Ana Rosa Viguera

The order of secondary structure elements does not determine the structure of a protein but does affect its folding kinetics.

We have analyzed the structure, stability and folding kinetics of circularly permuted forms of alpha-spectrin SH3 domain. All the possible permutations involving the disruption of the covalent linkage between two beta-strands forming a beta-hairpin have been done. The different proteins constructed here fold to a native conformation similar to that of wild-type protein, as demonstrated by nuclear magnetic resonance and circular dichroism. Although all the mutants have similar stabilities (they are 1 to 2 kcal mol-1 less stable than the wild-type) their rate constants for folding and unfolding are quite different. Protein engineering, in combination with kinetics indicates that the folding pathway has been changed in the circularly permuted proteins. We conclude that neither the order of secondary structure elements, nor the preservation of any of the beta-hairpins present in this domain, is crucial for the ability of the polypeptide to fold, but they influence the folding and unfolding kinetics and could determine its folding pathway.

Favourable native-like helical local interactions can accelerate protein folding

BACKGROUNDnExtensive studies of peptide conformation have provided reasonable knowledge of the rules determining helix stability. This knowledge can be used to stabilize proteins against chemical and thermal denaturation. This has been done in two proteins: the chemotactic protein from Escherichia coli, Che Y (a 129 aa alpha/beta parallel protein with five alpha-helices, which shows an accumulating intermediate during refolding) and the activation domain of human procarboxypeptidase A2, ADA2h (a 81 aa alpha + beta protein domain, with two alpha-helices, which follows a two-state mechanism). As the introduced stabilizing interactions are local in nature, the energy balance between the contribution of local and nonlocal interactions changes considerably. Recent theoretical analyses of protein folding using simplified models have indicated that optimization of folding speed requires this balance to be biased towards nonlocal interactions. To determine whether this is the case, we study here the folding kinetics of two ADA2h mutants in which alpha-helix 1 (mutant M1) or 2 (mutant M2) has been stabilized through local interactions, as well as the equilibrium and kinetic behaviour of a double mutant (DM) in which both helices have been stabilized.nnnRESULTSnThe stability of DM is considerably enhanced with respect to wild type (WI) and this mutant can be considered as a thermoresistant protein (Tm > 363 K). The thermodynamic parameters obtained by chemical denaturation (urea and GdnHCl) show that DM is approximately 2.6 kcal mol-1 more stable than WT. The effects on folding kinetics are different in each of the single mutants. M1 shows very little effect in refolding, while its unfolding is greatly decelerated with respect to WT. M2 shows, together with a deceleration in unfolding, a significant acceleration in refolding. As with equilibrium parameters, the kinetics of the double mutant can be explained by the simple addition of the effects found in each single mutant. Interestingly enough, the refolding slope mkf in mutants M2 and DM is smaller than in the wild-type and M1 mutant.nnnCONCLUSIONSnThermoresistance can be achieved, in some cases, by increasing favourable native local interactions. The balance between local and nonlocal interactions can be significantly changed in some proteins and still keep a cooperative unfolding transition similar to that of the wild type. The introduction of favourable local interactions by mutational redesign can also be used to increase the folding speed of certain proteins, showing that not all proteins in nature have been optimized for rapid folding, contrary to what has been theoretically indicated. This behaviour is probably also shared by other polypeptides with highly unstructured denatured states. All these phenomena have been shown experimentally in ADA2h by mutations that increase helix stability. However, the effects promoted for such an approach in proteins with residual structure and/or intermediates in the denatured ensemble could be different. This has been shown by experiments performed on CheY in which the cooperativity of the folding process was greatly affected.

Stabilization of proteins by rational design of α-helix stability using helix/coil transition theory

Backgound. Increasing protein stability is a major goal of protein engineering because of its potential industrial and pharmacological applications. Several different rule-of-thumb strategies have been employed for such a purpose, but a general rational method is still lacking. Recently, there has been significant progress in our understanding of the interactions responsible for helix stability in monomeric peptides and this information has been included in algorithms based on the helix/coil transition theory. We set out to investigate whether it is possible to use these algorithms to rationally increase protein stability. Results. Using a helix/coil transition algorithm, AGADIRms, we have designed mutations affecting solvent-exposed residues which, as predicted, significantly increase the helical stability in aqueous solution of peptides corresponding to the two alpha-helices of the activation domain of procarboxipeptidase A. Introduction of the same mutations in the protein results in proteins more resistant to urea or temperature denaturation, and there is a qualitative agreement between the expected and observed increases in stability. Conclusion. In this work we demonstrate that by using a helix/coil algorithm to design helix-stabilizing mutations on the solvent-exposed face of helices, it is possible to rationally increase the stability of proteins.

Unspecific hydrophobic stabilization of folding transition states.

Here we present a method for determining the inference of non-native conformations in the folding of a small domain, α-spectrin Src homology 3 domain. This method relies on the preservation of all native interactions after Tyr/Phe exchanges in solvent-exposed, contact-free positions. Minor changes in solvent exposure and free energy of the denatured ensemble are in agreement with the reverse hydrophobic effect, as the Tyr/Phe mutations slightly change the polypeptide hydrophilic/hydrophobic balance. Interestingly, more important Gibbs energy variations are observed in the transition state ensemble (TSE). Considering the small changes induced by the H/OH replacements, the observed energy variations in the TSE are rather notable, but of a magnitude that would remain undetected under regular mutations that alter the folded structure free energy. Hydrophobic residues outside of the folding nucleus contribute to the stability of the TSE in an unspecific nonlinear manner, producing a significant acceleration of both unfolding and refolding rates, with little effect on stability. These results suggest that sectors of the protein transiently reside in non-native areas of the landscape during folding, with implications in the reading of φ values from protein engineering experiments. Contrary to previous proposals, the principle that emerges is that non-native contacts, or conformations, could be beneficial in evolution and design of some fast folding proteins.

A thermodynamic analysis of a family of small globular proteins: SH3 domains

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied alpha-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, delta Cp,U, the proportionality factor, m, and the Gibbs energy, delta Gw298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on delta Cp,U and the m-factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The delta Gw298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.

The Calcium-binding C-terminal Domain of Escherichia coli α-Hemolysin Is a Major Determinant in the Surface-active Properties of the Protein

α-Hemolysin (HlyA) from Escherichia coli is a protein toxin (1024 amino acids) that targets eukaryotic cell membranes, causing loss of the permeability barrier. HlyA consists of two main regions, an N-terminal domain rich in amphipathic helices, and a C-terminal Ca2+-binding domain containing a Gly- and Asp-rich nonapeptide repeated in tandem 11–17 times. The latter is called the RTX domain and gives its name to the RTX protein family. It had been commonly assumed that membrane interaction occurred mainly if not exclusively through the amphipathic helix domain. However, we have cloned and expressed the C-terminal region of HlyA, containing the RTX domain plus a few stabilizing sequences, and found that it is a potent surface-active molecule. The isolated domain binds Ca2+ with about the same affinity (apparent K0.5 ≈150 μm) as the parent protein HlyA, and Ca2+ binding induces in turn a more compact folding with an increased proportion of β-sheet structure. Both with and without Ca2+ the C-terminal region of HlyA can interact with lipid monolayers spread at an air-water interface. However, the C-terminal domain by itself is devoid of membrane lytic properties. The present results can be interpreted in the light of our previous studies that involved in receptor binding a peptide in the C-terminal region of HlyA. We had also shown experimentally the distinction between reversible membrane adsorption and irreversible lytic insertion of the toxin. In this context, the present data allow us to propose that both major domains of HlyA are directly involved in membrane-toxin interaction, the nonapeptide repeat, calcium-binding RTX domain being responsible for the early stages of HlyA docking to the target membrane.

Reading protein sequences backwards

BACKGROUNDnReading a protein sequence backwards provides a new polypeptide that does not align with its parent sequence. The foldability of this new sequence is questionable. On one hand, structure prediction at low resolution using lattice simulations for such a protein provided a model close to the native parent fold or to a topological mirror image of it. On the other hand, there is no experimental evidence yet to tell whether such a retro protein folds (and to which structure) or not.nnnRESULTSnIn this work, we have analysed the possibility of a retro protein folding in two different ways. First, we modelled the retro sequence of the alpha-spectrin SH3 domain through distance geometry and molecular dynamics. This contradicted the plausibility of a mirror image of the native domain, whereas basic considerations opposed the likelihood of the native fold. Second, we obtained experimental evidence that the retro sequences of the SH3 domain, as well as the B domain of Staphylococcal protein A and the B1 domain of Streptococcal protein G, are unfolded proteins, even though some propensities for the formation of secondary structures might remain.nnnCONCLUSIONSnRetro proteins are no more similar to their parent sequences than any random sequence despite their common hydrophobic/hydrophilic pattern, global amino acid composition and possible tertiary contacts. Although simple folding models contribute to our global understanding of protein folding, they cannot yet be used to predict the structure of new proteins.

Hydrogen-exchange stability analysis of Bergerac-Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Amide hydrogen/deuterium exchange rates have been determined for two mutants of α-spectrin Src homology 3 domain (WT), containing an elongated stable (SHH) and unstable (SHA) distal loop. SHA, similarly to WT, follows a two-state transition, whereas SHH apparently folds via a three-state mechanism. Native-state amide hydrogen exchange is effective in ascribing energetic readjustments observed in kinetic experiments to species stabilized within the denatured base and distinguishing those from high-energy barrier crossings. Comparison of ΔGex and mex parameters for amide protons of these mutants demonstrates the existence of an intermediate and allows the identification of protons protected in this state. The consolidation of a form containing a prefolded long β-hairpin induces the switch to a three-state mechanism in an otherwise two-state folder. It can be inferred that the unbalanced high stability of individual elements of secondary structure in a polypeptide could ultimately complicate its folding mechanism.

The DSC data analysis for small, single-domain proteins. Application to the SH3 domain

Abstract In some cases, small globular domains can maintain their native three-dimensional structure when separated from the rest of the protein. But due to the small size of such domains and low heat effect accompanying their cooperative structure unfolding, this transition occurs in a very broad temperature range. It is shown that for such broad processes an accurate evaluation of the . parameters from the DSC data can be done only by a global curve-fitting analysis applied to the multiple DSC curves obtained under various solvent conditions.