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

Abstract

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 ( I eq) (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 I eq shows a circular dichroism measured at θ 222 = −10,300 deg.cm 2 dmol −1, indicating a 31% α-helix proportion, as estimated by the CONTIN program. X-ray scattering gave the radius of gyration for I eq 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 I eq too is compact. In these parameters, I eq closely resembles the kinetically-obtained intermediate I kin which we found on the folding pathway of wild-type SH3 at pH 3.0 (radius of gyration 18.7 Å and θ 222 = −8700 deg.cm 2dmol −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 I eq being nearly identical to the I kin, which appeared on the folding pathways of both wild-type SH3 and A45G at pH 3.