The Denatured State of N-PGK Is Compact and Predominantly Disordered

JOURNAL OF MOLECULAR BIOLOGY

卷:385

Article

Cliff, Matthew J.¹  Craven, C. Jeremy¹  Marston, James P.¹  Hounslow, Andrea M.¹  Clarke, Anthony R.²  Waltho, Jonathan P.^1,3,4 

[1] Univ Sheffield, Dept Mol Biol & Biotechnol, Western Bank, Sheffield S10 2TN, S Yorkshire, England

[2] Univ Bristol, Dept Biochem, Bristol BS8 1TD, Avon, England

[3] Univ Manchester, Fac Life Sci, Manchester M1 7DN, Lancs, England

[4] Univ Manchester, Manchester Interdisciplinary Bioctr, Manchester M1 7DN, Lancs, England

关键词: paramagnetic relaxation enhancement;    NMR;    folding intermediates;    protein folding;    phosphoglycerate kinase;   

DOI  :  10.1016/j.jmb.2008.10.004

来源: Elsevier

【 摘 要 】

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.

【 授权许可】

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