DEVELOPMENT OF "FRET-CAPTURE": A technique that exploits environmental-sensitive fluorophores as FRET donors to probe intermolecular protein-protein interactions and protein dynamics

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Fluorescence spectroscopy has become one of the most widely used tools for the development of new probes for ions, small molecules, and monitoring biological processes, such as protein folding, intracellular biomolecule distribution, protein-protein interactions, and phosphorylation events. An ideal approach for addressing these questions is the use of Förster resonance energy transfer (FRET), which is as one of the preferred methods for in cellulo studies. However, sometimes structural changes are too subtle to be detected with FRET. An alternative approach is the use of solvatochromic fluorophores which are a special class of molecules having spectroscopic properties that are dependent on the physicochemical properties of their environment. The group of Prof. Imperiali has developed new solvatochromic fluorophores as a powerful tool for the study of dynamic protein interactions. In this proposal the development of a fluorescence-based approach (FRET-CAPTURE) combining the principle of the solvatochromic fluorophores to induce energy transfer processes is described that could use the high apparent stokes shift of the FRET fluorescence for measurements with a high biological fluorescence background in living cells and could overcome the limitation of FRET.The initial model system for the methodology development will be based on the constructs that involve the calmodulin and the M13 peptide. Therefore several acceptor-donor combinations will be tested. Using the donor-acceptor combination showing the highest fluorescence increase for the FRET-CAPTURE signal it is planed to incorporate this tool in developing applications of the methodology for sensing various cytokines including EGF and TNF for application in 3D matrices.