Signal transduction in the visual system involves a series of interacting molecular switches that relay a signal from the receptor, rhodopsin, through various stages of regulation and amplification to a final target. The long-term objectives of this project are to elucidate the molecular mechanisms of activation in three key switches in the relay: rhodopsin, transducin and arrestin. The experimental approach is based on site-directed spin labeling (SDSL), a relatively recent technique developed in this laboratory. SDSL involves the selective placement of a nitroxide side chain in a protein and the analysis of the electron paramagnetic resonance (EPR) spectrum in terms of structure. The method provides structural information at the level of the backbone fold with a real-time resolution in the millisecond range.
A specific aim of the proposal is to advance SDSL to include: (1) analysis of backbone dynamics using multi-frequency EPR and spectral simulation; (2) identification of specific nitroxide side chain interactions using EPR and X-ray crystallography, and (3) the use of oriented systems for high-resolution topographical analysis. These advances will find application in studies of spin-labeled versions of rhodopsin, transducin and arrestin.
Specific aims i nclude: (1) a global description of the helix displacements that accompany the formation of activated rhodopsin (R*); (2) conformational changes induced in transducin and R* resulting from their mutual interaction, and (3) conformational changes induced in arrestin during transformation to the high affinity binding form by interaction with phosphorylated R*. A growing number of pathological conditions have been traced to genetic defects in the molecular switches of the visual and other signaling systems. In order to design pharmacological approaches for altering such pathological states, it is imperative to understand molecular mechanisms involved in the signal relay. Success in this project is anticipated to contribute to this understanding.
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