Dihydroxylation of four- and five-ring aromatic hydrocarbons by the naphthalene dioxygenase from Sphingomonas CHY-1

International audienceThe naphthalene dioxygenase from Sphingomonas CHY-1 exhibits extremely broad substrate specificity toward polycyclic aromatic hydrocarbons (PAHs). In a previous study, the catalytic rates of oxidation of 9 PAHs were determined using the purified dioxygenase, but the oxidation products formed from 4- to 5-ring hydrocarbons were incompletely characterized. Here, we reexamined PAH oxygenation reactions using Escherichia coli recombinant cells overproducing strain CHY-1 dioxygenase. Hydroxylated products generated by the dioxygenase were purified, and characterized by means of GC-MS, UV absorbance as well as 1H and 13C-NMR spectroscopy. Fluoranthene was converted to 3 dihydrodiols, the most abundant of which was identified as cis-7,8-dihydroxy-7,8-dihydrofluoranthene. This diol turned out to be highly unstable, converting to 8-hydroxyfluoranthene by spontaneous dehydration. The dioxygenase also catalyzed dihydroxylations on the C2-C3, and presumably the C1-C2 positions, although at much lower rates. Benz[a]anthracene was converted into three dihydrodiols, hydroxylated in positions C1-C2, C8-C9 and C10-C11, and one bis-cis-dihydrodiol. The latter compound was identified as cis,cis-1,2,10,11-tetrahydroxy-1,2,10,11-tetrahydrobenz[a]anthracene, which resulted from the subsequent dioxygenation of the 1,2- or 10,11-dihydrodiols. Chrysene dioxygenation yielded a single diol identified as cis-3,4-dihydroxy-3,4-dihydrochrysene, which underwent further oxidation to give cis,cis-3,4,9,10 chrysene tetraol. Pyrene was a poor substrate for the CHY-1 dioxygenase and gave a single dihydrodiol hydroxylated on C4 and C5, whereas benzo[a}pyrene was converted to two dihydrodiols, one of which was identified as cis-9,10-dihydrodiol. The selectivity of the dioxygenase is discussed in the light of the known 3D structure of its catalytic component, and compared to that of the few enzymes able to attack 4- and 5-ring PAHs

Quantum entanglement with acousto-optic modulators: 2-photon beatings and Bell experiments with moving beamsplitters

We present an experiment testing quantum correlations with frequency shifted photons. We test Bell inequality with 2-photon interferometry where we replace the beamsplitters by acousto-optic modulators, which are equivalent to moving beamsplitters. We measure the 2-photon beatings induced by the frequency shifts, and we propose a cryptographic scheme in relation. Finally, setting the experiment in a relativistic configuration, we demonstrate that the quantum correlations are not only independent of the distance but also of the time ordering between the two single-photon measurements.Comment: 14 pages, 16 figure

Détermination de la structure par RMN d'une protéine impliquée dans la biosynthèse de centres [Fe-S]: SufA

The SufA protein from Escherichia coli is a homodimeric scaffold protein, allowing the formation of iron-sulfur clusters [Fe-S] before forwarding them to a target protein. This protein belongs to the SUF (for sulfur mobilzation) operon which appears to be activited under oxidative stress. In order to have a better understanding of the coordination of the [Fe-S] by the SufA protein, we undertook the determination of the three-dimensional structure by nuclear magnetic resonance (NMR). Currently, we achieved on the monomeric form of the apo-protein. Preliminary results with the reconstituted [Fe-S] protein are also presented in this document. We were able to observe the presence of three cysteine residues whose role, which was determinated by biochemical studies, is to stabilize the [Fe-S] cluster. However the exact coordination of the [Fe-S] cluster by these cysteines is not well known. The structure resolution of the dimer (in progress), and complementary NMR studies on the holoprotein SufA should bring a better understanding of the coordination mode of its cluster. It is worth to note that structural differences, with regard to the X-ray structures (published during this thesis) were observed, located particulary in the C-terminal sequence who we find two over the three coordinating cysteines.La protéine SufA d'Escherichia coli est une protéine homodimérique, dite d'échafaudage, qui permet l'assemblage de centres [Fe-S] avant de les transmettre à une protéine cible possèdant une fonction biologique. Elle appartient à un ensemble de protéines organisées en opéron (SUF) qui semble activé dans le cadre de stress oxydatif. Afin de mieux comprendre le mode de fixation du centre [Fe-S] par la protéine SufA, la détermination de la structure tridimensionnelle par RMN a été entreprise. Nous avons obtenu la structure monomérique de la protéine sans son centre [Fe-S] (forme apo) et effectué des études de résonance magnétique nucléaire (RMN) préliminaires de la protéine SufA avec son centre métallique reconstitué. Nous avons pu ainsi observer trois résidus cystéine, que des études biochimiques ont montré comme étant impliqués dans la chélation du centre [Fe-S], et affirmer qu'ils étaient proches du site métallique sans toutefois définir exactement le mode de coordination du centre [Fe-S]. La détermination de la structure du dimère, en cours, et des études RMN complémentaires sur l'holoprotéine (avec son centre [Fe-S]) devrait nous permettre de répondre à ces questions toujours en suspens. D'autre part nous avons observé des différences structurales par rapport aux structures cristallographiques (publiées au cours de ce travail de thèse), particulièrement dans la partie C-terminale de la protéine où se situent deux des cystéines observées

Détermination de la structure par RMN d'une protéine impliquée dans la biosynthèse de centres [Fe-S]: SufA

The SufA protein from Escherichia coli is a homodimeric scaffold protein, allowing the formation of iron-sulfur clusters [Fe-S] before forwarding them to a target protein. This protein belongs to the SUF (for sulfur mobilzation) operon which appears to be activited under oxidative stress. In order to have a better understanding of the coordination of the [Fe-S] by the SufA protein, we undertook the determination of the three-dimensional structure by nuclear magnetic resonance (NMR). Currently, we achieved on the monomeric form of the apo-protein. Preliminary results with the reconstituted [Fe-S] protein are also presented in this document. We were able to observe the presence of three cysteine residues whose role, which was determinated by biochemical studies, is to stabilize the [Fe-S] cluster. However the exact coordination of the [Fe-S] cluster by these cysteines is not well known. The structure resolution of the dimer (in progress), and complementary NMR studies on the holoprotein SufA should bring a better understanding of the coordination mode of its cluster. It is worth to note that structural differences, with regard to the X-ray structures (published during this thesis) were observed, located particulary in the C-terminal sequence who we find two over the three coordinating cysteines.La protéine SufA d'Escherichia coli est une protéine homodimérique, dite d'échafaudage, qui permet l'assemblage de centres [Fe-S] avant de les transmettre à une protéine cible possèdant une fonction biologique. Elle appartient à un ensemble de protéines organisées en opéron (SUF) qui semble activé dans le cadre de stress oxydatif. Afin de mieux comprendre le mode de fixation du centre [Fe-S] par la protéine SufA, la détermination de la structure tridimensionnelle par RMN a été entreprise. Nous avons obtenu la structure monomérique de la protéine sans son centre [Fe-S] (forme apo) et effectué des études de résonance magnétique nucléaire (RMN) préliminaires de la protéine SufA avec son centre métallique reconstitué. Nous avons pu ainsi observer trois résidus cystéine, que des études biochimiques ont montré comme étant impliqués dans la chélation du centre [Fe-S], et affirmer qu'ils étaient proches du site métallique sans toutefois définir exactement le mode de coordination du centre [Fe-S]. La détermination de la structure du dimère, en cours, et des études RMN complémentaires sur l'holoprotéine (avec son centre [Fe-S]) devrait nous permettre de répondre à ces questions toujours en suspens. D'autre part nous avons observé des différences structurales par rapport aux structures cristallographiques (publiées au cours de ce travail de thèse), particulièrement dans la partie C-terminale de la protéine où se situent deux des cystéines observées

Détermination de la structure par RMN d'une protéine impliquée dans la biosynthèse de centres [Fe-S] (SufA)

La protéine SufA d'Escherichia coli est une protéine homodimérique, dite d'échafaudage, qui permet l'assemblage de centres [Fe-S] avant de les transmettre à une protéine cible possèdant une fonction biologique. Elle appartient à un ensemble de protéines organisées en opéron (SUF) qui semble activé dans le cadre de stress oxydatif. Afin de mieux comprendre le mode de fixation du centre [Fe-S] par la protéine SufA, la détermination de la structure tridimensionnelle par RMN a été entreprise. Nous avons obtenu la structure monomérique de la protéine sans son centre [Fe-S] (forme apo) et effectué des études de résonance magnétique nucléaire (RMN) préliminaires de la protéine SufA avec son centre métallique reconstitué. Nous avons pu ainsi observer trois résidus cystéine, que des études biochimiques ont montré comme étant impliqués dans la chélation du centre [Fe-S], et affirmer qu'ils étaient proches du site métallique sans toutefois définir exactement le mode de coordination du centre [Fe-S]. La détermination de la structure du dimère, en cours, et des études RMN complémentaires sur l'holoprotéine (avec son centre [Fe-S]) devrait nous permettre de répondre à ces questions toujours en suspens. D'autre part nous avons observé des différences structurales par rapport aux structures cristallographiques (publiées au cours de ce travail de thèse), particulièrement dans la partie C-terminale de la protéine où se situent deux des cystéines observéesThe SufA protein from Escherichia coli is a homodimeric scaffold protein, allowing the formation of iron-sulfur clusters [Fe-S] before forwarding them to a target protein. This protein belongs to the SUF (for sulfur mobilzation) operon which appears to be activited under oxidative stress. ln order to have a better understanding of the coordination of the [Fe-S] by the SufA protein, we undertook the determination of the three-dimensional structure by nuclear magnetic resonance (NMR). Currently, we achieved on the monomeric form of the apo-protein. Preliminary results with the reconstituted [Fe-S] protein are also presented in this document. We were able to observe the presence of three cysteine residues whose role, which was determinated by biochemical studies, is to stabilize the [Fe-S] cluster. However the exact coordination of the [Fe-S] cluster by these cysteines is not well known. The structure resolution of the dimer (in progress), and complementary NMR studies on the holoprotein SufA should bring a better understanding of the coordination mode of its cluster. It is worth to note that structural differences, with regard to the X-ray structures (published during this thesis) were observed, located particulary in the C-terminal sequence who we find two over the three coordinating cysteinesGRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

From artificial biomacromolecules to supramolecular hydrogels for bioelectrocatalysis.

Bio-hybrid artificial materials are being developed for the bioelectrochemical transformation of small gas molecules. Particular emphasis is put on energy storage and chemical energy conversion, with the oxidation of H2 to H+ and the selective reduction of CO2 to electrofuels and commodity chemicals as main targets.In particular, electron conducting supramolecular hydrogels have been developed that are capable to immobilize and stabilize metalloenzymes at the surface of an electrode while ensuring both their electrical connection and the transport of substrate/product molecules to and from the electrode. In one example, a self-assembling artificial proteins was prepared that is capable to self-organize into conducting nanofilaments and redox hydrogels: starting from a mutated prion-forming domain, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized and self-assembled into structurally homogenous nanofibrils. Molecular modeling confirmed that the redox groups are aligned along the fibril axis and are tethered to its core by a long, flexible polypeptide chain that allows close encounters between the fibril-bound oxidized or reduced redox groups. Redox hydrogel films capable of immobilizing a [NiFeSe] hydrogenase under mild conditions at the surface of carbon electrodes were obtained by simple pH jump. In this way, bioelectrodes for the electrocatalytic oxidation of H2 were fabricated that afforded catalytic current densities of up to 270 μA cm−2 at 45 °C under quiescent conditions, with an overpotential of 0.33 V

From artificial biomacromolecules to supramolecular hydrogels for bioelectrocatalysis.

Bio-hybrid artificial materials are being developed for the bioelectrochemical transformation of small gas molecules. Particular emphasis is put on energy storage and chemical energy conversion, with the oxidation of H2 to H+ and the selective reduction of CO2 to electrofuels and commodity chemicals as main targets.In particular, electron conducting supramolecular hydrogels have been developed that are capable to immobilize and stabilize metalloenzymes at the surface of an electrode while ensuring both their electrical connection and the transport of substrate/product molecules to and from the electrode. In one example, a self-assembling artificial proteins was prepared that is capable to self-organize into conducting nanofilaments and redox hydrogels: starting from a mutated prion-forming domain, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized and self-assembled into structurally homogenous nanofibrils. Molecular modeling confirmed that the redox groups are aligned along the fibril axis and are tethered to its core by a long, flexible polypeptide chain that allows close encounters between the fibril-bound oxidized or reduced redox groups. Redox hydrogel films capable of immobilizing a [NiFeSe] hydrogenase under mild conditions at the surface of carbon electrodes were obtained by simple pH jump. In this way, bioelectrodes for the electrocatalytic oxidation of H2 were fabricated that afforded catalytic current densities of up to 270 μA cm−2 at 45 °C under quiescent conditions, with an overpotential of 0.33 V

Supramolecular engineering of 1-D nanomaterials by self-assembly of artificial amyloid proteins

International audienceOne-dimensional (1-D) nanomaterials are ideally suited for exploring a number of physical phenomena at the nanoscale and for investigating the dependence of physical properties on size and dimensionality. They also hold much promise as interconnects and as key elements for the fabrication of electronic, optoelectronic and electrochemical energy devices with nanoscale dimensions.The lack of facile, simple, high-yield methods for the production of 1-D nanomaterials in large quantities is nonetheless a major obstacle to their widespread use and study, and development of bottom-up manufacturing processes based on self-assembly at room temperature in environmentally benign solvents remains a major challenge in nanoscience. Living systems provide numerous examples of self-organization in aqueous environment in the form of protein structures amyloid fibrils. The latter are self-assembled one-dimensional protein arrays with β-strands perpendicular to the growth axis . We now report a few examples of biohybrid protein nanofilaments obtained by conjugation of an artificial prosthetic group to HETPFD-the prion forming domain of the HET protein from the fungus P. anserina-followed by self-assembly of the resulting artificial protein in aqueous solution. The obtained nanofilaments show a high degree structural and morphological homogeneity and, if properly functionalized, can exchange electrons with an electrode in a reversible manner

Protein assembly for the design of new materials: characterization of interactions at amino-acid level betweenalpha-lactalbumin and lysozyme

The final structure and stability of supramolecular objects result from interactions between proteins at amino-acid level. Their characterization is the basis for the design of new proteinbased biomaterials. In this study we focus on the interactions and assembly of bovine alphalactalbumin (LAC) with hen egg white lysozyme (LYS). While LYS interacts with the apo and holo forms of LAC (Ca2+ depleted and loaded form respectively), these interactions lead to the formation of micro-spheres with apoLAC only. Our objectives are to identify the amino-acids involved in the interactions and to establish the mechanism of such self-assemblyinto suprastructures. The early step of the process is most probably the formation of heterodimers.A structural characterization of both holoLAC-LYS and apoLAC-LYS dimers was performed in order to understand why micro-spheres are formed with apoLAC only. The surfaces of interaction of holoLAC with LYS and LYS with the holo and apo forms of LAC were determined using (1H, 15N) Nuclear Magnetic Resonance. Interacting amino-acids were identified according to the chemical shift perturbations measured during titration of one 15Nlabelled protein by its non-labelled partner. The titration of 15N-holoLAC by LYS indicatesthat holoLAC interacts with LYS through its beta-domain (beta-sheet and flexible loop). The reverse titration indicates that LYS interacts with holoLAC mainly through its alpha-domain, including amino-acids of helices B and D and C- and N-terminal regions. Few residues of its beta-domain are also involved. In both cases the surfaces of interaction are localized on one side of the proteins and are composed of contiguous surface patches. LYS interacts with the apo form of LAC mainly through its alpha-domain and this surface patch is larger than with the holo form. This difference could be explained by the increased flexibility of the apoLACwhich is in its molten globule state at the working temperature (45°C). In order to know how proteins interact on an atom-to-atom basis, docking experiments were performed using the HADDOCK program. The interacting amino-acids were transformed into ambiguous intermolecular distance restraints to calculate hetero-dimers structures. This characterization is a prerequisite for the use of LAC and LYS micro-spheres as biomaterials, such as bioactive compound carrier. These findings give key elements to understand protein interaction and self-assembly process in mixed system

A Segmented Beam Dump for the CTS Line at CTF3

We propose a new segmented beam dump to be installed in thespectrometer line at the end of the CTF3 linac. The device will allowfor time-resolved energy distribution measurements in a single shotand would therefore be a useful tool in tuning the accelerator.CLI