A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding.

TitleA partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding.
Publication TypeJournal Article
Year of Publication2001
AuthorsReader, J. S., N. A. J. van Nuland, G. S. Thompson, S. J. Ferguson, C. M. Dobson, and S. E. Radford
JournalProtein Sci
Volume10
Issue6
Pagination1216-24
Date Published2001 Jun
ISSN0961-8368
KeywordsAnilino Naphthalenesulfonates, Azurin, Circular Dichroism, Cyclophilin A, Fluorescent Dyes, Hydrogen-Ion Concentration, Kinetics, Magnetic Resonance Spectroscopy, Models, Molecular, Proline, Protein Folding, Protein Structure, Secondary, Sodium, Stereoisomerism, Time Factors, Ultracentrifugation, Urea
Abstract

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.

DOI10.1110/ps.52801
Alternate JournalProtein Sci.
PubMed ID11369860
PubMed Central IDPMC2374025