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Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain. A study using electrospray ionization mass spectrometry and fluorescence binding studies

Ashcroft, A.E., Brinker, A., Coyle, J.E., Weber, F., Kaiser, M., Moroder, L., Parsons, M.R., Jager, J., Hartl, U.F., Hayer-Hartl, M. and Radford, S.E. (2002) Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain. A study using electrospray ionization mass spectrometry and fluorescence binding studies. Journal of Biological Chemistry, 277 (36). 33115-33126. ISSN 1083-351X

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Abstract

Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.

Item Type: Article
Copyright, Publisher and Additional Information: Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Academic Units: The University of Leeds > University of Leeds Research Centres and Institutes > Astbury Centre for Structural Molecular Biology (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute of Molecular and Cellular Biology (Leeds)
Depositing User: Repository Officer
Date Deposited: 13 Mar 2006
Last Modified: 08 Feb 2013 17:02
Published Version: http://www.jbc.org./cgi/content/full/277/36/33115
Status: Published
Refereed: Yes
Identification Number: 10.1074/jbc.M203398200
URI: http://eprints.whiterose.ac.uk/id/eprint/606

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