Probing the role of the conserved beta-II turn Pro-76/Gly-77 of mitochondrial cytochrome c.
The loop segment comprising residues 70-84 in mitochondrial cytochrome c serves to direct the polypeptide backbone to permit the functionally required heme Fe - S (Met-80) co-ordination. The primary sequence here is highly conserved, which is something rarely observed in surface loop segments and suggests that its purpose is more complex than its obvious structural role. The beta-II turn formed by Pro-76 and Gly-77 is postulated to be key to the redirection of the peptide backbone required to execute the loop. We assessed the importance of Pro-76 and Gly-77 by mutating 1 or both of these residues to alanine such that the range of allowable dihedral angles was altered, and this resulted in significant changes in physicochemical properties and biological activities. We observed structural perturbations using circular dichroism spectroscopy and thermal denaturation studies. Based on these changes, we propose that the Pro-76/Gly-77 beta-II turn precisely orients the 70s loop, not only to maintain the backbone orientation required for the formation of the axial heme ligand, but also to provide a complementary surface to physiological partners.
Black
Karen M
KM
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4H7, Canada.
Wallace
Carmichael J A
CJ
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017124:Conserved Sequence; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D006418:Heme / Q000737:chemistry; D066298:In Vitro Techniques; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
C014059:CYC1 protein, S cerevisiae; D017931:DNA Primers; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D045304:Cytochromes c