Similarities between the spectrin SH3 domain denatured state and its folding transition state.

We have expanded our description of the energy landscape for folding of the SH3 domain of chicken alpha-spectrin by a detailed structural characterization of its denatured state ensemble (DSE). This DSE is significantly populated under mildly acidic conditions in equilibrium with the folded state. Evidence from heteronuclear nuclear magnetic resonance (NMR) experiments on (2)H, (15)N-labeled protein suggests the presence of conformers whose residual structure bears some resemblence to the structure of the folding transition state of this protein. NMR analysis in a mutant with an engineered, non-native alpha-helical tendency shows a significant amount of local non-native structure in the mutant, while the overall characteristics of the DSE are unchanged. Comparison with recent theoretical predictions of SH3 domain folding reactions reveals an interesting correlation with the predicted early events. Based on these results and recent data from other systems, we propose that the DSE of a protein will resemble the intermediate or transition state of its nearest rate-limiting step, as a consequence of simple energetic and kinetic principles.

The design of linear peptides that fold as monomeric beta-sheet structures.

Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.

Beta-hairpin and beta-sheet formation in designed linear peptides.

Recent knowledge about the determinants of beta-sheet formation and stability has notably been improved by the structural analysis of model peptides with beta-hairpin structure in aqueous solution. Several experimental studies have shown that the turn region residues can not only determine the stability, but also the conformation of the beta-hairpin. Specific interstrand side-chain interactions, hydrophobic and polar, have been found to be important stabilizing interactions. The knowledge acquired in the recent years from peptide systems, together with the information gathered from mutants in proteins, and the analysis of known protein structures, has led to successful design of a folded three-stranded monomeric beta-sheet structure.

Design of a 20-amino acid, three-stranded beta-sheet protein.

A 20-residue protein (named Betanova) forming a monomeric, three-stranded, antiparallel beta sheet was designed using a structural backbone template and an iterative hierarchical approach. Structural and physicochemical characterization show that the beta-sheet conformation is stabilized by specific tertiary interactions and that the protein exhibits a cooperative two-state folding-unfolding transition, which is a hallmark of natural proteins. The Betanova molecule constitutes a tractable model system to aid in the understanding of beta-sheet formation, including beta-sheet aggregation and amyloid fibril formation.

Ionization-reactivity relationships for cysteine thiols in polypeptides.

Thiol-disulfide exchange reactions are required for many aspects of cellular metabolism including the folding of disulfide-bonded proteins, electron transfer, and numerous regulatory mechanisms. To identify factors influencing the rates of these reactions in polypeptides, the reactivities of Cys thiols in 16 model peptides were measured. For each of the peptides, which contained single Cys residues with thiol pKas ranging from 7.4 to 9.1, the rates of exchange with four disulfide-bonded compounds were measured. In reactions with two of the disulfide reagents, cystine and 2-hydroxyethyl disulfide, the peptide thiols displayed Bronsted correlations between reaction rate and pKa similar to those observed previously with model compounds (betanuc = 0.5 and 0.3, respectively). For two reagents with net charges, oxidized glutathione and cystamine, however, the apparent Bronsted coefficients were 0 and 0.8, respectively. These observations are in striking contrast with those obtained with model compounds, for which the Bronsted coefficients for the nucleophilic thiolates are largely independent of the disulfide-containing compound. The differences in the apparent Bronsted coefficients can be largely accounted for by electrostatic interactions between charged groups on the peptides and disulfide reagents and demonstrate that such interactions can play a dominant role in determining the rates of thiol-disulfide exchange in biological molecules. The results presented here provide an improved basis for predicting the rates of these reactions and suggest ways in which differences in the rates of competing reactions can be either minimized, to simplify the analysis of disulfide-coupled folding reactions, or enhanced, to favor formation of particular disulfides.

Electrostatic interactions in the active site of the N-terminal thioredoxin-like domain of protein disulfide isomerase.

Proteins with the thioredoxin fold have widely differing stabilities of the disulfide bond that can be formed between the two cysteines at their active site sequence motif Cys1-Xaa2-Yaa3-Cys4. This is believed to be regulated not by varying the disulfide bond itself, but by modulating the stability of the dithiol form of the protein through interactions with the ionized form of the Cys1 thiol group. A consistent relationship between disulfide bond stability and Cys1 thiol pKa value is found here for DsbA, thioredoxin, and the N-terminal thioredoxin-like domain of protein disulfide isomerase (PDI a), which has a very low thiol pKa value of 4.5. This thiolate anion is stabilized by 5.7 kcal/mol in the dithiol form, giving rise to the corresponding instability of the disulfide bond and the oxidizing properties of PDI a. Electrostatic interactions in the active site of the PDI a-domain have been characterized in order to understand the physical basis of this stabilization. Linkage with the ionization of the imidazole group of His3 in the active site demonstrates that this charge-charge interaction contributes 1.1 kcal/mol. The remainder of the stabilization is believed to be due primarily to interactions with the partial positive charges at the N-terminus of an alpha-helix, which are exceedingly sensitive to charges of surrounding residues.

Comparison of the (30-51, 14-38) two-disulphide folding intermediates of the homologous proteins dendrotoxin K and bovine pancreatic trypsin inhibitor by two-dimensional 1H nuclear magnetic resonance.

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) revealed that the native conformation is still stable in each intermediate state with two native disulphide linkages, in the absence of each of the corresponding third disulphide bonds. This is thought to be a consequence of the extreme stability of the native BPTI conformation. The current study addresses the question of whether the native-like conformation would be populated significantly at the two-disulphide stage in disulphide refolding if the final structure is less stable than in the case of BPTI. Dendrotoxin K from black mamba venom provides a good model to test this, since it contains the BPTI fold and was shown to fold predominantly via the same pathway, but its native conformation is stable than that of BPTI. The conformation of a chemically trapped two-disulphide intermediate in the disulphide refolding of dendrotoxin K, with blocking groups on Cys5 and Cys55 and disulphide bonds between Cys30 and Cys51, and Cys14 and Cys38, respectively, has been determined by 1H NMR spectroscopy and compared to those of the native protein and of the corresponding intermediate in BPTI. The analysis reveals that the dendrotoxin K intermediate adopts a partly-folded conformation, in contrast to the quasi-native conformation of the corresponding BPTI intermediate. It is similar to the partly-folded conformation of the BPTI intermediate with just the Cys30-Cys-51 disulphide bond, but with a more fixed conformation in the region of the Cys14-Cys38 disulphide bond. The destabilisation of the fully native conformation of the dendrotoxin K intermediate, relative to BPTI, appears to reduce the cooperativeity of the folding process.

Ionisation of cysteine residues at the termini of model alpha-helical peptides. Relevance to unusual thiol pKa values in proteins of the thioredoxin family.

The physical basis of the unusually low pKa values of an active site cysteine thiol group in proteins with the thioredoxin fold is unknown. The electrostatic field associated with an alpha-helix pointing with its N terminus towards the cysteine residue has been implicated to lower the thiol pKa value by up to 5 pH units in glutaredoxin and DsbA. Here, the influence of the presence of an alpha-helical conformation on the ionisation of a cysteine thiol group located at or near the helix terminus is investigated in highly helical synthetic peptides with the generic sequence Ac-AAAAAAAAARAAAARAAAARAA-(NH2). The thiol pKa values have been determined by monitoring the pH dependence of the absorbance at 240 nm, of the alpha-helix content measured by the mean residue ellipticity at 222 nm, and of the chemical shifts of protons close to the sulphur atom of the cysteine residue. The favourable interaction between the thiolate anion at the N terminus and the alpha-helix decreases the thiol pKa value by up to 1.6 pH units when compared to a normal thiol pKa value measured in an unfolded control peptide, corresponding to a stabilisation energy of 2.1 kcal/mol. At the C terminus, the thiol pKa value is increased, but by only 0.2 pH units. The observations are consistent with an interaction of the alpha-helix dipole with the cysteine thiolate anion, involving both its charge and hydrogen-bonding. Subtle conformational effects in different model peptides appear to influence the ionisation of the thiol group significantly, with an N terminal Cys-Pro sequence having the most favourable interaction with the alpha-helix.

Helix propensities of the amino acids measured in alanine-based peptides without helix-stabilizing side-chain interactions.

Helix propensities of the amino acids have been measured in alanine-based peptides in the absence of helix-stabilizing side-chain interactions. Fifty-eight peptides have been studied. A modified form of the Lifson-Roig theory for the helix-coil transition, which includes helix capping (Doig AJ, Chakrabartty A, Klingler TM, Baldwin RL, 1994, Biochemistry 33:3396-3403), was used to analyze the results. Substitutions were made at various positions of homologous helical peptides. Helix-capping interactions were found to contribute to helix stability, even when the substitution site was not at the end of the peptide. Analysis of our data with the original Lifson-Roig theory, which neglects capping effects, does not produce as good a fit to the experimental data as does analysis with the modified Lifson-Roig theory. At 0 degrees C, Ala is a strong helix former, Leu and Arg are helix-indifferent, and all other amino acids are helix breakers of varying severity. Because Ala has a small side chain that cannot interact significantly with other side chains, helix formation by Ala is stabilized predominantly by the backbone ("peptide H-bonds"). The implication for protein folding is that formation of peptide H-bonds can largely offset the unfavorable entropy change caused by fixing the peptide backbone. The helix propensities of most amino acids oppose folding; consequently, the majority of isolated helices derived from proteins are unstable, unless specific side-chain interactions stabilize them.

Aromatic side-chain contribution to far-ultraviolet circular dichroism of helical peptides and its effect on measurement of helix propensities.

Peptides of the sequence Ac-XKAAAAKAAAAKAAAAK-amide, where X is Tyr, Trp, or Ala, produce circular dichroism spectra that are typical of the alpha-helix; there are, however, significant differences between the Tyr, Trp, or Ala peptides in the magnitudes of the far-ultraviolet bands. A tyrosine or tryptophan residue is needed in each peptide in order to measure accurately the peptide concentration and the mean residue ellipticity. The N- or C-terminal position is chosen because helix fraying is greatest at each end and the Tyr or Trp residue should influence the helix content of the peptide least at these positions. Amide proton exchange measurements by proton nuclear magnetic resonance spectroscopy indicate that the Tyr, Trp, and Ala peptides possess similar amounts of H-bonded secondary structure. Comparison of the far-ultraviolet circular dichroism and absorption spectra of these peptides suggests that the differences in circular dichroism arise in each case from an induced aromatic circular dichroism band, which is positive for Tyr and negative for Trp. Insertion of one to three Gly residues between the aromatic residue and the rest of the helical sequence reduces the induced aromatic band to insignificant levels. Using this procedure of inserting Gly residues between the Tyr and the rest of the helical sequence, we remeasured the helix propensity of Gly. We find that the Ala:Gly ratio of helix propensities is 40, as opposed to our previous estimate of 100 determined using the Tyr peptide without considering the aromatic contribution of Tyr in the analysis [Chakrabartty, A., Schellman, J. A., & Baldwin, R. L. (1991) Nature 351, 586-588].