Dramatic stabilization of an SH3 domain by a single substitution: Roles ofthe folded and unfolded states

The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists i n equilibrium between folded and unfolded states under nondenaturing buffer conditions. In order to examine the origins of this instability, we have m ade mutations in the domain and characterized the thermodynamics and kineti cs of folding. Results of substitutions of negatively charged residues to n eutral amino acid residues suggest that the large electrostatic potential o f the domain does not play a dominant role in the instability of the domain . Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwi se highly conserved glycine residue in the diverging beta -turn connecting the beta3 and beta4 strands. Mutation of T22 to glycine results in signific ant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further chara cterize the basis for the stabilization of the T22 mutant relative to wild- type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native p-turn structure at this position. Correlation of fo lding rates with AGA-DIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, sug gests that the free energy of the unfolded state also plays a role in stabi lity. This result highlights the importance of both folded and unfolded sta tes for understanding protein stability. (C) 2001 Academic Press.