| Abstract
| - The N-terminal SH3 domain of the Drosophila adapter protein Drk (drkN SH3 domain) ismarginally stable (ΔGU = 1 kcal/mol) and exists in equilibrium between folded and highly populatedunfolded states. The single substitution T22G, however, completely stabilizes the protein (ΔGU = 4.0kcal/mol). To probe the causes of instability of the wild-type (WT) protein and the dramatic stabilizationof the mutant, we determined and compared nuclear magnetic resonance structures of the folded WT andmutant drkN SH3 domains. Residual dipolar coupling (RDC) and carbonyl chemical-shift anisotropy (C‘-CSA) restraints measured for the WT and T22G domains were used for calculating the structures. Thestructures for the WT and mutant are highly similar. Thr22 of the WT and Gly22 of the mutant are at thei + 2 position of the diverging, type-II β-turn. Interestingly, not only Gly22 but also Thr22 successfullyadopt an αL conformation, required at this position of the turn, despite the fact that positive φ values areenergetically unfavorable and normally disallowed for threonine residues. Forcing the Thr22 residue intothis unnatural conformation increases the free energy of the folded state of the WT domain relative to itsT22G mutant. Evidence for residual helix formation in the diverging turn region has been previouslyreported for the unfolded state of the WT drkN SH3 domain, and this, in addition to other residual structure,has been proposed to play a role in decreasing the free energy of the unfolded state of the protein. Togetherthese data provide evidence that both increasing the free energy of the folded state and decreasing thefree energy of the unfolded state of the protein contribute to instability of the WT drkN SH3 domain.
|
