The overall objective of this research is to understand the structural basis for the production of a functional cytochrome P450 (P450) molecule. Emphasis is on the cellular targeting to the endoplasmic reticulum (ER), folding of the protein and membrane topology, and amino acids involved in substrate interactions. Previous work has demonstrated that P450 is exclusively retained in the ER, that redundant ER retention signals are present in both the N-terminal signal anchor sequence and the cytoplasmic catalytic domain, that the sequence linking (""""""""linker"""""""") these two regions may be critical for folding of the protein, and that six amino acid substitutions confer a novel steroid hydroxylase activity to P450 2C2.
The specific aims are: 1) to examine the ER retention signals, 2) determine the requirements of the ER retention properties for the N-terminal signal anchor sequence, 3) examine the mechanism by which mutations in the """"""""linker"""""""" sequence reduce activity, 4) examine the kinetic and substrate binding properties of P450 2C2 mutants with novel steroid hydroxylase activity, and 5) express large amounts of soluble P45O-2C2 and variants for potential structural studies. Experiments will include the generation of mutants, the production of adequate protein in insect cells and bacteria for membrane and structural studies, and kinetic analysis of the different chimeric proteins to examine the effect of the modulations that are imposed on the leader sequences and other membrane retention signals.
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