The major goal of this research is to investigate the molecular basis for the triggering of a visual nerve impulse by the vertebrate rod. Knowledge of these molecular mechanisms can provide a conceptual basis for eventual therapeutic intervention with regard to visual disorders. The rod comprises a supramolecular assembly, in which the retinal disk membranes contain rhodopsin, an integral membrane protein, together with characteristic bilayer lipids whose composition is tightly regulated. The key signal transducing event in vision is the metarhodopsin I to metarhodopsin II conformational transition. They will test the hypothesis that upon photoexcitation of rhodopsin, the retinal Schiff base of Lys-296 is deprotonated, leading to breakage of the salt bridge to Glu-113 , which is propagated to the cytoplasmic loops of the receptor. As a result, the Meta I-Meta II transition is sensitive to the deformation energy of the membrane lipid bilayer. Diseases such as retinitis pigmentosa (RP) may be associated with perturbation of the Meta I-Meta II equilibrium, due either to mutation of rhodopsin, or alteration of the bilayer lipid environment. Two complementary biophysical methods will be employed: deuterium (2H) NMR spectroscopy and surface plasmon resonance (SPR) spectroscopy. (i) 2H NMR spectroscopy will investigate the role of the bilayer deformation energy in enabling the Meta I-Meta II conformation change of rhodopsin to occur, due to area or curvature frustration of the membrane free energy. (ii) The retinal chromophore of rhodopsin will be 2H-labeled, and 2H NMR studies will be conducted to elucidate the conformation and orientation of the retinal chromophore in the dark state. They will then investigate the changes that occur upon photoexcitation, leading to movement of the transmembrane helices and formation of the activated Meta II state. (iii) SPR spectroscopy of rhodopsin in supported planar bilayers will be used to determine how the rigid body movement of the helices influences the cytoplasmic loops of the receptor, yielding the exposure of recognition sites for the G protein (transducin). Additional SPR studies will investigate the binding constants and signaling states of the proteins involved in amplification and quenching of the visual response. Thus they intend to provide a comprehensive picture of how rhodopsin together with the bilayer lipids yields triggering of visual excitation in the vertebrate rod, which is a paradigm for membrane structure-function relationships and signal transduction in general.
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