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Site-directed mutagenesis has been used to produce local stability changes at two regions of the binding site surface of the alpha-spectrin SH3 domain (Spc-SH3) differing in their intrinsic stability. Mutations were made at residue 56, located at the solvent-exposed side of the short 3(10) helix, and at residue 21 in the tip of the flexible RT-loop. NMR chemical-shift analysis and X-ray crystallography indicated negligible changes produced by the mutations in the native structure limited to subtle rearrangements near the mutated residue and at flexible loops. Additionally, mutations do not alter importantly the SH3 binding site structure, although produce significant changes in its affinity for a proline-rich decapeptide. The changes in global stability measured by differential scanning calorimetry are consistent the local energy changes predicted by theoretical models, with the most significant effects observed for the Ala-Gly mutations. Propagation of the local stability changes throughout the domain structure has been studied at a per-residue level of resolution by NMR-detected amide hydrogen-deuterium exchange (HX). Stability propagation is remarkably efficient in this small domain, apparently due to its intrinsically low stability. Nevertheless, the HX-core of the domain is not fully cooperative, indicating the existence of co-operative subunits within the core, which is markedly polarized. An equilibrium phi-analysis of the changes in the apparent Gibbs energies of HX per residue produced by the mutations has allowed us to characterize structurally the conformational states leading to HX. Some of these states resemble notably the folding transition state of the Spc-SH3 domain, suggesting a great potential of this approach to explore the folding energy landscape of proteins. An energy perturbation propagates more effectively from a flexible region to the core than in the opposite direction, because the former affects a broader region of the energy landscape than the latter. This might be of importance in understanding the special thermodynamic signature of the SH3-peptide interaction and the relevance of the dual character of SH3 binding sites., (2007 Wiley-Liss, Inc.)

For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of alpha-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above approximately 0.5 mM the population of the intermediate state becomes significant at temperatures between 45 degrees C and 80 degrees C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.

We have studied the effects produced by site-directed mutagenesis upon energetic and structural cooperativity in the Src homology region 3 domain of alpha-spectrin. The mutation of Asn47 to Gly or Ala in the distal loop brings about significant changes to the global stability of the domain in spite of not affecting its structure to any great extent. The binding affinity for a proline-rich peptide is also largely diminished in both mutant domains. We have compared the apparent Gibbs energies of the amide hydrogen-deuterium exchange (HX) between the wild-type and the Gly47 mutant. The observed changes in the Gibbs energy of HX indicate a remarkable energetic cooperativity in this small domain. Regions of the domain's core have a high cooperativity with the position of the mutation, indicating that their HX occurs mainly in states in which the distal loop is unstructured. More flexible regions, which undergo HX mainly by local motions, show a lower but still considerable cooperativity with the distal loop. We conclude that there is an important correlation between regional stability and cooperativity in this small domain.

Using nuclear magnetic resonance we have measured the hydrogen exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin as a function of pH*. At very acidic pH* values the exchange of most residues appears to occur via global unfolding, although several residues show abnormally large Gibbs energies of exchange, suggesting the presence of some residual structure in the unfolded state. At higher pH* HX occurs mainly via local or partial unfoldings. We have been able to characterize the coupling between the electrostatic interactions in this domain and the conformational fluctuations occurring under native conditions by analyzing the dependence upon pH* of the Gibbs energy of exchange. The SH3 domain seems to be composed of a central core, which requires large structural disruptions to become exposed to the solvent, surrounded by smaller subdomains, which fluctuate independently.

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.