Rational Control of Off‐State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement**

Abstract

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 [a] Dr.

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism

“…The trans-PL and trans-TW species we observe here have been previously reported in room-temperature crystal structures of rsEGFP2 , and in cryostructures of Cl-rsEGFP2 (Table S3). Absorption spectra and quantum chemical calculations also point to the presence of these two forms in rsEGFP2 solutions . We expect that the ON-to-OFF reaction starting from the cis chromophore can therefore eventually result in either of these two trans conformations.…”

“…Further work, such as coherently controlling the photoisomerization and tracking its yield, would be required to investigate this. Interestingly, Adam and colleagues report that rsEGFP2 mutants in which the available binding pocket volume is decreased switch to their ON state much more efficiently than mutants where the available binding pocket volume is increased . It will be valuable to investigate whether a HT reaction pathway dictated by a volume-constraining protein cage is responsible for this increased switching capability and whether coherent CI crossing is important for the efficiency of photoactivation.…”

Light-Induced Forward and Reverse Intersystem Crossing in Green Fluorescent Proteins at Cryogenic Temperatures

“…Proteins were overexpressed and purified as previously described . A tiny fraction of a 2 μL drop of a 1:1 mixture of a 2 mg/mL (ca.…”

“…Proteins were overexpressed and purified as previously described. 22 A tiny fraction of a 2 μL drop of a 1:1 mixture of a 2 mg/mL (ca. 0.1 mM) protein solution (50 mM HEPES, pH 7.5) with glycerol (final glycerol concentration estimated to 50%) was placed in a crystallographic loop and mounted on the magnetic sample holder of our cryo-microspectrophotometer CAL(AI) 2 DOSCOPE.…”

A Photoswitchable Solvatochromic Dye for Probing Membrane Ordering by RESOLFT Super‐resolution Microscopy**

“…RESOLFT microscopy can be used for live-cell imaging without exogenous buffers, and with lower laser powers than the analogous depletion laser used in stimulated emission techniques (STED), which is a limiting factor in their application due to photobleaching. Switchable fluorescent proteins are commonly applied as imaging agents in RESOLFT microscopy, [37][38][39][40][41][42][43][44][45][46][47][48][49] however they suffer from poor photostability, low brightness, and the need to be introduced by genetic encoding. Switchable small-molecule dyes are a promising alternative to overcome these shortcomings, [50][51][52][53][54][55] and have the potential to incorporate environmental sensing capabilities alongside the photoswitching required for super-resolution imaging.…”