A Stable α-Helix-rich Intermediate Is Formed by a Single Mutation of the β-Sheet Protein, src SH3, at pH 3

Recently, we have found a transient intermediate on the folding pathway of src SH3. Intending to investigate the structure of the transient intermediate, we tested a mutant of src SH3, named A45G, using circular dichroism, fluorescence and X-ray solution scattering, and incidentally found that it forms a stable α-helix-rich intermediate (Ieq) (different from the native β-sheet-based secondary structure) at pH 3.0, but contains only β-sheets at pH 6.0, whereas wild-type SH3 forms only β-sheets at both pH 3.0 and pH 6.0. The intermediate Ieq shows a circular dichroism measured at θ222 = −10,300 deg.cm2 dmol−1, indicating a 31% α-helix proportion, as estimated by the CONTIN program. X-ray scattering gave the radius of gyration for Ieq as 19.1 Å at pH 3.0 and 15.4 Å at pH 6.0, and Kratky plots showed a clear peak at pH 3.0, 4.0 and 6.0, indicating that Ieq too is compact. In these parameters, Ieq closely resembles the kinetically-obtained intermediate Ikin which we found on the folding pathway of wild-type SH3 at pH 3.0 (radius of gyration 18.7 Å and θ222 = −8700 deg.cm2dmol−1), indicating a 26% α-helix proportion in our previous paper. Refolding experiments with A45G were done at pH 6.0 by stopped-flow apparatus monitored by circular dichroism, and compared to kinetic experiments with wild-type SH3 at pH 6.0. The result showed an α-helix-rich intermediate at the same dichroism amplitude, but nine times slower in formation-rate. A pH-jump experiment from pH 3.0 to pH 5.9 on A45G was also performed. This showed no bursts, and the rate of conformation-change was almost as fast as the refolding rate of A45G at pH 6.0. These kinetic experiment data would be consistent with Ieq being nearly identical to the Ikin, which appeared on the folding pathways of both wild-type SH3 and A45G at pH 3.