BIOORGANIC STUDIES OF RETINAL PROTEINS

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The studies on retinal proteins cover an extremely broad range of science because of its totally interdisciplinary nature; no single discipline can solve the numerous problems remaining to be answered. The main focus of our studies is to synthesize a number of tailored retinal analogs and use them to clarify specific aspects in the general area in which retinal proteins are involved. (1) Bleaching adaptation : This is a field which is much less understood than visual transduction on a molecular structural basis. This is because there are far fewer structural handles to deal with, and also because the field is far less advanced in comparison to visual transduction. However, starting with an analog which has a locked 11-cis-ene structure, we have found several structural clues which will allow us to design further molecules to clarify the mechanism of this important area. (2) The triggering process of visual transduction. During the last grant period, we studied the process and structural requirements of the transduction process. Our next aim is to explore the photochemistry and very early stage changes through very fast spectroscopic measurements. (3) A major effort is to streamline the process of photoaffinity labeling, particularly that of membrane proteins exemplified by the rhodopsins. Very few studies have led to the final sequencing of amino acids because of difficulties encountered in separation of the peptide fragments. We will continue to simplify the analytical protocols. We have managed to successfully characterize cross-linked amino acids both with rhodopsin (Rh) and bacteriorhodopsin (BR). However, this has led to further interesting questions. In the case of Rh, what is the absolute sense of twist of the chromophore, and in the case of BR, which is the correct orientation or the chromophore within the binding site. These problems will be addressed mainly through solid state NMR of analogs containing specific labels, and applying some non routine NMR techniques. (4) Most of the approximately 100 analogs made in our lab was for the purpose of spectroscopy. We will now measure enzymatic activities in order to gain further insight into the structural, electronic add other factors involved in visual transduction. (5) Retinal mimics will be used as templates to produce monoclonal antibodies that carry suitably distributed charges within artificial binding sites of 11-cis-retinal; this provides another approach for investigating electrostatic effects operating between the ligand and its binding site.