CURRENT PROGRESS ON CRYSTALLOGRAPHIC COMPLEXES OF TRANSDUCIN. Susan M. Redford, Heidi Hamm^{[[daggerdbl]]}, Joseph Noel, Structural Biology Lab, The Salk Institute, La Jolla, CA 92037, ^{[[daggerdbl]]}University of Illinois College of Medicine, Chicago, IL 60680

The opportunity to understand the structure and function of a G protein signal transduction cascade at the atomic level is the underlying goal of this research. The current studies employ peptides that mimic rhodopsin's functionally essential cytoplasmic face in complex with G_{t[[alpha]][[beta]][[gamma]]} to probe the structural consequences of receptor/G-protein binding. Recently the heterotrimer structure, G_{t[[alpha]][[beta]][[gamma]]} has been reported to 2.0 Å resolution. In these studies, a chimeric Gta Gta/_i[[alpha]], which has been E. Coli expressed, is substituted for G_t[[alpha]]. G_t[[alpha]]/_{i[[alpha]][[beta]][[gamma]]} crystals are then grown in complex with peptides which mimic rhodopsin's cytoplasmic loops 2 and 3.

Signaling cascades provide mechanisms to receive, transmit and amplify cellular messages, and thus are critical pathways for mounting a cellular response to an extra-cellular stimulus. Heterotrimeric G-proteins have evolved as ubiquitous solutions for signal transduction and amplification throughout many cells, and are involved in processes such as vision, olfaction, hormone signaling, and responses to cytokines. The [[alpha]] subunit has been studied extensively biochemically, as it contains the GTPase function, and binds rhodopsin, the [[beta]] and [[gamma]] subunits, and cGMP-PDE. Diffraction quality crystals of the heterotrimeric complex are grown in microseeded hanging drops containing 10 mg/ml protein, 10% PEG-8000, 50 mM Tris, pH 8.0, 50 mM NaCl, 10% glycerol, and 0.1% [[beta]]-mercaptoethanol. The space group is C2 with unit cell dimensions of a=133.4, b=91.4, c=83.2, [[beta]]=120.1deg.. Progress to date will be reported.