Cones must function over a wide range of light intensities and a broad span of the electromagnetic spectrum. The ability to perform under such diverse conditions arises in large part from the unique set of phototransduction proteins found in cone cells. Light responses initiate with the absorption of photons by the visual pigments (cone opsins). The long term goals of this project are to understand how the cone opsin family has attained absorption maxima from 350 nm to 650 nm while achieving the rapid photobleaching and regeneration properties that play a large role in shaping the light responses of cones. This proposal will combine molecular, biochemical and biophysical approaches to investigate the molecular mechanisms underlying the function of cone visual pigments. In our First Aim, the short wavelength-sensitive (SWS1) visual pigments will be purified from transfected COS cells in order to investigate how particular amino acid residues in the transmembrane helical segments and the extracellular domain regulate photoactivation and regeneration properties. The conformational changes that occur following isomerization of 11-cis retinal will be studied by low temperature UV/visible and fluorescence spectroscopy. The amino acids that regulate the formation and stability of the light-activated conformation (R*) will be studied in conjunction with molecular modeling. These experiments will allow us to address in a comprehensive way several long standing issues about visual pigment behavior: mechanisms controlling light-activation and pigment regeneration. In order to study the structure of cone visual pigments, in our Second Aim we will investigate in a systematic fashion the stability of cone opsins in a variety of solubilizing agents. To accomplish this we will use an inducible heterologous over-expression system to produce milligram quantities of cone pigment. The information gained in this Aim will allow for protein crystallization trials in the future. ? ? ?
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