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 from the phenolate to imidazolinone ring in the excited state of the chromophore 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 can involve vibrational relaxation without conformational change of the chromophore. In this case the nonradiative relaxation rate should follow the exponential “energy gap law”. If the twisted intramolecular charge transfer (TICT) governs nonradiative relaxation, strong local electric field directed from phenolate to imidazolinone can block this process resulting in high QY. 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. Finally, we apply this model to explain the changes of QY in red genetically encoded calcium indicators (GECIs) upon binding Ca2+ ions. Our model provides useful guidelines for engineering brighter RFP probes.

Role of local electric field in controlling fluorescence quantum yield of red fluorescent proteins

2019 Fall National Meeting

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