Ultrafast light-induced response of photoactive yellow protein chromophore analogues

The fluorescence decays of several analogues of the photoactive yellow protein (PYP) chromophore in aqueous solution have been measured by femtosecond fluorescence up-conversion and the corresponding time-resolved fluorescence spectra have been reconstructed. The native chromophore of PYP is a thioester derivative of p-coumaric acid in its trans deprotonated form. Fluorescence kinetics are reported for a thioester phenyl analogue and for two analogues where the thioester group has been changed to amide and carboxylate groups. The kinetics are compared to those we previously reported for the analogues bearing ketone and ester groups. The fluorescence decays of the full series are found to lie in the 1–10 ps range depending on the electron-acceptor character of the substituent, in good agreement with the excited-state relaxation kinetics extracted from transient absorption measurements. Steady-state photolysis is also examined and found to depend strongly on the nature of the substituent. While it has been shown that the ultrafast light-induced response of the chromophore in PYP is controlled by the properties of the protein nanospace, the present results demonstrate that, in solution, the relaxation dynamics and pathway of the chromophore is controlled by its electron donor–acceptor structure: structures of stronger electron donor–acceptor character lead to faster decays and less photoisomerisation

Ionized aminohydroxycarbene and its isomers: relative stability and unimolecular reactivity

International audienceAb initio molecular orbital calculations at the G2(MP2,SVP) level have been employed to explore a large part of the [H-3, C, N, O](.+) potential energy surface. Ionized aminohydroxycarbene, NH2-C.+-OH, 1, is found to correspond to the global minimum of the surface. The other stable species are also unconventional structures: ion-neutral complexes OC . . . NH3+., 2 and CO . . . NH3+., 2', and the distonic ion, (H3N+CO)-O-., 3. The more classical structures [HCONH2](.+), 4, and [HC(OH)NH](.+), 5 are higher in energy. The heat of formation of the five radical cations have been determined using their atomization energies. The various isomerization reactions connecting 1-5 as well as their dissociation by H or CO losses have been theoretically investigated and compared with the available experimental data. (C) 2001 Elsevier Science B.V. All rights reserved

Photoisomérisation ultrarapide du chromophore et de la protéine jaune photoactive (effets de structure et d' environnement)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Real-Time Monitoring of Chromophore Isomerization and Deprotonation During the Photoactivation of the Fluorescent Protein Dronpa

International audienceDronpa is a GFP-related photochromic fluorescent protein used as probe in superresolution microscopy. It is known that the photochromic reaction involves cis/trans isomerization of the chromophore and protonation/deprotonation of its phenol group, but the sequence in time of the two steps and their characteristic timescales are still the subject of much debate. We report here a comprehensive UV-visible transient absorption spectroscopy study of the photoactivation mechanism of Dronpa, covering all relevant timescales from ~100 fs to milliseconds. The Dronpa-2 variant was also studied and showed the same behavior. By carefully controlling the excitation energy to avoid multi-photon processes, we could measure both the spectrum and the anisotropy of the first photoactivation intermediate. We show that the observed few nanometer blue-shift of this intermediate is characteristic for a neutral cis chromophore, and that its anisotropy of ~0.2 is in good agreement with the reorientation of the transition dipole moment expected upon isomerization. These data constitute the first clear evidence that trans→cis isomerization of the chromophore precedes its deprotonation and occurs on the picosecond timescale, concomitantly to the excited-state decay. We found the deprotonation step to follow in ~10 μs and lead directly from the neutral cis intermediate to the final state

Multiscale transient absorption study of the fluorescent protein Dreiklang and two point variants provides insight into photoswitching and nonproductive reaction pathways

International audienceDreiklang is a reversibly photoswitchable fluorescent protein used as a probe in advanced fluorescence imaging. It undergoes a unique and still poorly understood photoswitching mechanism based on the reversible addition of a water molecule to the chromophore. We report the first comprehensive study of the dynamics of this reaction by transient absorption spectroscopy from 100 fs to seconds in the original Dreiklang protein and two point variants. The picture that emerges from our work is that of a competition between photoswitching and nonproductive reaction pathways. We found that photoswitching had a low quantum yield of 0.4%. It involves electron transfer from a tyrosine residue (Tyr203) to the chromophore and is completed in 33 ns. Nonproductive deactivation pathways comprise recombination of a charge transfer intermediate, excited-state proton transfer from the chromophore to a histidine residue (His145), and decay to the ground state via micro-/ millisecond-lived intermediates

Dynamic contrast with reversibly photoswitchable fluorescent labels for imaging living cells

International audienc

Dynamic contrast for overcoming spectral interferences in fluorescence imaging

International audienc

Ultrafast Photoisomerization of Photoactive Yellow Protein Chromophore Analogues in Solution: Influence of the Protonation State

We investigate solvent viscosity and polarity effects on the photoisomerization of the protonated and deprotonated forms of two analogues of the photoactive yellow protein (PYP) chromophore. These are trans-p-hydroxybenzylidene acetone and trans-p-hydroxyphenyl cinnamate, studied in solutions of different polarity and viscosity at room temperature, by means of femtosecond fluorescence up-conversion. The fluorescence lifetimes of the protonated forms are found to be barely sensitive to solvent viscosity, and to increase with increasing solvent polarity. In contrast, the fluorescence decays of the deprotonated forms are significantly slowed down in viscous media and accelerated in polar solvents. These results elucidate the dramatic influence of the protonation state of the PYP chromophore analogues on their photoinduced dynamics. The viscosity and polarity effects are, respectively, interpreted in terms of different isomerization coordinates and charge redistribution in S_1. A trans-to-cis isomerization mechanism involving mainly the ethylenic double-bond torsion and/or solvation is proposed for the anionic forms, whereas “concerted” intramolecular motions are proposed for the neutral forms

DNA Repair by Photolyase: A novel substrate with low background absorption around 265 nm for transient absorption studies in the UV

International audienceCPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH(-)) cofactor functions its photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present it novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 run. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH(-)). Flash-induced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase

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

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