The long term goal of this project is to elucidate the biochemical and biophysical mechanisms underlying visual excitation and photoreceptor metabolism. The approach is to attack important, related problems at several levels of organization of the photoreceptor cell starting at the level of the visual pigment and ranging to NMR studies of retina in vivo.
Specific aims i nclude (1) the structure of visual pigments: (A) Modeling the structure of visual pigments by (a) mapping out the amino acids lining the chromophore binding site by covalently linking the chromophore to specific cysteines; (b) characterizing the divalent cation binding site of rhodopsin, investigating if cone pigments have such a site, and determining if cation binding has any physiological consequences; (c) developing a detailed three dimensional structural model for visual pigments based on a combination of experimental constraints and computer modeling using semi-empirical observations on the placement of amino acids in membrane proteins. (B) Characterizing the properties of a cone- type pigment from gecko, P521 by (a) obtaining the vibrational spectrum of the chromophore of the pigment in its normal and Cl-depleted states; (b) determining the sequence and composition of carbohydrate moiety of the pigment; (c) localizing the Cl- binding site of P521 with respect to the chromophore; (d) characterizing the phosphorylation of P521 after light absorption in vivo. (2) Control of the pKa of the Schiff base and its Counter ion: (A) elucidating the role of water in determining the pK of the Schiff base; (B) determining the key amino acids in the retinal binding site which control the pK of the Schiff base; (C) determining the effect of protonation of the counter ion of both vertebrate and invertebrate visual pigments, and determining if anions can serve as a counter ion under some circumstances. (3) Photochemistry of Visual Pigments: (A) determining if the counter ion of octopus rhodopsin becomes protonated after light absorption, and resolving the protonation changes kinetically; (B) determining if deuteration affects the kinetics of the transitions between the bleaching intermediates, and clarifying the protonation changes that occur; and (C) elucidating the effect of chloride ions on the photobleaching of cone- type pigments. (4) Electrostatic Properties of Photoreceptor Membranes Containing a Cone Type Pigment and their Consequences: (A) measuring the sign and magnitude of the surface charge density of gecko P521-containing photoreceptor membranes, and determining if the membrane is positively charged (B) characterizing the transducin associated with gecko P521; sequencing it; comparing the part of its sequence which interacts with activated P521; and (5) in vivo NMR Studies of the Retina: (A) 31P-NMR studies to (a) observe GDP/GTP exchange on transducin in vivo; (b) observe cGMP in vivo and observe its modulation by light and calcium; (B) Proton NMR studies (a) to obtain water suppressed spectra of toad retinas and identify the major peaks, especially lactate; (b) to observe and quantify the effects of light on the species identified in 5a; and (c) to observe and quantify the effects of oxygen, glucose and calcium on the species identified in 5a.
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