The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved.

The protein engineering analysis of the alpha-spectrin SH3 domain at three different stability conditions (pH 7.0, 3.5 and 2.5) reveals a folding transition state structured around the distal loop beta-hairpin and the 310-helix. This region is impervious to overall changes in protein stability, suggesting a transition state ensemble with little conformational variability. Comparison with the Src SH3 domain (36% sequence homology) indicates that the transition state in this protein family may be conserved. Discrepancies at some positions can be rationalized in terms of the different interactions made by the different side chains in both domains. Brønsted plot analysis confirms the straight phi(doubledagger-U) results and shows two folding subdomains for this small protein. These results, together with previous data on circular permutants of the alpha-spectrin SH3 domain, indicate that polypeptide topology and chain connectivity play a major role in the folding reaction of this protein family.