NMR reveals light-induced changes in the dynamics of a photoswitchable fluorescent protein
The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature, and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in-situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a non-fluorescent state. Our results add a dynamic view to the static structures obtained by X-ray crystallography. Including NMR into the analytical toolbox used for fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins, and their correlation with the observed photochemical and photophysical properties.\n\nSignificancePhoto-transformable Fluorescent Proteins (PTFPs) are essential tools for super-resolution (SR) microscopy. In practical applications, however, researchers often encounter problems when using PTFPs in a particular cellular context, because the environmental conditions (pH, temperature, redox potential, oxygen level, viscosity, ...) affect their brightness, photostability, phototransformation kinetics, etc. Rational fluorescent protein engineering exploits the mechanistic information available from structural studies, mainly X-ray crystallography, in order to design new PTFP variants with improved properties for particular applications. Here we apply NMR spectroscopy in solution to investigate the light-induced changes in conformational dynamics of rsFolder, a reversibly switchable fluorescent protein. The dynamic view offered by NMR highlights protein regions that comprise potentially interesting mutation points for future mutagenesis campaigns.