Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A

E. Welker, Kosuke Maki, M. C Ramachandra Shastry, Darmawi Juminaga, Rajiv Bhat, Harold A. Scheraga, Heinrich Roder

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

The earliest folding events in single-tryptophan mutants of RNase A were investigated by fluorescence measurements by using a combination of stopped-flow and continuous-flow mixing experiments covering the time range from 70 μs to 10 s. An ultrarapid double-jump mixing protocol was used to study refolding from an unfolded ensemble containing only native proline isomers. The continuous-flow measurements revealed a series of kinetic events on the submillisecond time scale that account for the burst-phase signal observed in previous stopped-flow experiments. An initial increase in fluorescence within the 70-μs dead time of the continuous-flow experiment is consistent with a relatively nonspecific collapse of the polypeptide chain whereas a subsequent decrease in fluorescence with a time constant of ≈80 μs is indicative of a more specific structural event. These rapid conformational changes are not observed if RNase A is allowed to equilibrate under denaturing conditions, resulting in formation of nonnative proline isomers. Thus, contrary to previous expectations, the isomerization state of proline peptide bonds can have a major impact on the structural events during early stages of folding.

Original languageEnglish
Pages (from-to)17681-17686
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number51
DOIs
Publication statusPublished - Dec 21 2004

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Pancreatic Ribonuclease
Proline
Fluorescence
Peptides
Tryptophan

Keywords

  • Burst phase
  • Continuous-flow
  • Proline isomers
  • Stopped-flow

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A. / Welker, E.; Maki, Kosuke; Shastry, M. C Ramachandra; Juminaga, Darmawi; Bhat, Rajiv; Scheraga, Harold A.; Roder, Heinrich.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 51, 21.12.2004, p. 17681-17686.

Research output: Contribution to journalArticle

Welker, E. ; Maki, Kosuke ; Shastry, M. C Ramachandra ; Juminaga, Darmawi ; Bhat, Rajiv ; Scheraga, Harold A. ; Roder, Heinrich. / Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A. In: Proceedings of the National Academy of Sciences of the United States of America. 2004 ; Vol. 101, No. 51. pp. 17681-17686.
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AU - Bhat, Rajiv

AU - Scheraga, Harold A.

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