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The development of bright fluorescent proteins (FPs) emitting beyond 600 nm continues to be of interest both from a fundamental perspective in understanding protein-chromophore interactions and from a practical perspective as these FPs would be valuable for cellular imaging. We previously reported ultrafast spectral observations of the excited-state dynamics in mPlum resulting from interconversion between direct hydrogen bonding and water-mediated hydrogen bonding between the chromophore acylimine carbonyl and the Glu16 side chain. Here,
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