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

Tetsunari Kimura, Shuji Akiyama, Takanori Uzawa, Koichiro Ishimori, Isao Morishima, Tetsuro Fujisawa, Satoshi Takahashi

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40 Citations (Scopus)

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 μs 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 ∼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.

Original languageEnglish
Pages (from-to)349-362
Number of pages14
JournalJournal of Molecular Biology
Volume350
Issue number2
DOIs
Publication statusPublished - 2005 Jul 8
Externally publishedYes

Keywords

  • ANS fluorescence
  • Burst phase intermediate
  • Collapse
  • Protein folding
  • Ribonuclease A

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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