Decoding mEos4b day‐long maturation and engineering fast‐maturing variants

The maturation speed of fluorescent proteins is a crucial parameter that influences cellular brightness, effective labeling efficiency, and temporal resolution in fluorescence microscopy. Green-to-red photoconvertible fluorescent proteins (PCFPs) used in pulse-chase experiments and super-resolution techniques such as Photoactivated Localization Microscopy (PALM), single-particle-tracking PALM (sptPALM), and Minimal Fluorescence Photon Fluxes Microscopy (MINFLUX) may be hampered by slow maturation. We systematically characterized the maturation speed of mEos-derived PCFPs in Escherichia coli and found that, in contrast to pcStar and mEosEM, several variants such as mEos2, mEos3.1, mEos3.2, and mEos4b mature extremely slowly. Strikingly, the oxidation step in these PCFPs is fast and not rate-limiting. Through a structure-guided mutagenesis strategy, we developed a strategy to reduce the day-long maturation time of mEos4b by nearly two orders of magnitude without significantly impacting its molecular brightness and photophysical performance under single-molecule imaging conditions.