RMN study of rsFolder (2021)

Zooming into the chromophore environment of a fluorescent protein with solution NMR spectroscopy

Fluorescent proteins of the GFP family that change their fluorescent state upon illumination at specific wavelengths are widely used markers for super-resolution imaging modalities. The photophysical properties of these proteins, however, crucially depend on the environmental conditions in which they are expressed and used. Currently, strategies based on rational design to improve fluorescent proteins mainly exploit the mechanistic information available from X-ray crystallographic structures that lack important information on conformational dynamics, protonation states, and hydrogen-bonding, as well as their dependence on physicochemical conditions. In this collaborative work involving the NMR and I2SR groups, we focused on rsFolder, a reversibly switchable green fluorescent protein that has been designed at IBS. We demonstrated that solution NMR spectroscopy can detect subtle changes in the chromophore environment with atomic resolution, providing new insights into the pH-dependence of the observed photo-switching properties of rsFolder. Our results can be exploited to design and test new FP variants with higher robustness towards environmental changes. This work also introduces NMR spectroscopy into the field of fluorescent protein research as a new tool to probe chromophore state populations and dynamics as a function of a variety of environmental conditions.

Disentangling chromophore states in a reversibly switchable green fluorescent protein : mechanistic insights from NMR spectroscopy. Christou NE, Giandoreggio-Barranco K, Ayala I, Adam V, Bourgeois D, Brutscher B. Journal of the American Chemical Society 2021, 143, 19, 7521–7530 DOI : 10.1021/jacs.1c02442

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