title: Influence of point mutations on the flexibility of cytochrome b5: molecular dynamics simulations of holoproteins.
abstract: Two membrane-bound isoforms of cytochrome b5 have been identified in mammals, one associated with the outer mitochondrial membrane (OM b5) and the other with the endoplasmic reticulum (microsomal, or Mc b5). The soluble heme binding domains of OM and Mc b5 have highly similar three-dimensional structures but differ significantly in physical properties, with OM b5 exhibiting higher stability due to stronger heme association. In this study, we present results of 8.5-ns length molecular dynamics simulations for rat Mc b5, bovine Mc b5, and rat OM b5, as well as for two rat OM b5 mutants that were anticipated to exhibit properties intermediate between those of rat OM b5 and the two Mc proteins: the A18S/I32L/L47R triple mutant (OM3M) and the A18S/I25L/I32L/L47R/L71S quintuple mutant (OM5M). Analysis of the structure, fluctuations, and interactions showed that the five b5 variants used in this study differed in organization of their molecular surfaces and heme binding cores in a way that could be used to explain certain experimentally observed physical differences. Overall, our simulations provided qualitative microscopic explanations of many of the differences in physical properties between OM and Mc b5 and two mutants in terms of localized changes in structure and flexibility. They also reveal that opening of a surface cleft between hydrophobic cores 1 and 2 in bovine Mc b5, observed in two previously reported simulations (E. M. Storch and V. Daggett, Biochemistry, 1995, Vol. 34, pp. 9682-9693; A. Altuve, Biochemistry, 2001, Vol. 40, pp. 9469-9483), probably resulted from removal of crystal contacts and likely does not occur on the nanosecond time scale. Finally, the MD simulations of OM5M b5 verify that stability and dynamic properties of cytochrome b5 are remarkably resistant to mutations that dramatically alter the stability and structure of the apoprotein.
authors.affiliation: Department of Molecular Biosciences, University of Kansas, 1251 Wescoe Hall Drive, Room 2010, Malott Hall, Lawrence, KS 66045-7582, USA.
authors.forename: Qinyi
authors.initials: Q
authors.lastname: Cheng
authors.forename: David R
authors.initials: DR
authors.lastname: Benson
authors.forename: Mario
authors.initials: M
authors.lastname: Rivera
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Kuczera
mesh_terms: D000818:Animals; D002417:Cattle; D003198:Computer Simulation; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000737:chemistry; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D015394:Molecular Structure; D018583:Pliability; D017354:Point Mutation; D011487:Protein Conformation; D020033:Protein Isoforms; D051381:Rats
chemical_list: D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D020033:Protein Isoforms; D015786:Cytochromes b5