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

Details

Serval ID
serval:BIB_D834A3DCC4A6
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A
Journal
Proceedings of the National Academy of Sciences of the United States of America
Author(s)
Welker  E., Maki  K., Shastry  M. C., Juminaga  D., Bhat  R., Scheraga  H. A., Roder  H.
ISSN
0027-8424 (Print)
Publication state
Published
Issued date
12/2004
Volume
101
Number
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
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 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.
Keywords
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
Yes
Create date
24/01/2008 14:40
Last modification date
20/08/2019 15:57
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