Intracellular fluorescent probes for chemical processes in single living cells

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For nondestructive analysis of chemical processes in living cells, we have developed some general methods and new intracellular fluorescent probes for detecting 1) second messengers, cGMP, diacylglycerol and phosphatidylinositol-3,4,5-triphosphate, 2) protein phosphorylation, 3) protein confbrmational changes, 4) protein-protein interactions, and 5) protein localizations in live cells under a confocal laser microscope.The approaches for the present probe developments are use of fluorescence resonance energy transfer for reporting binding of substrates (analytes) to molecular recognition domains in dual-fluorophore conjugated probe molecules, and use of protein splicing chemistry for detecting protein-protein interactions.Key molecules and steps of cellular signaling pathways were visualized in relevant live cells using developed fluorescent probe molecules. These probes were found of general importance not only for fundamental biology studies, but also for assay and screening methods for chemicals that inhibit or facilitate cellular signaling pathways.