Spectrophotometric, mass spectrometeric and structural studies of the prototypical G protein coupled receptor rhodopsin

Abstract

Rhodopsin is the integral membrane protein responsible for black and white vision in low light conditions and is found at high concentration in the mammalian retina. Rhodopsin is a prototypical member of the G protein coupled receptor super family that control much of physiology. Improved understanding of rhodopsin signal transduction and amplification via coupling to the heterotrimeric G protein transducin may reveal conserved activation mechanisms that are relevant to other members of the GPCR super family. Described here are several studies that examine the molecular determinants responsible for heterotrimeric G protein coupling to metarhodopsin II, the active photointermediate of bovine rhodopsin. Employing uv-visible spectroscopy we have investigated the nature of the interaction between the C-terminal tail of transducin and metarhodopsin II. We have provided evidence that suggests the orientation of transducin when it interacts with metarhodopsin II.

Mass spectrometry is a powerful technique for characterizing intact and digested proteins. We have optimized mass spectral methods for investigating integral membrane proteins, utilizing rhodopsin as a model system. The mass spectrometric studies provide the foundation for future investigations into agonist and antagonist interactions and the related G protein coupled receptors using molecular crosslinking. Development and validation of new tools for generating structural constraints for conformational states of proteins that are not amiable to more traditional structural determination techniques are described. Antibody imprinting studies on rhodopsin were advanced with the work presented here. The x-ray crystal structure of the anti-rhodopsin antibody K42-41L in complex with a synthetic epitope mimetic peptide is described. These studies led to the generation of a model of the third cytoplasmic loop of the photointermediate metarhodopsin I and constraints on the conformational changes in metarhodopsin II.