Bioluminescent photoproteins afford high sensitivity of detection and have been employed as labels in bioanalysis. We propose to further expand the applications of these photoproteins by modifying them to possess unique bioluminescence properties. One of the goals of this work is to alter the electronic and H-bonding network within the chromophore-binding pocket of these photoproteins in order to shift their emission wavelengths. This will be achieved by incorporation of non-natural amino acids into the aequorin structure and by performing site-directed mutagenesis. The resulting proteins will be characterized in terms of their activity and structure-function relationship. We also propose to mimic the natural phenomenon that occurs in the jellyfish Aequorea Victoria where transfer of energy from aequorin to GFP results in the emission of green light. For that, we propose to prepare protein-based """"""""artificial jellyfish"""""""" by attaching a fluorophore to unique sites on aequorin close to the coelenterazine binding pocket. This will allow transfer of energy from aequorin to the fluorophore, thus, shifting the wavelength of emission of the protein. """"""""Molecular switches"""""""" that can be """"""""turned on"""""""" in the presence of a target analyte will be prepared by constructing hybrid proteins between aequorin variants and a binding protein for a specific analyte. The hybrid proteins will be constructed by inserting the gene of the binding protein into the gene of aequorin. The conformational changes that occur in the binding protein upon ligand binding will bring the two parts of aequorin together, allowing for the emission of bioluminescence. In addition, """"""""molecular switches"""""""" will be constructed by preparing fusion proteins of a dissected aequorin and genes that code for a pair of poplypeptides that can form a leucine zipper. The leucine zipper allows for aequorin to re-assembly and bioluminescence emission. When target DNA is present, the leucine zipper is pulled apart; aequorin is disassembled, with the subsequent loss of light. Applications of the above modified photoproteins in bioanalysis will be demonstrated by developing highly sensitive assays for panels of analytes that are important in disease diagnosis. Furthermore, these assays based on aequorin variants with different emission wavelengths will be incorporated into a microcentrifugal microfluidic platform, which should result in multiplex analysis with applications in point-of-care diagnostics and HTPS. ? ?
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