Unraveling the Reaction Mechanisms of Photoactive Proteins

 
Members involved:
Nadia DOZOVA, Agathe ESPAGNE, Fabien LACOMBAT, Lucie LUDVIKOVA, Pascal PLAZA, Emilie RENOUARD


(See our publications below)

Using femtosecond transient absorption spectroscopy as a tool, we aim at understanding the photoinduced dynamics and activation mechanisms of different types of photoactive proteins, either involved in natural biological functions or used in advanced imaging applications.
We are interested in the photolyases and cryptochromes superfamily (PCSf) of light-driven flavoproteins, either involved in DNA photorepair or signaling, and particularly in genetically divergent and/or bifunctional members of the PCSf. These « non-canonical » proteins are characterized by marked structural differences at the level of DNA recognition, electron transfer pathways and nature of the secondary cofactors. Our goal is to unravel their reaction mechanisms, in particular the photoreduction of their flavin cofactor (FAD), used either to activate DNA photorepair in photolyases or trigger signaling in cryptochromes.
In close thematic connection, we are studying other types of flavoprotein, in particular involved in post-transcriptional chemical modifications of tRNA, like the methyltransferase TrmFO. The excited-state dynamics of the flavin, key actor of the enzymatic activity, is here used as a reporter of its interactions with the local environment.

We are in addition interested in the photoreactivity of fluorescent proteins (FPs) of the Green Fluorescent Protein (GFP) family. FPs are ubiquitous tools in biological imaging, where they are used as genetically-encoded fluorescent markers. They are however not simple molecular beacons: they exhibit a variety of photochemical reactions, the mechanisms of which are only partially understood. We are more particularly studying the subclass of reversibly photoswitchable fluorescent proteins (RSFPs). According to structural studies, photoswitching involves either chromophore cis/trans isomerization coupled to proton transfer, or chromophore hydration. Our goal is to build a full picture of the photoswitching mechanisms and dynamics, from femtoseconds to hours. This fundamental work is exploited in the context of the development by colleagues from our pole of new protocols of dynamic contrast imaging.


Experimental approach
:

Our main tool is a homemade femtosecond transient absorption spectrometer, implementing a pump-continuum-probe configuration. The laser source is a Ti:Sapphire system from Spectra-Physics (Tsunami oscillator and Spitfire regenerative amplifier). It delivers 50-fs ~0.7 mJ pulses at 1 kHz around 800 nm. Tunable pump pulses in the visible are produced by a noncollinear optical parametric amplifier (NOPA, Clark-MXR). A broadband continuum probe is obtained by self-phase modulation of a 800-nm beam in a CaF2 plate. Its time delay with respect to the pump is adjusted in a motorized optical delay line. The pump and probe beams are focused with parabolic mirrors and overlapped in the sample, kept in a 1-mm cell. The sample may be constantly displaced in two directions to avoid the re-excitation of a previously excited area. The sample may also be thermostated with the help of a water bath circulated through the sample holder. The time resolution of our setup is of the order of 100 fs and it allows broadband probing over the entire visible region (~330 to 750 nm). Measurements with probe polarized parallel and perpendicular to the polarization of the pump give access to transient absorption anisotropy.

The same femtosecond laser source as above is used to power a commercial fluorescence up-conversion spectrometer (FOG100-DA, CDP Systems), operating à 1 kHz. In brief, the pump-induced fluorescence is collected and mixed with an ultrashort pulse (the gate) at 800 nm in a nonlinear optical crystal (BBO). An up-converted fluorescence beam is generated when the gate and fluorescence pulses temporally overlap. Fig 2 gives a schematic view of the setup. Its time resolution of the order of 200 fs. Spectral resolution is achieved by tuning the phase-matching condition of the nonlinear crystal.


Our publications
:

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2019

Photosensitized oxidative addition to gold(i) enables alkynylative cyclization of o-alkylnylphenols with iodoalkynes Article de journal

Zhonghua Xia; Vincent Corcé; Fen Zhao; Cédric Przybylski; Agathe Espagne; Ludovic Jullien; Thomas Le Saux; Yves Gimbert; Héloïse Dossmann; Virginie Mouriès-Mansuy; Cyril Ollivier; Louis Fensterbank

Nature Chemistry, 11 (9), p. 797-805, 2019, ISSN: 1755-4349.

Liens

Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome Article de journal

Fabien Lacombat; Agathe Espagne; Nadia Dozova; Pascal Plaza; Pavel Müller; Klaus Brettel; Sophie Franz-Badur; Lars-Oliver Essen

Journal of the American Chemical Society, 141 (34), p. 13394-13409, 2019, ISSN: 0002-7863.

Résumé | Liens

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

N Dozova; F Lacombat; C Bou-Nader; D Hamdane; P Plaza

Physical Chemistry Chemical Physics, 21 (17), p. 8743-8756, 2019, ISSN: 1463-9076.

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2018

A novel diarylethene-based photoswitchable chelator for reversible release and capture of Ca2+ in aqueous media Article de journal

N Dozova; G Pousse; B Barnych; J -M Mallet; J Cossy; B Valeur; P Plaza

Journal of Photochemistry and Photobiology A: Chemistry, 360 , p. 181–187, 2018.

Résumé | Liens

Delocalized hole transport coupled to sub-ns tryptophanyl deprotonation promotes photoreduction of class II photolyases Article de journal

F Lacombat; A Espagne; N Dozova; P Plaza; E Ignatz; S Kiontke; L O Essen

Physical Chemistry Chemical Physics, 20 (39), p. 25446-25457, 2018, ISSN: 1463-9076.

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Macroscale fluorescence imaging against autofluorescence under ambient light Article de journal

R Zhang; R Chouket; M -A Plamont; Z Kelemen; A Espagne; A G Tebo; A Gautier; L Gissot; J -D Faure; L Jullien; V Croquette; T Le Saux

Light: Science and Applications, 7 (1), 2018.

Résumé | Liens

2017

Loss of Fourth Electron-Transferring Tryptophan in Animal (6-4) Photolyase Impairs DNA Repair Activity in Bacterial Cells Article de journal

J Yamamoto; K Shimizu; T Kanda; Y Hosokawa; S Iwai; P Plaza; P Müller

Biochemistry, 56 (40), p. 5356–5364, 2017.

Résumé | Liens

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

F Lacombat; P Plaza; M -A Plamont; A Espagne

Journal of Physical Chemistry Letters, 8 (7), p. 1489–1495, 2017.

Résumé | Liens

Repair of (6-4) Lesions in DNA by (6-4) Photolyase: 20 Years of Quest for the Photoreaction Mechanism Article de journal

J Yamamoto; P Plaza; K Brettel

Photochemistry and Photobiology, 93 (1), p. 51–66, 2017.

Résumé | Liens

Ultrafast flavin photoreduction in an oxidized animal (6-4) photolyase through an unconventional tryptophan tetrad Article de journal

P R Martin; F Lacombat; A Espagne; N Dozova; P Plaza; J Yamamoto; P Müller; K Brettel; A De La Lande

Physical Chemistry Chemical Physics, 19 (36), p. 24493–24504, 2017.

Résumé | Liens

2016

Excited-state symmetry breaking of linear quadrupolar chromophores: A transient absorption study Article de journal

N Dozova; L Ventelon; G Clermont; M Blanchard-Desce; P Plaza

Chemical Physics Letters, 664 , p. 56–62, 2016.

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

T Chatterjee; F Lacombat; D Yadav; M Mandal; P Plaza; A Espagne; P K Mandal

Journal of Physical Chemistry B, 120 (36), p. 9716–9722, 2016.

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2015

Discovery and functional analysis of a 4th electron-transferring tryptophan conserved exclusively in animal cryptochromes and (6-4) photolyases Article de journal

P Müller; J Yamamoto; P R Martin; S Iwai; K Brettel

Chemical Communications, 51 (85), p. 15502-15505, 2015, ISSN: 1359-7345.

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Photo-induced cation translocation in a molecular shuttle based on a calix[4]-biscrown including DCM and DMABN chromophores Article de journal

N Dozova; R Kumar; T Pradhan; F Lacombat; B Valeur; J S Kim; P Plaza

Chemical Communications, 51 (80), p. 14859–14861, 2015.

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Photoswitching Kinetics and Phase-Sensitive Detection Add Discriminative Dimensions for Selective Fluorescence Imaging Article de journal

Jérôme Querard; Tal-Zvi Markus; Marie-Aude Plamont; Carole Gauron; Pengcheng Wang; Agathe Espagne; Michel Volovitch; Sophie Vriz; Vincent Croquette; Arnaud Gautier; Thomas Le Saux; Ludovic Jullien

Angewandte Chemie International Edition, 54 (9), p. 2633-2637, 2015, ISSN: 1433-7851.

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