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Many faces of the unfolded state: conformational heterogeneity in denatured yeast cytochrome C.

Authors
  • Pletneva, Ekaterina V
  • Gray, Harry B
  • Winkler, Jay R
Type
Published Article
Journal
Journal of molecular biology
Publication Date
Jan 28, 2005
Volume
345
Issue
4
Pages
855–867
Identifiers
PMID: 15588831
Source
Medline
License
Unknown

Abstract

We have measured fluorescence energy-transfer (FET) kinetics from a dansyl fluorophore (Dns) introduced by derivatization of a Cys side-chain to the Fe(III) heme covalently attached to unfolded yeast iso-1 cytochrome c (cyt). To gain a global picture of the unfolded state, we examined variants with the fluorophore attached on three different helices (K4C, E66C, K99C) and in three different loops (H39C, D50C, L85C). Analysis of the FET kinetics data gave distributions of distances between the fluorescent donor and acceptor; these distributions demonstrate that the guanidine hydrochloride (GuHCl)-denatured polypeptide ensemble is not a simple random coil. Although misligation imposes some constraints, it is not the only source of structural complexity in the unfolded protein. Our FET kinetics data reveal a high degree of heterogeneity in the unfolded ensemble of cytochrome c. We detect relatively large populations of compact structures in unfolded Dns(C50)cyt, Dns(C39)cyt, and Dns(C66)cyt. These structures likely play a role in forming a hydrophobic core during the folding process.

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