Photoconvertible fluorescent proteins (PCFPs) like mEos4b shift their fluorescence emission from green to red upon 405 nm illumination, making them essential markers for super-resolution techniques such as Single Molecule Localization Microscopy (SMLM). However, their photophysical properties continue to reveal surprises. Besides photoconversion, PCFPs can reversibly switch between fluorescent and nonfluorescent states. In its red form, mEos4b undergoes “negative switching” : it turns off under 561 nm light and reactivates under 405 nm light due to cis-trans isomerization of the chromophore.
In this collaboration between the I2SR and NMR groups of IBS, and the SyMMES laboratory, researchers identified a new, additional, “positive switching” mode : 405 nm light turns off red mEos4b, while 561 nm light switches it back on—the inverse of negative switching. Using UV-vis spectroscopy, fluorescence imaging, NMR, and EPR, they found that this arises from light-induced conformational heterogeneity. The red form of mEos4b exists in two fluorescent states, differing in their H-bonding networks around the chromophore. Under 405 nm light, the less fluorescent state builds up, while 561 nm light or darkness favors the brighter state. Notably, the less fluorescent state has a higher pKa, making it nearly dark and long-lived at physiological or low pH.
This positive switching leads to blinking in SMLM, highlighting how subtle light-induced conformational shifts, often undetectable in crystallography, profoundly impact single-molecule imaging.
Light-Induced Conformational Heterogeneity Induces Positive Photoswitching in Photoconvertible Fluorescent Proteins of the EosFP Family. Wulffelé J, Maity A, Ayala I, Gambarelli S, Brutscher B, Bourgeois D. J Am Chem Soc. 2025 ; 147(12):10357-10368. doi : 10.1021/jacs.4c17311.
Contact : Dominique Bourgeois (IBS/Integrated Imaging of Stress Response Group)