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The folding pathway...
The folding pathway of an FF domain : Characterization of an on-pathway intermediate state under folding conditions by N-15, C-13(alpha) and C-13-methyl relaxation dispersion and H-1/(2) H-exchange NMR Spectroscopy
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- Korzhnev, Dmitry M. (författare)
- University of Toronto, Ontario, Canada
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- Religa, Tomasz L. (författare)
- MRC, University of Cambridge, UK
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- Lundström, Patrik, 1971- (författare)
- University of Toronto, ON, Canada
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- Fersht, Alan R. (författare)
- MRC, University of Cambridge, UK
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- Kay, Lewis E. (författare)
- University of Toronto, Ontario, Canada
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(creator_code:org_t)
- Elsevier, 2007
- 2007
- Engelska.
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 372:2, s. 497-512
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
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- The FF domain from the human protein HYPA/FBP11 folds via a lowenergy on-pathway intermediate (. Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by N-15, C-13(alpha) and methyl C-13 Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by 15N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U-I-F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone N-15, C-13(alpha) and side-chain methyl 13C chemical shifts, with further information from protection factors for the backbone amide groups from H-1/(2) H-exchange. Notably, helices H1-H3 are at least partially formed in 1, while helix H4 is largely disordered. Chemical shift differences for the methyl 13 C nuclei suggest a paucity of stable, native-like hydrophobic interactions in 1. These data are consistent with (D-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and (1) data can elucidate whole experimental folding pathways.
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