Laboratoire P.A.S.T.E.U.R

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On the Mass of Atoms in Molecules: Beyond the Born-Oppenheimer Approximation

PHYSICAL REVIEW X 25 août 2017

Describing the dynamics of nuclei in molecules requires a potential energy surface, which is traditionally provided by the Born-Oppenheimer or adiabatic approximation. However, we also need to assign masses to the nuclei. There, the Born-Oppenheimer picture does not account for the inertia of the electrons, and only bare nuclear masses are considered. Nowadays, experimental accuracy challenges the theoretical predictions of rotational and vibrational spectra and requires the participation of electrons in the internal motion of the molecule. More than 80 years after the original work of Born and Oppenheimer, this issue has still not been solved, in general. Here, we present a theoretical and numerical framework to address this problem in a general and rigorous way. Starting from the exact factorization of the electron-nuclear wave function, we include electronic effects beyond the Born-Oppenheimer regime in a perturbative way via positiondependent corrections to the bare nuclear masses. 

Loss of fourth electron-transferring tryptophan in animal (6–4) photolyase impairs DNA repair activity in bacterial cells.

(6–4) photolyases ((6–4)PLs) are flavoproteins that use blue light to repair the UV-induced pyrimidine(6–4)pyrimidone photoproduct in DNA. Their FAD cofactor can be reduced to its repair-active FADH form by a photoinduced electron transfer reaction. In animal (6–4)PLs, a chain of four Trp residues was suggested to be involved in a step-wise transfer of an oxidation hole from the flavin to the surface of the protein. Here, we investigated the effect of mutation of the fourth Trp on the DNA photorepair activity of Xenopus laevis (6–4)PL (Xl64) in bacterial cells. The photoreduction and photorepair properties of this mutant protein were independently characterized in vitro. Our results demonstrate that the mutation of the fourth Trp in Xl64 drastically impairs the DNA repair activity in cells, and that this effect is due to the inhibition of the photoreduction process. We thereby show that the photoreductive formation of FADH through the Trp tetrad is essential for the biological function of the animal (6–4)PL. The role of the Trp cascade, and of the fourth Trp in particular, are discussed.


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

Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) photolyases (repairing pyrimidine-pyrimidone (6–4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues. Using femtosecond polarized transient absorption spectroscopy, we performed a detailed analysis of the photoactivation reaction in the (6-4) photolyase of Xenopus laevis with oxidized FAD. We showed that the excited flavin is very quickly reduced (~0.5 ps) by a nearby tryptophan residue, yielding FAD●– and WH●+ radicals. Subsequent kinetic steps in the picosecond regime were assigned to the migration of the positive charge along the Trp tetrad, in competition with charge recombination. We propose that the positive charge is actually delocalized over various Trp residues during most of the dynamics and that charge recombination essentially occurs through the proximal tryptophanyl radical. Oxidation of the fourth tryptophan is thought to be reached about as fast as that of the third one (~40 ps), based on a comparison with a mutant protein lacking the distal Trp, implying ultrafast electron transfer between these two residues. This unusual mechanism sheds light on the rich diversity of electron transfer pathways found in various photolyases, and evolution-related cryptochromes alike.


Dynamic multicolor protein labeling in living cells

Chem. Sci.2017, Advance Article 


Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST, hereafter called FAST) is a 14-kDa protein tag giving a bright green-yellow fluorescent complex upon interaction with the fluorogenic dye 4-hydroxy-3-methylbenzylidene rhodanine (HMBR). Here, we report a collection of fluorogens enabling to tune the fluorescence color of FAST from greenyellow to orange and red. Beyond allowing multicolor imaging of FAST-tagged proteins in live cells, these fluorogens enable dynamic color switching because of FAST’s reversible labeling. This unprecedented behavior allows selective detection of FAST-tagged proteins in cells expressing both green and red fluorescent species through two-color crosscorrelation, opening exciting prospects to overcome spectral crowding and push the frontiers of multiplexed imaging.

Intramolecularly Protein-Crosslinked DNA Gels: New Biohybrid Nanomaterials with Controllable Size and Catalytic Activity

Small 2017, 1700706


DNA micro- and nanogels—small-sized hydrogels made of a crosslinked DNA backbone—constitute new promising materials, but their functions have mainly been limited to those brought by DNA. Here a new way is described to prepare submicrometer-sized DNA gels of controllable crosslinking density that are able to embed novel functions, such as an enzymatic activity. It consists of using proteins, instead of traditional base-pairing assembly or covalent approaches, to form crosslinks inside individual DNA molecules, resulting in structures referred to as intramolecularly protein-crosslinked DNA gels (IPDGs). It is first shown that the addition of streptavidin to biotinylated T4DNA results in the successful formation of thermally stable IPDGs with a controllable crosslinking density, forming structures ranging from elongated to raspberry-shaped and pearl-necklace-like morphologies. Using reversible DNA condensation strategies, this paper shows that the gels can be reversibly actuated at a low crosslinking density, or further stabilized when they are highly crosslinked. Finally, by using streptavidin–protein conjugates, IPDGs with various enzymes are successfully functionalized. It is demonstrated that the enzymes keep their catalytic activity upon their incorporation into the gels, opening perspectives ranging from biotechnologies (e.g., enzyme manipulation) to nanomedicine (e.g., vectorization).