Specifically collapsed intermediate in the early stage of the folding of ribonuclease A.


Abstract

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. Study holds ProTherm entries: 19216, 19217, 19218, 19219 Extra Details: protein folding; burst phase intermediate; collapse; ribonuclease A; ANS fluorescence

Submission Details

ID: HDPQQcuP3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Kimura T;Akiyama S;Uzawa T;Ishimori K;Morishima I;Fujisawa T;Takahashi S,J. Mol. Biol. (2005) Specifically collapsed intermediate in the early stage of the folding of ribonuclease A. PMID:15935376
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