W0287

Structure Determination of Visual Rhodopsin. Ronald E. Stenkamp^1,2, Krzysztof Palczewski^3,4,5, Takashi Kumasaka^7,8, Tetsuya Hori^7,8, Craig A. Behnke^2,6, Hiroyuki Motoshima⁷, Brian A. Fox^2,6, Isolde Le Trong^1,2, David C. Teller^2,6, Tetsuji Okada³, Masaki Yamamoto⁷, Masashi Miyano⁷. ¹Dept. of Biological Structure, ²Biomolecular Structure Center, ³Dept. of Ophthalmology, ⁴Dept. of Pharmacology, ⁵Dept. of Chemistry, ⁶Dept. of Biochemistry, Univ. of Washington, Seattle, WA 98195, USA, ⁷Structural Biophysical Lab, RIKEN Harima Inst, Hyogo 679-5148, Japan, 8, Graduate School of Bioscience and Biotechnology, Tokyo Inst. of Technology, Yokohama 226-8501, Japan.

Rhodopsin is the membrane protein responsible for the first step in vision, the absorption of light. It is a G-protein coupled receptor (GPCR) which responds to a conformational change in its retinal chromophore brought about by absorption of a photon. Rhodopsin activates its G-protein, transducin, to initiate a signal transduction cascade that results in a nervous signal to the brain. To better understand the structural changes involved in this and other signal transduction processes, we have determined the structure of ground-state rhodopsin using X-ray crystallographic methods. The emphasis of the talk in this session with be on technical difficulties faced in growing crystals, dealing with light-sensitive proteins, solving the twinned crystal structure, and refining it. Stabilization of the protein with zinc appeared to be crucial for obtaining crystals. The light sensitivity of the protein, even at liquid nitrogen temperatures, was dealt with by judicious applications of black plastic and duct tape to the experimental hutches at the synchrotrons. MAD techniques were applied to a mercury derivative to solve the structure of the natural protein. Various techniques were used to handle the merohedrally twinned crystals, but the best MAD phaseswere obtained from a crystal with a low twinning fraction. Refinement using detwinned data and refinement using a twinned model gave comparable results. In addition to the technical issues, the structure will be discussed. In particular, the seven transmembrane helices common to all GPCRs will be described with an emphasis on the kinks and bends that might have physiological significance. The results of comparing the structure with that of bacteriorhodopsin will be presented, as will structural details concerning the retinal chromophore.

This work has been supported by the NIH grant EY09339, Research to Prevent Blindness, Inc., Foundation Fighting Blindness, Inc., the Ruth and Milton Steinback Fund, and the E.K. Bishop Foundation.