Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration

Abstract

Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the first time-resolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the picosecond-nanosecond regime that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule.

Interplay Between Locally Excited and Charge Transfer States Governs the Photoswitching Mechanism in Fluorescent Protein Dreiklang

“…However, in contrast to many other FPs, the excitation of peak A leads to very weak (albeit non-negligible 10 ) fluorescence. This weak steady-state fluorescence is identical 10 to the fluorescence produced by excitation of peak B. These observations suggest that ESPT in Dreiklang is strongly suppressed and happens with a very small quantum yield.…”

“…Fig. 2 shows absorption spectra of the ON-and OFF-forms of Dreiklang 7,10 . The absorption spectrum of the ON-form features two bands: one at ∼ 3.01 eV (411-413 nm) and a twice more intense one at 2.43 eV (511 nm), with a shoulder at 2.58 eV (480 nm).…”

Ultrafast photoinduced flavin dynamics in the unusual active site of the tRNA methyltransferase TrmFO

“…In turn, c* was estimated to 10.9 µM using the transient spectra of the [Ru(bpy) 3 ] 2+ complex recorded under the same conditions, as previously detailed. 63 The obtained spectrum (blue line in Figure 7; called for short RP 3 ) should of course be taken with some care but we note that changing c rp by ±20% did not alter much the overall shape of the spectrum. Two bands may be seen: the one peaking at 377 nm is assigned to FAD •and the one at 445 nm to Y 346 OH •+ .…”

Computational Modeling Reveals the Mechanism of Fluorescent State Recovery in the Reversibly Photoswitchable Protein Dreiklang

“…2)is located in the cavity sufficiently close to the carbon atom C65 (3.12 Å in the model, 3.38 Å in the crystal) to form the hydrated chromophore moiety. While simulations of the photo-induced chemical reaction of formation of the hydrated chromophore are beyond the scope of this study, we simply point out that our model system for the ON-state (the neutral Chro, deprotonated His145 and protonated Glu222) does not contradict the early mechanistic hypotheses 6.…”

“…In this subsection, we validate our models against the available experimental data. 3,6 The original experimental study 3…”