Maity Arijit, Glushonkov Oleksandr, Ayala Isabel, Tacnet Pascale, Wulffelé Jip, Frachet Philippe, Brutscher Bernhard, Bourgeois Dominique, Adam Virgile
Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, Grenoble, France.
Integrated Structural Biology Grenoble (ISBG), Université Grenoble Alpes, CNRS, CEA, EMBL, Grenoble, France.
Protein Sci. 2025 Aug;34(8):e70234. doi: 10.1002/pro.70234.
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.
荧光蛋白的成熟速度是一个关键参数，它会影响细胞亮度、有效标记效率以及荧光显微镜中的时间分辨率。用于脉冲追踪实验和超分辨率技术（如光激活定位显微镜（PALM）、单粒子追踪PALM（sptPALM）和最小荧光光子通量显微镜（MINFLUX））的绿到红光可转换荧光蛋白（PCFP）可能会因成熟缓慢而受到阻碍。我们系统地表征了大肠杆菌中mEos衍生的PCFP的成熟速度，发现与pcStar和mEosEM不同，mEos2、mEos3.1、mEos3.2和mEos4b等几种变体成熟极其缓慢。引人注目的是，这些PCFP中的氧化步骤很快，不是限速步骤。通过结构引导的诱变策略，我们开发了一种策略，可以将mEos4b长达一天的成熟时间缩短近两个数量级，而不会显著影响其在单分子成像条件下的分子亮度和光物理性能。
