The native state conformational ensemble of the SH3 domain from alpha-spectrin

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential s canning 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) he lix, and part of the long RT loop, whereas the loops connecting secondary s tructure elements show no measurable protection. Apparent stability constan ts 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 re gion, 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 l oop). All residues in the domain have apparent Gibbs energies lower than th e global unfolding Gibbs energy measured by differential scanning calorimet ry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reacti ons. A structure-based thermodynamic analysis has allowed us to predict cor rectly the thermodynamics of the global unfolding of the domain and to defi ne the ensemble of conformational states that quantitatively accounts for t he observed pattern of hydrogen exchange protection. These results demonstr ate that under native conditions the SH3 domain needs to be considered as a n ensemble of conformations and that the hydrogen exchange data obtained un der those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independ ent local folding/unfolding reactions indicates that under native condition s the scale of cooperative interactions is regional rather than global.