NMR REVEALS NEW SECRETS OF FLUORESCENT PROTEINS USED IN SUPER-RESOLUTION MICROSCOPY
Photoconvertible fluorescent proteins (PCFPs) such as mEos4b change their fluorescence color from green to red upon illumination with UV light. They are popular markers for super-resolution imaging modalities such as quantitative and single particle tracking Single Molecule Localization Microscopy (SMLM). The photophysical properties of these proteins, however, are exceedingly complex. In this collaborative work involving the NMR and I2SR groups of the IBS, researchers observed by multidimensional NMR spectroscopy that mEos4b exhibits two distinct conformations in the green state that slowly interconvert. NMR also revealed that these conformations differ in the protonation states of two amino-acid side chains in the chromophore pocket, resulting in an altered hydrogen bond network. These subtle rearrangements were not visible in high-resolution x-ray structures of mEos4b. Importantly, only one of these conformations photoconverts efficiently to the red state, while the other one appears to be more susceptible to photobleaching. This study helps to explain the observed complex photophysical behavior of mEos4b and related PCFPs. More generally, it reveals how conformational dynamics of fluorescent proteins affect their photophysics, and in particular the photoconversion mechanisms of mEos-derived PCFPs. Finally, their results open the door for designing new PCFP variants with superior photoconversion efficiency.
Structural Heterogeneity in a Phototransformable Fluorescent Protein impacts its Photochemical Properties. Arijit Maity, Jip Wulffelé, Isabel Ayala, Adrien Favier, Virgile Adam, Dominique Bourgeois*, and Bernhard Brutscher*. Adv. Sci. (2023), 2306272 Doi : 10.1002/advs.202306272