Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching

Abstract

Recently, reversible photoswitching in bulk samples or in individual molecules of Dronpa, a mutant of a green fluorescent protein (GFP)-like fluorescent protein, has been demonstrated. Intense irradiation at 488 nm changed Dronpa in a dim protonated form, and weak irradiation at 405 nm restored it to the bright deprotonated form. Here, we report on the mechanism of photoswitching of Dronpa by means of ensemble and single-molecule spectroscopy. Ensemble spectroscopy shows that the photoswitching can be described, in first approximation, by a three-state model including a deprotonated (B), a protonated (A1), and a photoswitched protonated (A2) forms of the chromophore. While the B and the A1 forms are in a ground state acid-base equilibrium, the B and the A2 forms are reversibly photoswitched upon irradiation with 488 and 405 nm light. At the single-molecule level, the on-times in fluorescence intensity trajectories excited at 488 nm decrease with increasing the excitation power, consistent with the photoswitching from the B to A2 form. The on-times agree well with expected values, which are calculated based on the ensemble spectroscopic properties of Dronpa. The fluorescence trajectory obtained with simultaneous dual-color excitation at 488 and 405 nm demonstrates reversible photoswitching between the B and the A2 forms at the single-molecule level. The efficiency of the photoswitching from the A2 to B form increased with increasing the excitation power of the 405 nm light. Our results demonstrate that Dronpa holds its outstanding photoswitching properties, based on a photo-induced protonation/deprotonation process, even at the single-molecule level.

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

“…The switching mechanism of Skylan-NS is likely due to a cis/trans isomerization of the chromophore, accompanied by a change of the chromophoric protonation /deprotonation states (22)(23)(24). However, the precise mechanism linking its molecular structure to the preferred characteristics is unknown and needs to be further studied.…”

Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations

“…Rather, absorption of a violet photon may lead to deprotonation of the phenol oxygen (via excited-state proton transfer), favoring back-isomerization to the cis state, from which photoconversion to the red form may readily occur. The excited-state behavior of photoactivatable proteins is to a large extent determined by the protonation states of the chromophore and its environment (23,26). Therefore, more detailed investigations of these protonation states and their interplay with isomerization will be required to decipher the photoactivation mechanisms of IrisFP.…”

“…In the absence of light, the 4-(p-hydroxybenzylidene)-5-imidazolinone chromophores of Dronpa and mTFP0.7 assume a cis conformation, in which the anionic, fluorescent state of the chromophore is predominant at physiological pH. Light-induced off-switching of the fluorescence was proposed to arise from a cis-trans photoisomerization of the chromophore accompanied by a change of its protonation state (22)(23)(24), a loss of chromophore planarity and, for mTFP0.7, a more disordered chromophore structure in the trans conformation (9). Alternatively, the loss of fluorescence in the off state was recently explained by light-induced protonation of the Dronpa chromophore, associated with increased chromophore flexibility, which enhances nonradiative deactivation pathways (25).…”

Fluorescence nanoscopy. Methods and applications

“…PSmOrange and EosFP [138,144]). Photoswitching involves cis-trans isomerisation of the chromophore accompanied by a change of its protonation state (the anionic state being the fluorescent state and the protonated the nonfluorescent-interestingly, the chromophore can be fluorescent in both the cis and the trans conformations as long as the protein scaffold keeps it in a planar conformation) [145][146][147]. When kept in the dark, these proteins relax to the thermodynamically stable cis conformation within minutes to several hours; in thermal equilibrium, the photoswitchable proteins predominately populate the cis conformation with the chromophore in the anionic state.…”