Light-InducedConformational Heterogeneity InducesPositive Photoswitching in Photoconvertible Fluorescent Proteins ofthe EosFP Family

Green-to-red photoconvertible fluorescent proteins (PCFPs) of the EosFP family are commonly used in ensemble pulse-chase and single-molecule localization or tracking approaches. However, these fluorescent proteins exhibit highly complex photophysical behaviors. In the green-form, recent NMR experiments revealed that mEos4b and other PCFP variants exist in two different conformational states at thermal equilibrium, which limits their effective photoconversion efficiency. Here, we investigate the conformational heterogeneity of mEos4b in the photoconverted red-form, employing a combination of solution NMR, UV–vis spectroscopy and fluorescence imaging. Only a single red population of mEos4b is observed at thermal equilibrium. However, a second population emerges under illumination with 405 or 488 nm light, which slowly decays in the dark or can be swiftly reverted under 561 nm light. This second population manifests itself through a pH-dependent positive photoswitching mechanism that adds to the already characterized negative photoswitching assigned to <i>cis</i>–<i>trans</i> isomerization of the chromophore. Our data indicate that positive photoswitching, instead, results from the light-induced formation of a second fluorescent state with a <i>cis</i> configuration of the chromophore that exhibits a substantially increased p<i>K</i>_a. Such a mechanism, suggested to result from rewiring of the H-bonding network around the first amino acid of the chromophore, adds to the panoply of switching scenarios observed in fluorescent proteins. It bears consequences for the spectroscopic characterization of PCFPs, reduces their apparent brightness and generates short-lived off-times perturbing single-molecule localization microscopy applications.