A stable alpha-helix-rich intermediate is formed by a single mutation of the beta-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 alpha-helix-rich intermediate (I(eq)) (different from the native beta-sheet-based secondary structure) at pH 3.0, but contains only beta-sheets at pH 6.0, whereas wild-type SH3 forms only beta-sheets at both pH 3.0 and pH 6.0. The intermediate I(eq) shows a circular dichroism measured at theta(222)=-10,300 deg.cm(2) dmol(-1), indicating a 31% alpha-helix proportion, as estimated by the CONTIN program. X-ray scattering gave the radius of gyration for I(eq) as 19.1 A at pH 3.0 and 15.4 A 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 A and theta(222)=-8700 deg.cm(2)dmol(-1)), indicating a 26% alpha-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 alpha-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.
Department of Physics, Kansai Medical University 18-89 Uyama-Higashi, Hirakata 573-1136, Japan.
Jinsong
J
Li
Yoshitaka
Y
Matsumura
Masaji
M
Shinjo
Masaki
M
Kojima
Hiroshi
H
Kihara
D000596:Amino Acids; D002942:Circular Dichroism; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012542:Scattering, Radiation; D012996:Solutions; D014965:X-Rays; D018909:src Homology Domains
D000596:Amino Acids; D050505:Mutant Proteins; D012996:Solutions; D016392:Proto-Oncogene Proteins pp60(c-src); D019791:Guanidine