In vivo imaging of single molecules, organelles, live cells, intact tissues and whole organisms using the green fluorescent protein (GFP) from the Aequorea jellyfish and its fluorescent homologues from Anthozoa corals has become an invaluable approach in cell biology, biotechnology, biomedical research and drug discovery. Photoactivatable, """"""""kindling"""""""" fluorescent proteins (termed KFPs) are a novel type of genetically encoded light-switchable reporters derived from Anthozoa GFP-like proteins. KFP turns from an absorbing but non-emitting chromoprotein (""""""""Off' state) to a fluorescent protein (""""""""On"""""""" state) upon an external photoactivation at one wavelength, and turns back into the chromoprotein after quenching at another wavelength. Transitions between """"""""Off' and """"""""On"""""""" states differing by about two orders of fluorescence intensity magnitude result from an intramolecular cis-trans isomerization of the KFP chromophore and can be modulated by its amino acid microenvironment. Depending on the light intensity and its duration the kindling may be reversible or irreversible. We have found that the photoconversion can also be caused by KFP conformational changes. This fact creates the possibility to construct KFP-based intracellular sensors, in which conformational changes of biochemically responsive protein domain(s) inserted into the KFP will directly affect the KFP conformation and, thus, the discrete """"""""On""""""""/""""""""Off' chromophore states. These KFP properties offer a number of unique features for engineering advanced biotechnological systems, such as """"""""pulse-chase"""""""" molecular labels and spatio-restricted fluorescence resonance energy transfer (FRET)- reporters for protein-protein interactions, and precisely localized intracellular biosensors for biomedical imaging in normal or diseased cells and tissues. We will create a set of fully-optimized monomeric multicolor KFPs with different ranges of photoconversion sensitivities and/or induced fluorescent state life-times, and use them for construction of several types of genetically encoded molecular biosensors suitable for live cell signaling applications and high-throughput screening of novel drugs. In-depth characterization of the light-induced photodynamic mechanisms of Anthozoa GFP-like proteins will be an additional benefit of the project.
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