The long term goal of this project is to understand how ions affect the molecular structure of bacteriorhodopsin (bR). This is a membrane protein found in the halophilic Halobacterium halobium, contains the chromophore retinal which undergoes a photocycle of reversible structural and environmental changes upon absorption of light. The protein's similarity to visual rhodopsin which also contains a retinal chromophore makes an excellent model for the visual process. Thus, a better understanding of the molecular processes of cation association with bR will provide an important step in determining if a similar mechanism applies to the visual rhodopsin cycle. Similarly, bR can also be used as a model for Gi regulatory protein involved in signal transduction pathways affecting intracellular calcium levels. The bR system to be used in these experiments is a straightforward and inexpensive model that can be used to gain a fundamental understanding of the molecular mechanisms involved in mammalian systems. The immediate focus of this proposal is to better understand the function of cations in the proton pump mechanism during the photocycle and specifically, to test the hypothesis that cations are displaced from the active site during this mechanism. The three specific aims designed to test this hypothesis are: (1) To determine the steady state intracellular and surface concentration of cations. Initial work will focus on measuring steady state cytosolic calcium ion by using a calcium ion sensitive probe which is loaded into the cells as a methyl ester. Once inside of the cells it is de-esterified by esterases near the cell surface and is able to bind cytosolic calcium. This binding cause the dye to fluoresce, and the fluorescence can be used to measure the concentration of calcium in the cytosol. (2) To determine the changes in intracellular concentration of cations and correlate these changes to the formation of photocycle intermediates. This will allow us to test the present theories regarding the environment at the active site during the photocycle. (3) To determine the changes in the surface concentration of cations and correlate these changes to the formation of photocycle intermediates. By using new lipophilic fluorescent probes, which measure surface cation concentrations, we expect to determine whether cation displacement at the active site results in a movement of the cation to the surface of the cell membrane where it can be taken up again by the protein at a later stage in the photocycle. An integral aim of this research is the training of minority students in biomedically related scientific techniques. This project is particularly suitable for this type of training because it involves an integrated multi-disciplinary approach, and the research can be divided into specific projects which would provide information that is not already known about this bacteria's photocycle and proton pump mechanism. This is an area of bR research which is not actively being studied; nevertheless, it would provide very important information relevant to various biological fields. Each project is focused, result oriented, and can be divided into individual, small or large, subprojects based on each student's background and available time commitments (e.g. undergraduate vs. graduate students.
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