STRUCTURE, FUNCTION, & DYNAMICS OF P-450 CYTOCHROMES

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Structure, function, and conformational dynamics of the cytochrome P-450 monooxygenase system are to be studied in the proposed research. (1) Topology of membrane-bound rabbit microsomal isozymes 2 and 4 will be probed via polyclonal and monoclonal antibodies as well as by specific and non-specific proteolysis. Immobilized polyclonal antibodies will be used to purify specific peptides released upon protease treatment of microsomes; HPLC-isolated peptides will be submitted to micro- sequence analysis to reveal sites within the primary structures available at the external (cytoplasmic) surface of the endoplasmic reticulum. A panel of monoclonal antibodies will be raised to synthetic peptides selected from each sequence by MSEQ computer analysis; these site-specific antibodies will be utilized to probe native and protease-digested microsomes via ELISA and Western-blotting procedures. Together, the results obtained will permit reconstruction of the membrane topological features of each cytochrome. Preliminary data and calculations suggest, in contrast to current theory, that the topology of the P-450's is simple and is similar to that known for other microsomal proteins. Isozymes 2 and 4 were chosen for the proposed studies as they are the major forms present in phenobarbital-induced (isozyme 2) and aryl hydrocarbon-induced (isozyme 4) animals, they represent two major gene sub-families, they typify low- and high-spin cytochrome types, they are involved in drug disposition tolerance, and they participate in the oxidative metabolism of physiological lipids, numerous drugs, and other xenobiotics; furthermore, they are highly similar to forms known in the human. (2) Conformational dynamics of isozyme 2 will be studied with site- specific chemical modification of Cys-152, a residue shown to serve as a "reporter" for binding events at the active site. Modification kinetics, carbon-13 NMR spectroscopy, and circular dichroism will be utilized to examine the role and importance of conformational changes in the function of the cytochrome. Functional aspects to be examined will include substrate binding, heme reduction kinetics, and protein-protein interactions. (3) Crystallization of many of the microsomal monooxygenase system components will be attempted with the ultimate aim of three- dimensional structure elucidation. Exploration of the "small Amphiphile" concept and co-crystallization will be major thrusts. A long-term objective of these studies is to achieve a sophisticated molecular view of the P-450 system that will serve to guide rational drug design.