title: Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562.
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-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros 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 micros 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.
authors.affiliation: Instituto de Química Física Rocasolano, CSIC, C/Serrano, 119, 28006 Madrid, Spain.
authors.forename: Pascal
authors.initials: P
authors.lastname: Garcia
authors.forename: Marta
authors.initials: M
authors.lastname: Bruix
authors.forename: Manuel
authors.initials: M
authors.lastname: Rico
authors.forename: Simone
authors.initials: S
authors.lastname: Ciofi-Baffoni
authors.forename: Lucia
authors.initials: L
authors.lastname: Banci
authors.forename: M C
authors.initials: MC
authors.lastname: Ramachandra Shastry
authors.forename: Heinrich
authors.initials: H
authors.lastname: Roder
authors.forename: Thierry
authors.initials: T
authors.lastname: de Lumley Woodyear
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Johnson
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
authors.forename: Paul D
authors.initials: PD
authors.lastname: Barker
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
chemical_list: D001059:Apoproteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D006418:Heme; D014364:Tryptophan; D014508:Urea; C017850:cytochrome b562, E coli; D019791:Guanidine