title: Structural model for an alkaline form of ferricytochrome C.
abstract: An (15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH approximately equal to 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 +/- 0.2 A. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70-80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.
authors.affiliation: CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Michael
authors.initials: M
authors.lastname: Assfalg
authors.forename: Ivano
authors.initials: I
authors.lastname: Bertini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Dolfi
authors.forename: Paola
authors.initials: P
authors.lastname: Turano
authors.forename: A Grant
authors.initials: AG
authors.lastname: Mauk
authors.forename: Federico I
authors.initials: FI
authors.lastname: Rosell
authors.forename: Harry B
authors.initials: HB
authors.lastname: Gray
mesh_terms: D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012996:Solutions
chemical_list: D012996:Solutions; D045304:Cytochromes c