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13 March 2019 Role of local electric field in controlling fluorescence quantum yield of red fluorescence proteins (Conference Presentation)
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Red fluorescent proteins (RFPs) and genetically encoded biosensors built upon them present additional imaging wavelengths and can report from deeper layers of tissues compared to green fluorescent proteins (GFPs). However, the fluorescence quantum yield (QY) of the popular red variants such as mCherry and mPlum are low (0.1 – 0.2). Same is true for a number of red genetically encoded calcium indicators (GECIs) even in their bright state. The theoretically proposed physical mechanism of nonradiative relaxation involves intramolecular charge transfer (CT) from the phenolate to imidazolinone ring in the excited state that is coupled to the chromophore twisting about the methine bridge bonds. Such twisting can result in transition (hopping) between potential energy surfaces of the excited state and the ground state at their conical intersection. An alternative pathway (supported by our MD simulations) can involve accidental drops of the CT dark excited state (S2) energy below the bright S1 state in the course of temporal fluctuations of the protein surrounding. If the CT state governs nonradiative relaxation, strong local electric field directed from phenolate to imidazolinone can block this process resulting in the QY increase. We use two-photon spectroscopy to evaluate the components of the protein electric field in the plane of the RFP chromophore and demonstrate that in a series of RFPs nonradiative relaxation rate is correlated with the field component along the long chromophore axis. This provides useful guidelines for engineering brighter RFP probes.
Conference Presentation
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Mikhail Drobizhev, J. Nathan Scott, Patrik R. Callis, Rosana Molina, and Thomas Hughes "Role of local electric field in controlling fluorescence quantum yield of red fluorescence proteins (Conference Presentation)", Proc. SPIE 10893, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI, 108930I (13 March 2019);

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