The general objective of this program is to understand protein methylation from three perspectives: (i) The role of methylation in the dynamics of protein biosynthesis and intracellular processing and trafficking, (ii) the physico-chemical and functional modulation of protein by enzyme-catalyzed methylation, and (iii) a study of the protein methyltransferases themselves, looking specifically at substrate-binding and/or active sites. In order to achieve the above, the following specific research objectives must be met. (1) Elucidation of possible roles for methylation in the dynamics of cytochrome c biosynthesis (elongation, termination, proteolytic resistance of the nascent protein), utilizing in vitro translation systems containing exogenously added mRNA which is transcribed in vitro from plasmid containing yeast iso-l-cytochrome c gene and purified protein-lysine N-methyltransferase. (2) Utilizing in vitro systems containing isolated mitochondria, examination of possible roles for methylation in the posttranslational uptake of cytochrome c apoprotein into the mitochondria and the subsequent attachment of the heme prosthetic group. (3) Utilizing physical methods, characterization of the precise physico-chemical effects exerted by methylation on cytochrome c, and characterization of the effects of methylation on the molecular interaction of cytochrome c with cytochrome c oxidase, reductase, and peroxidase. (4) Identification and subsequent purification of two enzymes implicated to exist from observations during the past grant period. (a) An enzyme which dealkylates methylated cytochrome c in vivo. (b) An enzyme which methylates the Res-86 lysine of cytochrome c. (5) Identification and characterization of the protein structure contributing to the active sites of highly purifiedprotein methyltransferase: Cytochrome c-specific protein-lysine N- methyltransferase from Neurospora crassa and protein-carboxyl O- methyltransferase from calf brain by photoaffinity labeling with analogues of S-adenosyl-L-methionine, and subsequent analysis of labeled peptides generated by enzymatic degradation of the modified methyltransferases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK009602-21
Application #
3224627
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1977-12-01
Project End
1992-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
21
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Temple University
Department
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122