Abstract
Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single-particle-tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side-chain protonation of the conserved residues E212 and H62, altering the hydrogen-bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green-to-Red photoconversion properties differ between the A and B states. The chromophore in the A-state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B-state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.