title: NMR investigation of ferricytochrome c unfolding: detection of an equilibrium unfolding intermediate and residual structure in the denatured state.
abstract: Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used (1)H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small DeltaG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger DeltaG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.
authors.affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Russell
authors.forename: R
authors.initials: R
authors.lastname: Melenkivitz
authors.forename: K L
authors.initials: KL
authors.lastname: Bren
mesh_terms: D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006736:Horses; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D003574:Cytochrome c Group; D008024:Ligands; D006418:Heme