Traditionally, significant progress towards a comprehensive understanding of the mechanisms by which a protein's structure subserves its function, a prerequisite for the practical control of its physiological activities, has come from studies which assay the changes in a particular activity, resulting either from specific amino acid chemical modifications or from amino acid substitutions created by a genetic lesions. However chemical modifications of amino acid side chains are limited to a small proportion of residues, while surviving genetic modifications are random and necessarily limited to non-lethal mutations. These classical approaches are now being dramatically extended through the use of recombinant DNA techniques to obtain cytochromes c with any desired amino acid sequence. Site-directed mutagenesis of cloned eukaryotic cytochrome c genes and the production of the protein in heterologous expression systems, will be performed to study how particular amino acid substitutions affect protein structure and stability its biosynthesis, as well as electron transport and binding affinities with various physiological reaction partner, such as the mitochondrial cytochrome c oxidase cytochrome c reductase and yeast cytochrome c peroxidase. Since our present technique for the production of mutant cytochromes c requires them to be at least partially functional an important secondary objective is the development of procedures for obtaining the expression in yeast of functionless mutants. The use of mutants of the apoprotein, the biosynthetic intermediate, opens the door to the examination of several physiologically relevant processes that characterize the life cycle of the protein. These include the recognition of the apoprotein by the outer mitochondrial membrane, its transport through the membrane, the enzyme-catalyzed covalent binding of the heme prosthetic group to the apoprotein, the release of the holoprotein into the mitochondrial intermembrane space and the mechanism by which the level of cytochrome c in mitochondria is regulated. Finally, our present system in yeast produces both N-terminally acetylated and non-acetylated rat cytochrome c, both carrying a fully trimethylated lysine 72; it also yields Drosophila melanogaster cytochrome c which has that lysine in tri-, di-, mono- and unmethylated forms, which have been separated by HPLC. These proteins provide a so far unique opportunity for studying the structural and the functional effects of these well known secondary modifications of cytochrome c.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029001-13
Application #
2175357
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1990-07-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1995-06-30
Support Year
13
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Hess, R A; Miller, L A; Kirby, J D et al. (1993) Immunoelectron microscopic localization of testicular and somatic cytochromes c in the seminiferous epithelium of the rat. Biol Reprod 48:1299-308
Koshy, T I; Luntz, T L; Garber, E A et al. (1992) Expression of recombinant cytochromes c from various species in Saccharomyces cerevisiae: post-translational modifications. Protein Expr Purif 3:441-52
Koshy, T I; Luntz, T L; Schejter, A et al. (1990) Changing the invariant proline-30 of rat and Drosophila melanogaster cytochromes c to alanine or valine destabilizes the heme crevice more than the overall conformation. Proc Natl Acad Sci U S A 87:8697-701
Garber, E A; Margoliash, E (1990) Interaction of cytochrome c with cytochrome c oxidase: an understanding of the high- to low-affinity transition. Biochim Biophys Acta 1015:279-87
Luntz, T L; Schejter, A; Garber, E A et al. (1989) Structural significance of an internal water molecule studied by site-directed mutagenesis of tyrosine-67 in rat cytochrome c. Proc Natl Acad Sci U S A 86:3524-8
Garber, E A; Luntz, T L; Margoliash, E (1988) The interaction of cytochrome c with cytochrome oxidase. Prog Clin Biol Res 274:749-69
Armstrong, G D; Chapman, S K; Sisley, M J et al. (1986) Preferred sites on cytochrome c for electron transfer with two positively charged blue copper proteins, Anabaena variabilis plastocyanin and stellacyanin. Biochemistry 25:6947-51
Swanson, M S; Zieminn, S M; Miller, D D et al. (1985) Developmental expression of nuclear genes that encode mitochondrial proteins: insect cytochromes c. Proc Natl Acad Sci U S A 82:1964-8
Hannum, C H; Matis, L A; Schwartz, R H et al. (1985) The B10.A mouse B cell response to pigeon cytochrome c is directed against the same area of the protein that is recognized by B10.A T cells in association with the Ek beta:Ek alpha Ia molecule. J Immunol 135:3314-22
Hannum, C H; Margoliash, E (1985) Assembled topographic antigenic determinants of pigeon cytochrome c. J Immunol 135:3303-13