Chemphyschem (2022) "Rational control of off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement"
Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its p-HBI chromophore, corresponding to the cis and trans isomers, with each one either protonated (neutral) or deprotonated (anionic) at the benzylidene ring. The relative populations and interconversion kinetics of these chromophore species depend on sample pH and buffer composition that alter in a complex way the strength of H-bonds that contribute in stabilizing the chromophore within the protein scaffold. We show in particular the important role of histidine-149 in stabilizing the neutral trans chromophore at intermediate pH values, leading to ground-state cis–trans isomerization with a peculiar pH dependence. We discuss the potential implications of our findings on the pH dependence of the photoswitching contrast, a critical parameter in nanoscopy applications.