Reversibly photo-switchable fluorescent proteins (RSFPs) are important tools for microscopy and other biotechnological applications. They are currently routinely used for Near-Field Super Resolution Microscopy techniques, e.g. RESOLFT (1). Their characteristic switching between a fluorescent “on” state and a non-fluorescent “off” state, combined with signal processing algorithms has allowed to improve the resolution of cellular imaging down to of a few nanometers. Crystallographic studies of such RSFPs have provided crucial insights on their atomic structure, that has guided the field of fluorescent protein engineering in the search for better tags. However, the crystal forms of such proteins studied at cryogenic temperatures does not provide a realistic picture of the conformational dynamics, and how they influence the photophysics of the RSFP. So far, only a single NMR study for the RSFP Dronpa has been reported in the literature (2). Here, we present a comprehensive NMR study of rsFolder, a green negative RSFP (3). Using a portable in-situ laser illumination device, we were able to perform NMR assignments and derive local dynamic information for both, the fluorescent “on” and “off” states of rsFolder, aided by Laser-driven Exchange NMR experiments. After photo-switching, rsFolder experiences significantly enhanced millisecond time scale dynamics, affecting mostly the environment of the chromophore as detected by extensive line broadening of backbone and side chain resonances. H/D exchange measurements also revealed a global destabilization of the β-barrel region facing the phenol ring of the endogenous chromophore, as well as the chromophore-connecting helical structures. Single-point mutants of rsFolder have been used in an attempt to make correlations between the NMR-observed features and the different photophysical properties of these mutant RSFPs.
(1) Hell, S.W., 2003. Nat.Biotechnol. 21, 1347–1355.
(2) Mizuno, H. et al, 2010. J.Biomol.NMR. 48, 237-246.
(3) El Khatib, M. et al, 2016. Sci.Reports. 6:18459(1-12)