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International audience; RsEGFP2 is a reversibly photoswitchable fluorescent protein used in super-resolved optical microscopies, which can be toggled between a fluorescent On state and a nonfluorescent Off state. Previous time-resolved ultraviolet-visible spectroscopic studies have shown that the Off-to-On photoactivation extends over the femto- to millisecond time scale and involves two picosecond lifetime excited states and four ground state intermediates, reflecting a trans-to-cis excited state isomerization, a millisecond deprotonation, and protein structural reorganizations. Femto- to millisecond time-resolved multiple-probe infrared spectroscopy (TRMPS-IR) can reveal structural aspects of intermediate species. Here we apply TRMPS-IR to rsEGFP2 and implement a Savitzky-Golay derivative analysis to correct for baseline drift. The results reveal that a subpicosecond twisted excited state precursor controls the trans-to-cis isomerization and the chromophore reaches its final position in the protein pocket within 100 ps. A new step with a time constant of 42 ns is reported and assigned to structural relaxation of the protein that occurs prior to the deprotonation of the chromophore on the millisecond time scale.

International audience; To determine phospholipase D (PLD) activity, an infrared spectroscopy assay was developed, based on the phosphate vibrational mode of phospholipids such as dimyristoylphophatidylcholine (DMPC), lysophosphatidylglycerol (lysoPG), dipalmitoylphosphatidylethanolamine (DPPE), and lysophosphatidylserine (lysoPS). The phosphate bands served to monitor the hydrolysis rates of phospholipids with PLD. The measurements could be performed within less than 20min with 10μl of buffer containing 2 to 40mM DMPC and 10 to 200ng of Streptomyces chromofuscus PLD (corresponding to 350-7000pmol of DMPC hydrolyzed per minute). The limit of sensitivity was approximately 10ng of PLD at 100mM Tris-HCl (pH 8.0) with 10mM Ca(2+) and 2.5mgml(-1) Triton X-100. Reproducible specific activity of PLD (35±5nmol of hydrolyzed DMPCmin(-1)μg(-1) PLD) measured by the infrared assay remained stable over 50 to 200ng of PLD and over 5 to 40mM DMPC. The feasibility of this assay to determine the hydrolysis rate of other phospholipids such as lysoPG, DPPE, and lysoPS was confirmed. The IC(50) of cobalt (800±200μM), a known S. chromofuscus PLD inhibitor, was measured by means of the infrared assay, demonstrating that this assay can be used to screen PLD activity and/or the specificity of its inhibitors.

International audience; New hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones were prepared in two steps from ethyl 4-phenyl-6-methyl-2-oxo tetrahydropyrimidine-5-carboxylates or 4-phenyl-6-methyl-2-thioxotetrahydropyrimidine-5-carboxylates, previously prepared by Biginelli reaction using appropriate aldehyde, urea derivatives and ethyl acetoacetate. Their antioxidant properties were evaluated by two methods: scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and scavenging effect on hydroxyl radicals. The results show that the compounds containing thiourea moiety have better activity.

International audience; Iron chelation in tumoral cells has been reported as potentially useful during antitumoral treatment. Our aim was to develop new polyaminoquinoline iron chelators targeting tumoral cells. For this purpose, we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, which we named Quilamines, based on an 8-hydroxyquinoline (8-HQ) scaffold linked to linear polyamine vectors. These were designed to target tumor cells expressing an overactive polyamine transport system (PTS). A set of Quilamines bearing variable polyamine chains was designed and assessed for their ability to interact with iron. Quilamines were also screened for their cytostatic/cytotoxic effects and their selective uptake by the PTS in the CHO cell line. Our results show that both the 8-HQ moiety and the polyamine part participate in the iron coordination. HQ1-44, the most promising Quilamine identified, presents a homospermidine moiety and was shown to be highly taken up by the PTS and to display an efficient antiproliferative activity that occurred in the micromolar range. In addition, cytotoxicity was only observed at concentrations higher than 100 μM. We also demonstrated the high complexation capacity of HQ1-44 with iron while much weaker complexes were formed with other cations, indicative of a high selectivity. We applied the density functional theory to study the binding energy and the electronic structure of prototypical iron(III)-Quilamine complexes. On the basis of these calculations, Quilamine HQ1-44 is a strong tridentate ligand for iron(III) especially in the form of a 1:2 complex.

International audience; Carbon monoxide recombination dynamics upon photodissociation with visible light has been characterized by means of ultrafast visible-pump/MidIR probe spectroscopy for the truncated hemoglobins from Thermobifida fusca and Bacillus subtilis. Photodissociation has been induced by exciting the sample at two different wavelengths: 400 nm, corresponding to the heme absorption in the B-band, and 550 nm, in the Q-bands. The bleached iron-CO coordination band located at 1850-1950 cm(-1) and the free CO absorption band in the region 2050-2200 cm(-1) have been observed by probe pulses tuned in the appropriate infrared region. The kinetic traces measured at 1850-1950 cm(-1) reveal multiexponential subnanosecond dynamics that have been interpreted as arising from fast geminate recombination of the photolyzed CO. A compared analysis of the crystal structure of the two proteins reveals a similar structure of their distal heme pocket, which contains conserved polar and aromatic amino acid residues closely interacting with the iron ligand. Although fast geminate recombination is observed in both proteins, several kinetic differences can be evidenced, which can be interpreted in terms of a different structural flexibility of the corresponding heme distal pockets. The analysis of the free CO band-shape and of its dynamic evolution brings out novel features about the nature of the docking site inside the protein cavity.

International audience; The interactions between synthetic polycations and phospholipid bilayers play an important role in some biophysical applications such as gene delivery or antibacterial usage. Despite extensive investigation into the nature of these interactions, their physical and molecular bases remain poorly understood. In this Article, we present the results of our studies on the impact of a hydrophobically modified strong polycation on the properties of a zwitterionic bilayer used as a model of the mammalian cellular membrane. The study was carried out using a set of complementary experimental methods and molecular dynamic (MD) simulations. A new polycation, poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (polymer 3), was synthesized, and its interactions with liposomes composed of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were examined using dynamic light scattering (DLS), zeta potential measurements, and cryo-transmission electron microscopy (cryo-TEM). Our results have shown that polymer 3 can efficiently associate with and insert into the POPC membrane. However, it does not change its lamellar structure, as was demonstrated by cryo-TEM. The influence of polymer 3 on the membrane functionality was studied by leakage experiments applying a fluorescence dye (calcein) encapsulated in the phospholipid vesicles. The MD simulations of model systems reveal that polymer 3 promotes formation of hydrophilic pores in the membrane, thus increasing considerably its permeability.

International audience; Adsorption of purified apo-ovotransferrin at the air-water interface was studied by ellipsometry, surface tension, polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS), and shear elastic constant measurements. No significant difference was observed between pH 6.5 and 8.0 as regards the final value of surface concentration and surface pressure. However at low concentration, a weak barrier to adsorption is evidenced at pH 6.5 and confirmed by PM-IRRAS measurements. At a pH where the protein net charge is negative (pH 8.0), the behavior of ovotransferrin at the air-water interface is more influenced by charge effects rather than bulk concentration effects. At this pH, the interface exhibits a low shear elastic constant and a spectral signature not usual for globular proteins.

International audience; Apolipoprotein A-I (ApoA-I) is a protein implicated in the solubilization of lipids and cholesterol from cellular membranes. The study of ApoA-I in phospholipid (PL) monolayers brings relevant information about ApoA-I/PL interactions. We investigated the influence of PL charge and acyl chain organization on the interaction with ApoA-I using dipalmitoyl-phosphatidylcholine, dioleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylglycerol monolayers coupled to ellipsometric, surface pressure, atomic force microscopy and infrared (polarization modulation infrared reflection-absorption spectroscopy) measurements. We show that monolayer compressibility is the major factor controlling protein insertion into PL monolayers and show evidence of the requirement of a minimal distance between lipid headgroups for insertion to occur, Moreover, we demonstrate that ApoA-I inserts deepest at the highest compressibility of the protein monolayer and that the presence of an anionic headgroup increases the amount of protein inserted in the PL monolayer and prevents the steric constrains imposed by the spacing of the headgroup. We also defined the geometry of protein clusters into the lipid monolayer by atomic force microscopy and show evidence of the geometry dependence upon the lipid charge and the distance between headgroups. Finally, we show that ApoA-I helices have a specific orientation when associated to form clusters and that this is influenced by the character of PL charges. Taken together, our results suggest that the interaction of ApoA-I with the cellular membrane may be driven by a mechanism that resembles that of antimicrobial peptide/lipid interaction.

International audience; Far infrared spectra of zwitterionic, cationic, and anionic forms of aliphatic amino acids in solid state have been studied experimentally. Measurements were done on glycine, L-alanine, L-valine, L-leucine, and L-isoleucine powder samples and film samples obtained from dried solutions prepared at pH ranging from 1 to 13. Solid state density functional theory calculations were also performed, and detailed potential energy distributions were obtained from normal mode results. A good correspondence between experimental and simulated spectra was achieved and this allowed us to propose an almost complete band assignment for the far infrared spectra of zwitterionic forms. In the 700-50 cm(-1) range, three regions were identified, each corresponding to a characteristic set of normal modes. A first region between 700 and 450 cm(-1) mainly contained the carboxylate bending, rocking, and wagging modes as well as the ammonium torsional mode. The 450-250 cm(-1) region was representative of backbone and sidechain skeletal bending modes. At last, the low wavenumber zone, below 250 cm(-1), was characteristic of carboxylate and skeletal torsional modes and of lattice modes. Assignments are also proposed for glycine cationic and anionic forms, but could not be obtained for all aliphatic amino acids due to the lack of structural data. This work is intended to provide fundamental information for the understanding of peptides vibrational properties.

International audience; The growth of human primary keratinocytes and fibroblasts on PLA-PEO-PLA copolymer films was investigated as an intermediate stage of a strategy aimed at making implantable dermo-epidermal substitutes. Four PLA-PEO-PLA triblock copolymers with the same PEO block and different DL-lactic acid/ethylene oxide molar ratios (LA/EO) (0.8, 1.4, 1.8 and 2), were synthesized and characterized by 1H-nuclear magnetic resonance and infrared spectroscopy. The films made of these copolymers were more hydrophilic than PLA50 and than tissue culture polystyrene controls according to contact angles with water. Proliferation and adhesion of human skin cells were evaluated by MTT assay and by scanning electron microscopy. The presence of PEO in the triblock copolymers influenced cell adhesion and proliferation of fibroblasts, whereas keratinocyte adhesion and proliferation were not affected. These features emphasize the interest of PLA-PEO-PLA triblock copolymers to serve as better compounds than the racemic PLA previously investigated to make supports for human skin primary cells and scaffolds for skin engineering.

International audience; The leaves of the Phoebe scortechinii (Gamb.) Kochummen Comb. Nov. (Lauraceae), afforded one new proaporphine-tryptamine dimer; (-)-phoebescortechiniine (1), along with two known ones; phoebegrandine A and phoebegrandine B. The proaporphine, tetrahydropronuciferine (2), was isolated for the first time as a natural product. The alkaloids were elucidated primarily by means of high field NMR and HRMS.

Cyclodepsipeptides incorporating a sugar amino acid, e.g., I, have been synthesized. A three-component reaction of a sugar amino acid (SAA) deriv., an aldehyde, and a dipeptide isonitrile in refluxing methanol afforded the corresponding 5-aminooxazole which, after sapon., underwent a trifluoroacetic acid promoted macrocyclization to furnish the cyclic sugar amino acids. [on SciFinder (R)]

International audience; The alkaloidal extract of the leaves of Phoebe grandis (nees) merr. have provided two new minor alkaloids; phoebegrandine D (1), a proaporphine-tryptamine dimer, and phoebegrandine E (2), an indoloquinolizidine. This is the first report on the occurrence of an indoloquinolizidine in the Phoebe species. The crude extract also exhibited antiplasmodial activity (IC50

International audience; From the seeds of Annona glauca and A. cherimola, two novel cyclic peptides, glaucacyclopeptide B and cherimolacyclopeptide G, have been isolated, respectively. Their structures were elucidated by chemical and spectral methods.

International audience; The adk gene from Mycobacterium tuberculosis codes for an enzyme of 181 amino acids. A sequence comparison with 52 different forms of adenylate kinases (AK) suggests that the enzyme from M. tuberculosis belongs to a new subfamily of "short" bacterial AKs. The recombinant protein, overexpressed in Escherichia coli, exhibits a low catalytic activity and an unexpectedly high thermal stability (Tm = 64.8 degrees C). Based on various spectroscopic data, on the known three-dimensional structure of the AK from E. coli and on secondary structure predictions for various sequenced AKs, we propose a structural model for AK from M. tuberculosis (AKmt). Proteins 1999;36:238-248.

International audience; Analysis of the nature of calculi provides a valuable mean for the diffential diagnosis of urinary lithiasis. In this work, we report a comparative study of the nature of 31 calculi performed by infra red and the classical chemical techniques.; L'analyse du calcul est un élément fondamental pour orienter le diagnostique étiologique de la lithiase. Les auteurs comparent à travers une série de 31 calculs les résultats obtenus par l'analyse infra rouge à ceux de la méthode chimique classique. Les résultats montrent que la technique chimique donne sur le plan analytique des faux positifs pour la struvite, la whewellite et des faux négatifs pour la weddellite, le phosphate amorphe de calcium carbonaté, la whitlockite, les protéines, l'urate d'ammonium et l'acide urique. Sur le plan clinique 83,9% des cas sont en discordance avec l'analyse infra rouge. La méthode chimique est donc une méthode non fiable et inadaptée à l'étude des calculs urinaires.

International audience; Nucleoside Diphosphate Kinases (NDPKs) have long been considered merely as housekeeping enzymes. The discovery of the NME1 gene, an anti-metastatic gene coding for NDPK-A, led the scientific community to re-evaluate their role in the cell. It is now well established that the NDPK family is more complex than what was first thought, and despite the increasing amount of evidence suggesting the multifunctional role of nm23/NDPKs, the specific functions of each family member are still elusive. Among these isoforms, NDPK-D is the only one to present a mitochondria-targeting sequence. It has recently been shown that this protein is able to bind and cross-link with mitochondrial membranes, suggesting that NDPK-D can mediate contact sites and contributes to the mitochondrial intermembrane space structuring. To better understand the influence of NDPK-D on mitochondrial lipid organisation, we analysed its behaviour in different lipid environments. We found that NDPK-D not only interacts with CL or anionic lipids, but is also able to bind in a non negligible manner to zwitterionic PC. NDPK-D alters membrane organisation in terms of fluidity, hydration and lipid clustering, effects which depend on lipid structure. Changes in the protein structure after lipid binding were evidenced, both by fluorescence and infrared spectroscopy, regardless of membrane composition. Taking into account all these elements, a putative mechanism of interaction between NDPK-D and zwitterionic or anionic lipids was proposed.

International audience

International audience; It has been recently shown that mitochondrial creatine kinase (mtCK) organizes mitochondrial model membrane by modulating the state and fluidity of lipids and by promoting the formation of protein-cardiolipin clusters. This report shows, using Brewster angle microscopy, that such clustering is largely dependent on the acyl chain composition of phospholipids. Indeed, mtCK-cardiolipin domains were observed not only with unsaturated cardiolipins, but also with the cardiolipin precursor phosphatidylglycerol. On the other hand, in the case of saturated dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin, mtCK was homogeneously distributed underneath the monolayer. However, an overall decrease in membrane fluidity was indicated by infrared spectroscopy as well as by extrinsic fluorescence spectroscopy using Laurdan as a fluorescent probe, both for tetramyristoylcardiolipin and bovine heart cardiolipin containing liposomes. The binding mechanism implicated the insertion of protein segments into monolayers, as evidenced from alternative current polarography, regardless of the chain unsaturation for the phosphatidylglycerols and cardiolipins tested.

International audience; We present here an improvement in the classical molybdate method for inorganic phosphate determination. This method has high sensitivity, with 1 nmol of Pi giving an absorbance change of 0.060 A850 unit. It is highly reproducible and the color remains stable for at least 3.5 h. In addition the use of sodium dodecyl sulfate makes it possible to stop enzymatic reactions without organic phosphate hydrolysis. It also shows an extremely low interference by highly concentrated solutions of different origins. Of special interest is its high tolerance to protein, permitting as much as 50 mg/ml of human serum protein in the sample without precipitation or color interference. For these reasons, it proves to be very useful in the determination of ATPases in tissues such as the ocular lens with low specific activity.

International audience; The conformations of the intrinsic membrane protein, rhodopsin, in its membrane-bound and detergent-solubilised states have been compared by hydrogen isotope exchange measurements. The infrared peptide exchange data show that the highly hydrophobic nature of rhodopsin is conserved in the presence of the two detergents used: Cemulsol LA 90 and Ammonyx LO. Only about 50% of the peptide hydrogens exchange under conditions where about 80% would exchange in most soluble proteins. The conformational stability of rhodopsin in these two detergents is also demonstrated by the similarity of the tritium exchange-out kinetics and the infrared amide I band frequencies for both membrane-bound and detergent-solubilised rhodopsin. Upon illumination of rhodopsin (bleaching) in the presence of detergents, the hydrogen exchange rates are greatly increased and shifts in the amide I band frequencies are observed, indicative of a large conformation change. No such change occurs upon bleaching membrane-bound rhodopsin. We conclude that the conformation of rhodopsin is not altered by solubilisation in non-ionic detergents. However, in agreement with previously published results, bleached rhodopsin is stabilised by the membrane but does not retain a native conformation in these detergents.