Light-Induced Conformational Heterogeneity Induces Positive Photoswitching in Photoconvertible Fluorescent Proteins of the EosFP Family
Year: 2025
Type: article
Abstract: 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 cis – trans isomerization of the chromophore. Our data indicate that positive photoswitching, instead, results from the light-induced formation of a second fluorescent state with a cis configuration of the chromophore that exhibits a substantially increased p K 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.
DOI: 10.1021/jacs.4c17311
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: green
Awards: CBH-EUR-GS, NODES: Nanoclusters and Oligomers dynamics for Decision-making and intracellular Signaling rewiring, Photoswitching mechanisms and environmental dependencies of fluorescent protein markers, Infrastructure for transnational access and discovery in structural biology, Infrastructure for transnational access and discovery in structural biology, Next generation of ultrastable fluorescent proteins for quantitative super-resolution microscopy, Infrastructure Française pour la Biologie Structurale Intégrée
OpenAlex ID: W4408444547