The goal of this work is to understand the visual-transduction system at a molecular level to define the mechanisms of both normal and abnormal vision. Definition of these mechanisms will provide a basis for design of new therapies for visual diseases. Understanding visual transduction is also of broader biological significance because the visual receptor rhodopsin is the prototype of the large family of G-protein couple receptors.
Two specific aims are proposed. The first is to characterize the three-dimensional structure of rhodopsin and define the structural changes that occur upon rhodopsin photoactivation. Because rhodopsin is a membrane protein, it is not readily amenable to direct structural approaches such as x-ray crystallography and NMR spectrometry. Instead, indirect methods such as chemical modification topography, chemical cross linking, and tethered reagent cleavage studies in conjunction with mass spectrometry will be used to gain information on solvent-exposed surfaces and distance geometry. Reconstituted membranes containing phospholipids synthesized with photolabeling groups will be used to study motion in the transmembrane region. The results will contribute to the understanding of the structure of rhodopsin and the conformational changes in its cytoplasmic domain that occur upon receptor activation to enable binding of the G protein transducin.
The second aim i s to characterize structurally the binding of transducin to the activated receptor. These studies will employ surface modification """"""""footprinting"""""""" and cross linking to explore how activation of the receptor enables G-protein binding. This work will provide structural information critical to defining the molecular mechanisms of visual transduction and the action of G-protein-coupled receptor systems and will also result in the development of methodology that can be applied to the study of other membrane protein systems.
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