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Garcia P , Bruix M , Rico M , Ciofi-Baffoni S , Banci L , Ramachandra Shastry MC , Roder H , De Lumley Woodyear T , Johnson CM , Fersht AR , Barker PD
Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562
Journal of Molecular Biology. 2005 ;346(1) :331-344
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Abstract
Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-?-helical bundle. The holo protein apparently refolds with a half-life of 4 ?s in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 ?s that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins. © 2005 Published by Elsevier Ltd.
Notes
00222836 (ISSN) Cited By: 4; Export Date: 25 May 2006; Source: Scopus CODEN: JMOBA; DOI: 10.1016/j.jmb.2004.11.044 Language of Original Document: English Correspondence Address: Barker, P.D.; Department of Chemistry; University of Cambridge; Lensfield Road Cambridge, CB2 1EW, United Kingdom; email: pdb30@cam.ac.uk Chemicals/CAS: cytochrome b562, 9064-79-3; guanidine hydrochloride, 50-01-1; heme, 14875-96-8, Apoproteins; Cytochrome b Group; cytochrome b562, E coli, 9064-79-3; Escherichia coli Proteins; Guanidine, 113-00-8; Heme, 14875-96-8; Tryptophan, 73-22-3; Urea, 57-13-6 References: Wittung-Stafshede, P., Role of cofactors in protein folding (2002) Accts Chem. 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