Structural constraints for the folding, stability and function of cytochrome C
Creators: name: Murphy, Michael E. P.
Publisher: name: University of British Columbia
Year: 2008

description: The relationship between structure, stability and function in yeast (Saccharomyces cerevisiae) cytochrome c was studied through the investigation of mutant proteins. Two isozymes of cyto-chrome c can be isolated from yeast, iso-1 and iso-2-cytochrome c. The structure of iso-l-cyto-chrome c had been previously determined in this laboratory using X-ray diffraction analyses. In the first part of this study, the structures of wild-type yeast iso-2-cytochrome c and a composite mutant protein (B-2036) composed of segments derived from the genes of both isozymes were structurally characterized by X-ray analyses. The overall fold of the composite is similar to the two native isozymes, however, specific intra-molecular atomic interactions are altered bythe presence of amino acid substitutions. These alterations are proposed to explain the loss ofthermodynamic stability of the composite relative to the wild-type proteins. In a second study, a-Loop A of iso-l-cytochrome c was replaced with the corresponding loop from cytochrome c2of Rhodospirillum rubrum to produce a mutant protein called RepA2 cytochrome c. Structural analysis of the RepA2 protein revealed that a-Loop A had folded in a conformation more similar to the original loop it replaced than the conformation of a-Loop A in cytochrome c2. Two substitutions, Va120Phe and His26Asn, that result from the a-Loop A replacement were shown to cause structural perturbations. Phe20 was back substituted to Va120 to produce Rep A2(Va120) cytochrome c. In vivo functional studies and reduction potential measurements of these S2-Loop A replacements demonstrate that Phe20 lowers the reduction potential by 19 mV at 25°C and diminishes cytochrome c function at higher temperatures. A third aspect of this work was to investigate the role of Pro71. The structures of four partially functional Pro71 substitutions were determined: Pro7lAla, Pro7lIle, Pro7lSer and Pro71Val cytochromes c. Analysis of these structures revealed that loss of function could be explained by the disruption of residues 82 and 83 which have been shown to be important in the binding and transfer of electrons to complexed electron transfer partners. Larger side chain replacements, Pro71Val and Pro7lIle, result in additional disruptions at the site of mutation which is part of a highly conserved segment of polypeptide chain. A comparison of all the mutant structures investigated in this work revealed that the overall fold of the protein was preserved at the expense of disrupting the packing of the hydrophobic core and highly mobile surface loops. Amino acid substitutions that cannot be incorporated into the cytochrome c fold without structural perturbations occur in conformationally restricted regions. In addition, the displacement of groups by these re-placements indicates the limits present in specific regions in terms of conformational flexibility. The structural perturbations that result from the replacement of conformationally restricted residues have been shown to diminish both the reduction potential and in vivo function of cytochrome c.