Reversibly photo-switchable fluorescent proteins (RSFPs), which can reversibly switch between a fluorescent and a non-fluorescent state upon illumination, are currently routinely used for Super Resolution Microscopy techniques. Despite the significant contribution of crystallographic studies to the understanding of RSFPs mechanistic properties and how these inform their photophysical behaviour, crystal forms of such proteins studied at cryogenic temperatures fail to capture potentially important dynamics present in RSFPs. During my PhD thesis, I used multidimensional solution NMR spectroscopy to add a dynamic perspective on the study of rsFolder, a green RSFP. Using a portable in-situ laser illumination device coupled with the NMR spectrometer, I was able to extract quantitative local dynamic information for both the fluorescent “on”- and “off”-states of rsFolder, characterized by a primarily cis and trans chromophore, respectively. NMR signatures of residues in the non-fluorescent “off”-state were identified using LASER-driven Exchange NMR experiments. The metastable photo-induced “off”- state of rsFolder appears more dynamic on the millisecond timescale than the fluorescent “on”-state. NMR investigations of the chromophore resulted in the deciphering of four configurations, populated in a pH-dependent fashion. Moreover, pH-induced cis-trans isomerization of the chromophore was observed, in the absence of light. NMR-derived values of activation energies for isomerization and free energy differences between the cis and trans chromophore enabled the mapping of the ground-state free energy landscape of rsFolder at different pH values and buffer compositions. Lastly, comparing NMR observables with optical measurements on rsFolder and mutants highlights the potential role that MR can play in the field of RSFP engineering. Altogether, my PhD work yielded in not only a reliable in-situ illumination set-up accompanied with relevant NMR experiments to study RSFPs, but also highlighted the importance of dynamics in understanding RSFPs’ photophysical properties.

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