop signal. The implications of this work for protein folding and stability are discussed.     

Introduction of unnatural amino acids into chalcone isomerase.

The active site cysteine residue of chalcone isomerase was rapidly and selectively modified under denaturing conditions with a variety of electrophilic reagents. These denatured and modified enzyme were renatured to produce enzyme derivatives containing a series of unnatural amino acids in the active site. Addition of methyl, ethyl, butyl, heptyl, and benzyl groups to the cysteine sulfur does not abolish catalytic activity, although the activity decreases as the steric bulk of the amino acid side-chain increases. Modification of the cysteine to introduce a charged homoglutamate or a neutral homoglutamine analogue results in retention of 22% of the catalytic activity. Addition of a methylthio group (SMe) to the cysteine residue of native chalcone isomerase preserves 85% of the catalytic activity measured with 2',4',4-trihydroxychalcone, 2',4',6',4-tetrahydroxychalcone, or 2'-hydroxy-4-methoxychalcone as substrates. The competitive inhibition constant for 4',4-dihydroxychalcone, the substrate inhibition constant for 2',4',4-trihydroxychalcone, and other steady-state kinetic parameters for the methanethiolated enzyme are very similar to those of the native enzyme. The strong binding of 4',4-dihydroxychalcone to the methanethiolated enzyme shows that there is no steric repulsion between this modified amino acid residue and the substrate analogue. This structure-activity study clearly demonstrates that the active site cysteine residue does not function as an acid-base or nucleophilic group in producing the catalysis or substrate inhibition observed with chalcone isomerase. The method presented in this paper allows for the rapid introduction of a series of unnatural amino acids into the active site as a means of probing the structure-function relationship.     

Hydrogen-bonding classes in proteins and their contribution to the unfolding reaction

This paper proposes to assess hydrogen-bonding contributions to the protein stability, using a set of model proteins for which both X-ray structures and calorimetric unfolding data are known. Pertinent thermodynamic quantities are first estimated according to a recent model of protein energetics based on the dissolution of alkyl amides. Then it is shown that the overall free energy of hydrogen-bond formation accounts for a hydrogen-bonding propensity close to helix-forming tendencies previously found for individual amino acids. This allows us to simulate the melting curve of an alanine-rich helical 50-mer with good precision. Thereafter, hydrogen-bonding enthalpies and entropies are expressed as linear combinations of backbone-backbone, backbone-side-chain, side-chain-backbone, and side-chain-side-chain donor-acceptor contributions. On this basis, each of the four components shows a different free energy versus temperature trend. It appears that structural preference for side-chain-side-chain hydrogen bonding plays a major role in stabilizing proteins at elevated temperatures.     

Hydrophilicity of polar amino acid side-chains is markedly reduced by flanking peptide bonds

Several amino acid side-chain hydropathy scales have been devised on the basis of solubility and water/organic solvent partitioning data obtained with free amino acids or side-chain analogs. In nearly all cases, these scales are based upon the structure-additivity assumption; it has been assumed that the transfer free energies of the amino acid side-chains are the same in these model compounds as they are in a polypeptide. This assumption is probably wrong. In the present study, deviations from additivity for amino acid side-chains are demonstrated by comparing a theoretically derived scale, which N-acetylamino acid amides. The results show that the flanking peptide bonds dramatically reduce the hydrophilicity of the polar side-chains, with deviations up to several kilocalories (1 kcal = 4.184 kJ) for the charged side-chains at pH 7.0. Further calculation shows that these deviations are due to reductions of 40 to 85% in the unfavorable transfer free energy of the polar functional groups. In addition, proximity of the neighboring amide bonds in the parent molecule (N-acetylglycine amide) decreases the hydrophilicity of the -CONH-backbone unit by 36%. This decrease is expected to be twice as large for -CONH- units in the interior of a polypeptide backbone. The significance of these observations is: (1) valid hydropathy scales can be obtained only with model peptides; (2) deviations from additivity are expected in all solvent systems, including non-polar solvents that are thought to mimic the interior of a membrane; (3) the spontaneous insertion of polypeptides into membranes is likely to occur much more readily than has been previously thought. In order to estimate the free energy of transferring the side-chains and the polypeptide backbone from water to the interior of a lipid bilayer, the results of this study are used to construct a hydropathy scale based upon the partitioning of solutes between water and non-polar solvents. The validity of hydropathy scales that are based on criteria other than solubility and water/organic solvent partitioning data is also discussed.     

Amino acid propensities are position-dependent throughout the length of alpha-helices

The 20 commonly occurring amino acids have been shown to have distinct position-dependent, helix-forming propensities near the ends of alpha-helices. Here, we show that the amino acids also have very strong position-dependent propensities throughout the length of a helix. Most helices are amphiphilic, and they have a strong tendency to both begin and end on the solvent-inaccessible face of the helix. These position-specific propensities should provide valuable parameters to guide de novo protein design, and should allow more precise prediction of helical topology in natural proteins.     

Protein design simulations suggest that side-chain conformational entropy is not a strong determinant of amino acid environmental preferences

Loss of side-chain conformational entropy is an important force opposing protein folding and the relative preferences of the amino acids for being buried or solvent exposed may be partially determined by which amino acids lose more side-chain entropy when placed in the core of a protein. To investigate these preferences, we have incorporated explicit modeling of side-chain entropy into the protein design algorithm, RosettaDesign. In the standard version of the program, the energy of a particular sequence for a fixed backbone depends only on the lowest energy side-chain conformations that can be identified for that sequence. In the new model, the free energy of a single amino acid sequence is calculated by evaluating the average energy and entropy of an ensemble of structures generated by Monte Carlo sampling of amino acid side-chain conformations. To evaluate the impact of including explicit side-chain entropy, sequences were designed for 110 native protein backbones with and without the entropy model. In general, the differences between the two sets of sequences are modest, with the largest changes being observed for the longer amino acids: methionine and arginine. Overall, the identity between the designed sequences and the native sequences does not increase with the addition of entropy, unlike what is observed when other key terms are added to the model (hydrogen bonding, Lennard-Jones energies, and solvation energies). These results suggest that side-chain conformational entropy has a relatively small role in determining the preferred amino acid at each residue position in a protein.     

Partial molar volumes of proteins: amino acid side-chain contributions derived from the partial molar volumes of some tripeptides over the temperature range 10-90 degrees C

The partial molar volumes of tripeptides of sequence glycyl-X-glycine, where X is one of the amino acids alanine, leucine, threonine, glutamine, phenylalanine, histidine, cysteine, proline, glutamic acid, and arginine, have been determined in aqueous solution over the temperature range 10-90 degrees C using differential scanning densitometry . These data, together with those reported previously, have been used to derive the partial molar volumes of the side-chains of all 20 amino acids. The side-chain volumes are critically compared with literature values derived using partial molar volumes for alternative model compounds. The new amino acid side-chain volumes, along with that for the backbone glycyl group, were used to calculate the partial specific volumes of several proteins in aqueous solution. The results obtained are compared with those observed experimentally. The new side-chain volumes have also been used to re-determine residue volume changes upon protein folding.     

Nature disfavors sequences of alternating polar and non-polar amino acids: implications for amyloidogenesis

Recent experiments with combinatorial libraries of de novo proteins have demonstrated that sequences designed to contain polar and non-polar amino acid residues arranged in an alternating pattern form fibrillar structures resembling beta-amyloid. This finding prompted us to probe the distribution of alternating patterns in the sequences of natural proteins. Analysis of a database of 250,514 protein sequences (79,708,024 residues) for all possible binary patterns of polar and non-polar amino acid residues revealed that alternating patterns occur significantly less often than other patterns with similar compositions. The under-representation of alternating binary patterns in natural protein sequences, coupled with the observation that such patterns promote amyloid-like structures in de novo proteins, suggests that sequences of alternating polar and non-polar amino acids are inherently amyloidogenic and consequently have been disfavored by evolutionary selection.     

EFFECTS OF DIPEPTIDES CONTAINING THE AMINO ACID,PROLINE ON THE CHEMOTAXIS OF TETRAHYMENA PYRIFORMIS. EVOLUTIONARY CONCLUSIONS ON THE FORMATION OF HORMONE RECEPTORS AND HORMONES

Our investigations demonstrate that proline-containing dipeptides can provoke a chemosensory response from the unicellular Tetrahymena pyriformis The chemotactic effects of the dipeptides have a close relationship with the side chain and the lipophilicity of the amino-terminal amino acid. Comparison of ‘mirror’ variants of proline-containing dipeptides points to the fact that dipeptides with small side chain and non-polar character amino acids (Gly-Pro, Ala-Pro) are preferred on the amino-terminal end. In the case of amino acids with very variable side chains, small (Pro-Gly) and the large side chain and non-polar character amino acids (Pro-Leu, Pro-Phe) on the carboxyl-terminal end can induce significant chemotactic responses. With valine on any terminus the proline-containing dipeptide induced a weak repellent effect.     

Periodicity in alpha-helix lengths and C-capping preferences.

We surveyed 299 high resolution, non-homologous protein crystal structures for alpha-helix lengths and capping preferences. We find that helices show a preference to have close to an integral number of turns. Helices can be usefully subdivided into either "favoured length" with 6, 7, 10, 11, 13, 14, 17, 18, 21, 22, 24, 25, 28, 29 or 31 residues, or "disfavoured length" with 8, 9, 12, 15, 16, 19, 20, 23, 26, 27 or 30 residues. Favoured length helices have their N and C-caps on the same side of the helix so they can lie on the protein surface. There is no significant difference in amino acid preferences at the N terminus between favoured and disfavoured length helices. At the C terminus, favoured length helices prefer non-polar side-chains at C4 and polar amino acid residues at C2, while disfavoured length helices prefer non-polar amino acid residues at C2. There are strong periodic trends in the likelihood of terminating a helix with a Schellman or alphaL C-capping motif. These can be rationalised by the preference for a non-polar side-chain at C3 with these motifs, favouring placing C3 on the buried side of the helix. We suggest that algorithms aiming to predict helices or C-capping in proteins should include a weight for the helix length.     

The use of natural and unnatural amino acid substrates to define the substrate specificity differences of Escherichia coli aspartate and tyrosine aminotransferases.

The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate diacarboxylic amino acids with similar rate constants. However, eTATase exhibits approximately 10(2)-10(4)-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to alpha-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L-Asp and L-Glu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidino group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acid.     

De novo design and structure-activity relationships of peptide emulsifiers and foaming agents

A series of eight amphipathic peptides (8, 11, 15, 2 x 18, 22, 26, 29 amino acids in length) were designed to investigate the effects of amino acid composition, peptide length and secondary structure on surface activity assessed as emulsification and foaming activity. The potential for alpha-helix formation at the hydrophobic/hydrophilic interface was maximized through the use of helix-forming amino acids, a relatively large hydrophobic surface of 200 degrees of arc and ion pairs between basic and acidic amino acids on the hydrophilic surface. Emulsification activity increased rapidly between 11 and 22 residues as alpha-helicity in aqueous solution increased. Despite their small size, the peptides produced exceptionally stable emulsions, compared with proteins. Foaming activity was enhanced by the presence of aromatic amino acids and the activity of the best peptide examined was superior to that of bovine serum albumin and beta-lactoglobulin.     

Helix propagation and N-cap propensities of the amino acids measured in alanine-based peptides in 40 volume percent trifluoroethanol.

The helix propagation and N-cap propensities of the amino acids have been measured in alanine-based peptides in 40 volume percent trifluoroethanol (40% TFE) to determine if this helix-stabilizing solvent uniformly affects all amino acids. The propensities in 40% TFE are compared with revised values of the helix parameters of alanine-based peptides in water. Revision of the propensities in water is the result of redefining the capping statistical weights and evaluating the helix nucleation constant with N-capping explicitly included in the helix-coil model. The propagation propensities of all amino acids increase in 40% TFE relative to water, but the increases are highly variable. In water, all beta-branched and beta-substituted amino acids are helix breakers. In 40% TFE, the propagation propensities of the nonpolar amino acids increase greatly, leaving charged and neutral polar, beta-substituted amino acids as helix breakers. Glycine and proline are strong helix breakers in both solvents. Free energy differences for helix propagation (delta delta G) between alanine and other nonpolar amino acids are twice as large in water as predicted from side-chain conformational entropies, but delta delta G values in 40% TFE are close to those predicted from side-chain entropies. This dependence of delta delta G on the solvent points to a specific role of water in determining the relative helix propensities of the nonpolar amino acids. The N-cap propensities converge toward a common value in 40% TFE, suggesting that differential solvation by water contributes to the diversity of N-cap values shown by the amino acids.     

Physico-chemical basis of the genetic code origin: stereochemical analysis of interactions of amino acids and nucleotides based on the progene hypothesis

A progene hypothesis has been proposed earlier to explain the mechanism of origin of the self-reproducing genetic system. Progenes (precursors of the genetic system) are mixed anhydrides of an amino acid and deoxyribotrinucleotide at the 3'-gamma-terminal phosphate (NpNpNppp-AA); they are produced from dinucleotides (NpNp) and 3'-gamma-aminoacylnucleotidylates (Nppp-AA) as a result of specific interaction between amino acid and dinucleotide. The postulated mechanism of progene formation accounts for the selection of substances, including chirality, the origin of the genetic code as well as for the mechanisms of formation, self-reproduction and evolution of the simpliest genetic system ("gene--polypeptide"). A stereochemical analysis of the progene formation mechanism has allowed us to support the main statements of the hypothesis that relate to the origin of the genetic code and to selection of substances. Atomic groups that could be responsible for the specificity of interaction between dinucleotides and amino acids in progene formation have been revealed. Stereochemical evidence for the physicochemical basis of the origin of the existing genetic code have been produced: 1) a special role of the second nucleotide in the codon is demonstrated in amino acid coding by the progene hypothesis principle; 2) an advantage of T against U in such coding is demonstrated; 3) for 16 amino acids out of 20 an agreement has been obtained between the optimal dinucleotide as revealed by the stereochemical analysis and the codon dinucleotides; 4) an explanation for the third nucleotide selection mechanism is offered. A restoration of the prebiotic code, based on these results, has indicated that the code contains 32 codons, is statistical and group-wise. It encodes 7 groups of isofunctional amino acids: 3 overlapping groups of non-polar amino acids 1) medium-size hydrophobic amino acids (chiefly Val, n-Val and a-But), 2) small and medium-size non-polar amino acids (chiefly Ala Val, n-Val a-But and Gly), 3) small non-polar amino acids (Gly, Ala, a-But) and 4 groups of polar amino acids--1) hydroxy--+dicarbonic (Asp, Glu, Ser and Thr), 2) dicarbonic (Asp and Glu), 3) hydroxy (Ser and Thr) and 4) basic (Arg and Lys). The code includes about 20 amino acids among which are 15-17 canonical and a few common non-canonical. The prebiotic code explains many properties of the existing genetic code and is capable of evolving into the latter by way of a gradual replacement of the physicochemical coding mechanism by the enzymatic coding mechanism.     

Conformational constraints of amino acid side chains in alpha-helices

The conformational freedom of amino acid side chains is strongly reduced when the side chains occur on an α-helix. A quantitative evaluation of this freedom has been carried out by means of conformational energy computations for all naturally occurring amino acids and for α-aminobutyric acid when they are placed in the middle of a right-handed poly(L-alanine) α-helix. One of the three possible rotameric states for rotation around the C^α ? C^β bond (viz. g⁺) is excluded completely on the helix because of steric hindrance, and the relative populations of the other two rotamers (t and g^?) are altered because of steric interactions and the reduction of hydrogen-bonding possibilities. The computed tendencies of the changes in distributions of rotamers, on going from an ensemble of all backbone conformations to the α-helix, agree with the observed tendencies in proteins. Minimum-energy side-chain conformations in an α-helix have been tabulated for use in conformational energy computations on polypeptides.     

Thermodynamic consequences of burial of polar and non-polar amino acid residues in the protein interior

Effects of amino acid substitutions at four fully buried sites of the ubiquitin molecule on the thermodynamic parameters (enthalpy, Gibbs energy) of unfolding were evaluated experimentally using differential scanning calorimetry. The same set of substitutions has been incorporated at each of four sites. These substitutions have been designed to perturb packing (van der Waals) interactions, hydration, and/or hydrogen bonding. From the analysis of the thermodynamic parameters for these ubiquitin variants we conclude that: (i) packing of non-polar groups in the protein interior is favorable and is largely defined by a favorable enthalpy of van der Waals interactions. The removal of one methylene group from the protein interior will destabilize a protein by approximately 5 kJ/mol, and will decrease the enthalpy of a protein by 12 kJ/mol. (ii) Burial of polar groups in the non-polar interior of a protein is highly destabilizing, and the degree of destabilization depends on the relative polarity of this group. For example, burial of Thr side-chain in the non-polar interior will be less destabilizing than burial of Asn side-chain. This decrease in stability is defined by a large enthalpy of dehydration of polar groups upon burial. (iii) The destabilizing effect of dehydration of polar groups upon burial can be compensated if these buried polar groups form hydrogen bonding. The enthalpy of this hydrogen bonding will compensate for the unfavorable dehydration energy and as a result the effect will be energetically neutral or even slightly stabilizing.     

Synthesis and modification of dibenzylglycine derivatives via the Suzuki-Miyaura cross-coupling reaction.

The objective of this paper is to describe in details of various available methods to prepare C(alpha,alpha)-dibenzylglycine (Dbzg) and then include our work involving the synthesis of side chain Dbzg derivatives. alpha,alpha-Disubstituted amino acids (alpha,alphaAAs) are important members in the family of modified amino acids. Replacement of the alpha-hydrogens of glycine 1 by alkyl groups leads to alpha,alphaAAs. The steric hindrance of the quaternary centre of Aib 2 combined with the helix-forming capacity has attracted the attention of structural biologists and protein crystallographers. Dbzg 3 is a special structural variant of Aib. The presence of two benzyl groups at C(alpha)-position not only impart rigidity to the peptide backbone in which it is incorporated, but also acts as a useful vehicle for studying pi-pi interactions. Although several C(alpha,alpha)-disubstituted glycines such as C(alpha,alpha)-diethyl glycine (Deg), C(alpha,alpha)-dipropyl glycine (Dpg) etc. have been studied in detail, not much has been known about Dbzg because of limited availability of synthetic procedures. Various Dbzg derivatives 19a-f have been prepared using ethyl isocyanoacetate 14 as a glycine equivalent (eq.). A useful and simple methodology has been developed using the Suzuki-Miyaura cross-coupling reaction for the modification of Dbzg derivatives 17d, 19d, 22. Using this 'Building Block Approach' (Accounts of Chemical Research 36, 2003, 342) one can generate a variety of Dbzg derivatives 20a-f and 23a-e, which may find useful applications in combinatorial synthesis and QSAR studies.     

Derivation and testing of pair potentials for protein folding. When is the quasichemical approximation correct?

Many existing derivations of knowledge-based statistical pair potentials invoke the quasichemical approximation to estimate the expected side-chain contact frequency if there were no amino acid pair-specific interactions. At first glance, the quasichemical approximation that treats the residues in a protein as being disconnected and expresses the side-chain contact probability as being proportional to the product of the mole fractions of the pair of residues would appear to be rather severe. To investigate the validity of this approximation, we introduce two new reference states in which no specific pair interactions between amino acids are allowed, but in which the connectivity of the protein chain is retained. The first estimates the expected number of side-chain contracts by treating the protein as a Gaussian random coil polymer. The second, more realistic reference state includes the effects of chain connectivity, secondary structure, and chain compactness by estimating the expected side-chain contrast probability by placing the sequence of interest in each member of a library of structures of comparable compactness to the native conformation. The side-chain contact maps are not allowed to readjust to the sequence of interest, i.e., the side chains cannot repack. This situation would hold rigorously if all amino acids were the same size. Both reference states effectively permit the factorization of the side-chain contact probability into sequence-dependent and structure-dependent terms. Then, because the sequence distribution of amino acids in proteins is random, the quasichemical approximation to each of these reference states is shown to be excellent. Thus, the range of validity of the quasichemical approximation is determined by the magnitude of the side-chain repacking term, which is, at present, unknown. Finally, the performance of these two sets of pair interaction potentials as well as side-chain contact fraction-based interaction scales is assessed by inverse folding tests both without and with allowing for gaps.