Biosensors designed to detect and provide a visible indication by changes in fluorescence of a variety of intracellular conditions will be developed by genetic engineering using the green fluorescent protein from the jellyfish Aequorea victoria as a template. Sensors have already been developed that respond to changes in pH and halide (chloride) concentration, which are essential for proper cellular function. These will be refined in function for use by cell and molecular biologists in single living cells, including human cells, in order to ease study of cellular processes in normal and diseased cells. In addition it is proposed to develop variants that are sensitive to changes in redox potential via reversible formation or breakage of disulfide bridges for use of detecting redox potentials in subcellular compartments such as the endoplasmic reticulum. Variants will be characterized by absorbance and fluorescence spectroscopy and the structures of potentially useful biosensors will be determined by X-ray crystallography in order to assess the structural basis for the molecular properties. Collaborators have agreed to test useful constructions for research in living organisms. The complex photodynamic behavior of the molecule will be investigated. The molecule shows reversible and irreversible changes in the absorption spectrum depending on the wavelength of irradiation. It also shows on/off switching behavior under appropriate conditions and in the absence of oxygen, irreversibly becomes red fluorescent when irradiated. The structural basis for these four transformations will be determined on flash-frozen crystals by crystallographic techniques for potential biological and/or commercial uses.
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