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The denatured state of N-PGK is compact and predominantly disordered

Cliff, M.J., Craven, C.J., Marston, J.P., Hounslow, A.M., Clarke, A.R. and Waltho, J.P. (2009) The denatured state of N-PGK is compact and predominantly disordered. Journal of Molecular Biology, 385 (1). pp. 266-277. ISSN 0022-2836

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Abstract

The Organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I-b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I-k) and denatured (D)]. The transition to I-b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I-b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I-b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics Of I-b indicate that it forms from the fully denatured state within 100 mu s, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I-b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology. (C) 2008 Elsevier Ltd. All rights reserved.

Item Type: Article
Copyright, Publisher and Additional Information: © 2009 Elsevier. This is an author produced version of a paper subsequently published in the Journal of Molecular Biology. Uploaded in accordance with the publisher's self-archiving policy.
Keywords: paramagnetic relaxation enhancement; NMR; folding intermediates; protein folding; phosphoglycerate kinase
Institution: The University of Sheffield
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > School of Biological Sciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield)
Depositing User: Miss Anthea Tucker
Date Deposited: 04 Mar 2009 10:03
Last Modified: 08 Feb 2013 16:57
Published Version: http://dx.doi.org/10.1016/j.jmb.2008.10.004
Status: Published
Publisher: Elsevier
Refereed: Yes
Identification Number: 10.1016/j.jmb.2008.10.004
URI: http://eprints.whiterose.ac.uk/id/eprint/7921

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