Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A
Détails
ID Serval
serval:BIB_D834A3DCC4A6
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A
Périodique
Proceedings of the National Academy of Sciences of the United States of America
ISSN
0027-8424 (Print)
Statut éditorial
Publié
Date de publication
12/2004
Volume
101
Numéro
51
Pages
17681-6
Notes
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S. --- Old month value: Dec 21
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S. --- Old month value: Dec 21
Résumé
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 micros 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-micros 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 approximately 80 micros 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.
Mots-clé
Kinetics
Models, Molecular
Mutation/genetics
Protein Conformation
Protein Denaturation
*Protein Folding
Ribonuclease, Pancreatic/*chemistry/genetics/*metabolism
Spectrometry, Fluorescence
Time Factors
Tyrosine/genetics/metabolism
Pubmed
Web of science
Open Access
Oui
Création de la notice
24/01/2008 15:40
Dernière modification de la notice
20/08/2019 16:57