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Role of local electric field in controlling fluorescence quantum yield of red fluorescence proteins (Conference Presentation)
Author(s): Mikhail Drobizhev; J. Nathan Scott; Patrik R. Callis; Rosana Molina; Thomas Hughes

Paper Abstract

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.

Paper Details

Date Published: 13 March 2019
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Proc. SPIE 10893, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI, 108930I (13 March 2019); doi: 10.1117/12.2510931
Show Author Affiliations
Mikhail Drobizhev, Montana State Univ. (United States)
J. Nathan Scott, Montana State Univ. (United States)
Patrik R. Callis, Montana State Univ. (United States)
Rosana Molina, Montana State Univ. (United States)
Thomas Hughes, Montana State Univ. (United States)


Published in SPIE Proceedings Vol. 10893:
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI
Samuel Achilefu; Ramesh Raghavachari, Editor(s)

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