Development of NMR as a tool for the structural and dynamic high-resolution characterization of phototransformable fluorescent proteins

The discovery of Phototransformable Fluorescent proteins (PTFPs) over the last decades has revolutionized the field of microscopy. Reversibly photo-switchable fluorescent proteins (RSFPs), in particular, are currently routinely used for Super Resolution Microscopy techniques, such as RESOLFT (Reversible Saturable Optical Fluorescence Transitions). Photo-induced switching between a fluorescent 'on'- and a dark 'off'-state, in combination with advanced illumination schemes has allowed for imaging nanometer sized compartments in biological cells. Crystallographic studies of such RSFPs have provided useful mechanistic explanations for their photophysical behaviour and has guided fluorescent protein engineering into designing better tags. However, the crystal forms of such proteins studied at cryogenic temperatures fail to capture dynamics present in RSFPs which could potentially play a significant role in their photophysics. So far, only a single NMR study for the RSFP Dronpa has been reported in the literature (Mizuno, 2008). During my PhD thesis, I was able to complement crystallographic studies of rsFolder, a green RSFP, with a dynamic perspective using multidimensional solution NMR spectroscopy. 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 NMR 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. (author)