Specifically collapsed intermediate in the early stage of the folding of ribonuclease A.
Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 micros after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at approximately 22 ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations.
Kimura
Tetsunari
T
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo, Kyoto 615-8510, Japan.
Akiyama
Shuji
S
Uzawa
Takanori
T
Ishimori
Koichiro
K
Morishima
Isao
I
Fujisawa
Tetsuro
T
Takahashi
Satoshi
S
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002942:Circular Dichroism; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014961:X-Ray Diffraction
D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D012259:Ribonuclease, Pancreatic; D019791:Guanidine