The objective of this research program is to contribute to an understanding of the molecular basis of the biological activities of membrane-bound proteins. These studies will focus on one group of electron transport enzymes of endoplasmic reticulum involving cytochrome b5 interactions with two electron donors, cytochrome b5 reductase and cytochrome P-450 reductase and mainly one of a number of electron acceptors, stearyl-CoA desaturase. By limiting the scope of the experimental system, we can achieve a detailed description of the structures of each enzyme, the mechanisms of the electron transfer reactions, the structures of both active, catalytic domains and segments that participate in interactions with the lipid bilayer, the mobility and flexibility of each enzyme in lipid bilayers, and the orientation of each protein in bilayers that yields catalytically productive interactions between the components of these interacting enzymes. These objectives will require not only application of the methodology of protein chemistry to examine nonpolar peptide primary, secondary and tertiary structure but also increased use of a variety of physical, chemical, immunological and molecular biological methods to achieve a relatively complete definition of these protein-protein and protein lipid interactions. The spectrum of techniques will include fluorescence energy transfer, x-ray diffraction, high resolution NMR, bifunctional crosslinking reagents, construction of expression vectors for these enzymes and site-directed mutagenesis. In addition to the basic information concerning membrane structure and function that these studies on one model system should generate, the system has more direct implications for health related problems since the finding that saturated fatty acids have been implicated in hypocholesterolemia, a constant finding in atherosclerosis, emphasizes the need for detailed information on the desaturase mechanism by which the human might regulate fatty acid desaturation, and cytochrome b5 also functions as an electron donor for cytochromes P-450 which are involved in liver detoxication reactions and in the conversion of certain drugs to carcinogens.
