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Structural features of cytochrome c' folding intermediates revealed by fluorescence energy-transfer kinetics.

Journal article
Authors Jennifer C Lee
Cecilia Engman
F Akif Tezcan
Harry B Gray
Jay R Winkler
Published in Proceedings of the National Academy of Sciences of the United States of America
Volume 99
Issue 23
Pages 14778-82
ISSN 0027-8424
Publication year 2002
Published at Department of Chemistry
Pages 14778-82
Language en
Links dx.doi.org/10.1073/pnas.192574099
Keywords Amino Acids, analysis, Bacterial Proteins, chemistry, metabolism, Crystallography, X-Ray, methods, Cytochromes c1, chemistry, metabolism, Energy Transfer, Guanidine, Kinetics, Protein Conformation, Protein Folding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, chemistry, metabolism, Spectrometry, Fluorescence
Subject categories Chemical Sciences, Biochemistry

Abstract

We employed fluorescence energy-transfer probes to investigate the polypeptide dynamics accompanying cytochrome c' folding. Analysis of fluorescence energy-transfer kinetics from wild-type Trp-72 or Trp-32 in a crystallographically characterized (1.78 A) Q1A/F32W/W72F mutant shows that there is structural heterogeneity in denatured cytochrome c'. Even at guanidine hydrochloride concentrations well beyond the unfolding transition, a substantial fraction of the polypeptides ( approximately 50%) adopts compact conformations (tryptophan-to-heme distance, approximately 25 A) in both pseudo-wild-type (Q1A) and mutant proteins. A burst phase (< or =5 ms) is revealed when stopped flow-triggered refolding is probed by tryptophan intensity: measurements on the Q1A protein show that approximately 75% of the Trp-72 fluorescence (83% for Trp-32) is quenched within the mixing deadtime, suggesting that most of the polypeptides have collapsed.

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