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

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

Research output: Contribution to journalArticle

44 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

Keywords

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

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A'. Together they form a unique fingerprint.

  • Cite this