The proposed research is a continuation of an interdisciplinary collaboration on the structure, dynamics, and physiology of proteins which bind and degrade juvenile hormone (JH) and JH mimics (JHM). The original goals of this collaborative research were to develop chemical and biochemical techniques to approach the molecular biology of juvenile hormone (JH) reception and catabolism. These initial objectives have been acheived, and in this renewal request, further goals are proposed: First, to study the structure of the JH and JHM binding proteins (JHBP an RP) in Manduca larval epidermal cell cytosol and nuclei, and of JH esterases (JHE) and epoxide hydrolases (JHEH) through (a) synthesis of radioligands, (b) JHBP/RP and JHE/EH purification and sequencing, (c) active site labeling and sequencing, (d) antibody production, (e) gene isolation and sequencing, and (f) overproduction of proteins; second, to examine the dynamics of the ligands and macromolecules, including (a) timing and location of JHBP/RP, JHE/EH, (b) movement of hormones and proteins between cell compartments, and (c) protein-ligand, protein-protein, and protein-DNA interactions. A key to the structure and dynamics goals will be the development of a baculovirus expression system in Bombyx for protein overproduction. The third goal is to correlate structure and dynamics with physiology by characterizing, (a) the appearance of JHBP/RP and JHE/EH in the life cycle, and (b) comparative biochemistry of JHBP/RP and JHE/EH in insects, with respect to ligand recognition and protein similarities. The characterization of receptor proteins for juvenile hormone (JH) and JH mimics (JHM) and knowledge of the pathways of their degradation are major keys to understanding the molecular basis for hormonal regulation of gene expression in insects. This research is an interdisciplinary collaboration which combines expertise in molecular biology and physiological manifestations of gene expression in insects (L. M. Riddiford), biochemical and molecular studies of JH metabolism (B. D. Hammock), and synthesis and use of high specific activity labeled JHs and JHMs (G. D. Prestwich). This proposal was submitted under the NSF initiative in the Chemistry of Life Processes.
