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, we 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, our results open the door for designing new PCFP variants with superior photoconversion efficiency.
Publication: Maity et al. Adv. Sci. 2023, 2306272, doi: 10.1002/advs.202306272
Collaboration: D. Bourgeois (IBS)
Conformational heterogeneity in mEos4b affects its photophyscial properties (2023)
mEOS4b_Green_heterogeneity