The goal of this work is to understand the visual transduction system at a molecular level in order 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 in that the visual receptor rhodopsin is the prototype of the large family of G-protein coupled receptors. This work will employ state of the art mass spectrometry technologies in conjunction with chemical modification and crosslinking experiments to characterize covalent and higher order structures of the receptor protein rhodopsin, its G-protein transducin, its effector enzyme cyclic GMP phosphodiesterase, and the sites of interaction of these proteins in the signal transduction process. There are four Specific Aims: 1.
The first aim i s to refine methodology for mass spectrometric characterization of covalent modifications on rhodopsin and other membrane proteins. These methods will employ protein cleavage on blotting membranes and on a new type of sample preparation column in conjunction with fragment isolation by both conventional reversed phase HPLC and the newer method of hydrophilic interaction chromatography. 2.
The second aim i s to probe the three dimensional structure of rhodopsin and the conformational changes which occur upon photoactivation via chemical modification and intramolecular cross-linking experiments. These experiments will employ both photoactivated and heterobifunctional chemical cross-linking reagents to define intramolecular distances, as well as chemical modification experiments to define surface exposed residues, to gain information on the structural changes which occur in rhodopsin upon photoactivation and mutation induced constitutive activation. 3.
The third aim i s to extend the applicants' previous work on rhodopsin phosphorylation by characterizing all of the sites of phosphorylation at both high and low bleaching levels. 4.
The fourth aim i s to define the sites of protein-protein interactions in the visual transduction pathway using chemical cross-linking experiments in conjunction with mass spectrometric analysis. These studies will define the sites of interaction between rhodopsin and transducin, and between the transducin alpha subunit and cyclic GMP phosphodiesterase. This work will provide structural information critical to defining the molecular mechanisms of visual transduction, as well as G-protein coupled receptor systems more generally, and will also provide methodology applicable to study of other integral membrane protein systems.
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