Agathe Espagne

Excited-state relaxation dynamics of a PYP chromophore model in solution: influence of the thioester group

Abstract Cis–trans photoisomerization of a photoactive yellow protein chromophore model, the deprotonated trans S -phenyl thio- p -hydroxycinnamate, is studied in aqueous solution by subpicosecond transient absorption and gain spectroscopy. The excited-state deactivation is found to involve the formation, in 1.7 ps, of an intermediate state which decays in 2.8 ps. A persistent bleaching signal is observed at longer times indicating that the excited state not only relaxes to the ground state but also partly forms a stable photoproduct, possibly the cis isomer. This behavior is analogous to that of the native photoactive yellow protein.

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

Dronpa is a photochromic green fluorescent protein (GFP) homologue used as a probe in super-resolution 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 time scales 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 time scales 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 multiphoton 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 time scale, 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.

Very Fast Product Release and Catalytic Turnover of DNA Photolyase

and enters into the binding pocket of the enzyme, thus approaching the FAD cofactor (3.1 in CPD photolyase and even 2.7 in the closely related (6–4) photolyase.) Following excitation of FAD in its fully reduced state (FADH ), an electron is transferred to the CPD, thereby initiating the splitting of the dimer, and subsequently returned to the flavin. The overall rate krepair of these photochemical repair steps is ~10 s . Despite the fast repair reaction, the catalytic turnover number of photolyase under saturating continuous light has been reported to be only in the order of 0.1 to 1 s ; this suggests that exchange of repaired DNA (product) for damaged DNA (substrate) is slow and rate limiting. While substrate binding was found to be rather fast (k1~10 m 1 s 1 [12, ), the rate of product release (k2) has not been established as yet, and this step might well be rate limiting. Interestingly, in an X-ray diffraction experiment at 100 K, the restored pyrimidines remained in the binding pocket in close proximity to the flavin throughout data collection. Here, we accessed product-forsubstrate exchange directly with a time-resolved experiment based on the well-established 14, 15] quenching of FADH fluorescence by electron transfer to a T<>T–CPD present in the binding pocket. Surprisingly, following an intense repair flash, the flavin fluorescence did not recover immediately, but rather with a time constant of ~50 ms (at 10 8C). This observation suggests that the restored thymines act as electron acceptors and hence as fluorescence quenchers of nearby excited FADH ; the hitherto unobserved 50 ms kinetics then reflect product release from the binding pocket and set an upper limit of 2 10 s 1 to the catalytic turnover number of photolyase. To verify this prediction, we re-examined the turnover number of photolyase under strong continuous laser light and a high substrate concentration. A rate of 260 s 1 was observed, more than 100 times faster than previously reported. The quenching of FADH fluorescence by the CPD has been used for titration of binding processes by applying steadystate fluorescence spectroscopy. Time-resolved fluorescence studies revealed that the fluorescence decay of FADH accelerates from 1.4 ns in the absence of substrate to 160 ps when a CPD is bound. Based on this difference in lifetime, we designed an experiment of the pump–probe principle: a strong actinic flash is applied to repair the substrate bound at the ACHTUNGTRENNUNGenzyme’s active site, and a weak probe flash at variable time delay serves to read out the fluorescence intensity and kinetics as a measure for the occupation of the substrate binding pocket. To avoid complications due to the antenna chromophore present in photolyases, we used an apophotolyase from Anacystis nidulans that was overexpressed in E. coli and is devoid of the 8-HDF antenna chromophore. The substrate was a UV-irradiated dT18 oligonucleotide that contained on average six randomly distributed CPDs (see the Supporting Information for details on enzyme and substrate preparation). Note that our substrate is heterogeneous with respect to the number and distribution of the CPDs per strand. Presence of this substrate quenched the steady-state fluorescence of FADH in photolyase by 83 % (Figure 1, inset). Time-resolved FADH fluorescence traces induced by a weak probe flash of 100 ps duration are presented in the main panel of Figure 1. In the absence of substrate (thick black trace), the fluorescence had a lifetime of 1.3 ns (thin black line; see SupScheme 1. Schematic representation of the catalytic cycle of DNA photolyase.

Photoswitching Kinetics and Phase‐Sensitive Detection Add Discriminative Dimensions for Selective Fluorescence Imaging

Non-invasive separation-free protocols are attractive for analyzing complex mixtures. To increase selectivity, an analysis under kinetic control, through exploitation of the photochemical reactivity of labeling contrast agents, is described. The simple protocol is applied in optical fluorescence microscopy, where autofluorescence, light scattering, as well as spectral crowding presents limitations. Introduced herein is OPIOM (out-of-phase imaging after optical modulation), which exploits the rich kinetic signature of a photoswitching fluorescent probe to increase selectively and quantitatively its contrast. Filtering the specific contribution of the probe only requires phase-sensitive detection upon matching the photoswitching dynamics of the probe and the intensity and frequency of a modulated monochromatic light excitation. After in vitro validation, we applied OPIOM for selective imaging in mammalian cells and zebrafish, thus opening attractive perspectives for multiplexed observations in biological samples.

Ultrafast Dynamics of a Green Fluorescent Protein Chromophore Analogue: Competition between Excited-State Proton Transfer and Torsional Relaxation

The competition between excited-state proton transfer (ESPT) and torsion plays a central role in the photophysics of fluorescent proteins of the green fluorescent protein (GFP) family and their chromophores. Here, it was investigated in a single GFP chromophore analogue bearing o-hydroxy and p-diethylamino substituents, OHIM. The light-induced dynamics of OHIM was studied by femtosecond transient absorption spectroscopy, at different pH. We found that the photophysics of OHIM is determined by the electron-donating character of the diethylamino group: torsional relaxation dominates when the diethylamino group is neutral, whereas ultrafast ESPT followed by cis/trans isomerization and ground-state reprotonation are observed when the diethylamino group is protonated and therefore inactive as an electron donor.

Excited-state dynamics of the PYP chromophore in solution. Environment and structure effects

This chapter demonstrates that the photo-induced behavior of five analogues of the photoactive yellow protein (PYP) chromophore is strongly dependent on their electron donor–acceptor structure with the help of ultrafast spectroscopy experiments. Compounds for which the carbonyl group is substituted with a strong electron acceptor, in particular the thioester derivative that models the native chromophore of PYP, do not isomerize in aqueous and alcoholic solutions. Their relaxation pathway involves a new transient, the formation of which is sensitive to the polarity and viscosity of the solvent. Hypothetical relaxation mechanisms are discussed in the chapter. The preferred one involves formation of a spectroscopically detectable ground state with a 90o twisted conformation of the ethylenic bond. The effect of the solvent on the excited-state relaxation is analyzed to stress further the existence of different relaxation pathways in these compounds.

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.

Isomerization process in the native and denatured photoactive yellow protein probed by subpicosecond absorption spectroscopy.

The Photoactive Yellow Protein (PYP) is the blue-light photoreceptor that presumably mediates negative phototaxis of the purple bacterium Halorhodospira halophila. Its chromophore is the deprotonated trans-p-cowmaric acid covalently linked, via a thioester bond, to the unique cystein residue of the protein. Like for rhodopsins, the trans to cis isomerization of the chromophore is shown to be the first overall step of the PYP photo-cycle, but the reaction path that leads to the formation of the cis isomer is not clear. From time resolved spectroscopy measurements on native PYP in solution, it comes out that the excited state deactivation involves a series of fast events on the subpicosecond and picosecond timescales correlated to the chromophore reconfiguration. On the other hand, chromophore H-bonding to the nearest amino acids is shown to play a key role in the trans excited state decay kinetics. In an attempt to evaluate further the role of the mesoscopic environment in the photophysics of PYP, this chapter makes a comparative study of the native and denatured PYP. The excited-state relaxation path and kinetics are monitored by subpicosecond time-resolved absorption and gain spectroscopy.

Chapter 82 - Chemical structure effect on the excited-state relaxation dynamics of the PYP chromophore

In order to better understand the early photophysics of PYP, this chapter compares three model chromophores, the deprotonated trans-p-coumaxic acid (pCA 2- ) and its amide (pCM - ) and phenyl thioester (pCT - ) analogues, in aqueous solution. The excited-state relaxation dynamics is followed by subpicosecond transient absorption and gain spectroscopy. The excited-state deactivation pathway of PYP model chromophores is found to depend strongly on the substituent adjacent to the carbonyl group. The photoisomerization reaction of the deprotonated p-coumaric acid (pCA 2- ) and of its amide analogue (pCM - ) in solution does not show any spectroscopically detectable intermediate, which is quite different from PYP. On the contrary, the phenyl thioester derivative pCT - exhibits a photo-physical behavior in solution surprisingly close to that of the protein during its initial deactivation step. This chapter highlights the determining role of the thioester bond in the primary molecular events in PYP.