M Thierry PrangéProfessor – Emeritus University Paris CitéCiTCoM
Three main topics in macro molecular structural biochemistry, using X-ray diffraction as the main technique:
1- Investigation of LTPs (or lipid Transfer Proteins) from plants or fungus, and their mode of transport. (Collaboration with three INRA groups (Dijon, Nantes, and Nice).
2- Studies on gas-protein interactions (range 1 to 50 Bars). Analysis and elucidation of the general anesthesia mechanism. Using neutral gas like Ar, Xe, Kr, N2 or reactive species like O2, CO, N2O. (In Collaboration with a group of the CYCERON Lab Caen, and the Sanofi Co).
3- Analyses of protein and nucleic acids 3D structures under very high hydrostatic pressures (from 1 to 20 kBars). (Collaboration with two groups at ESRF, Grenoble, and SOLEIL, Orsay, Gif)
1- Investigation of LTPs (or lipid Transfer Proteins) from plants or fungus, and their mode of transport.
LTP’s are ubiquitous proteins playing an important role in plant signaling processes. They are especially involved in the SAR mechanism (Systemic Acquired Resistance). Some elicitins from fungi or oomycetes belong to this class of 10k proteins. Protein – lipid associations were investigated by X-ray diffraction on monocrystals and reveal the broad diversity in the binding modes, usually hydrophobic, but covalent in some rare cases:
Left: The DIR-1, protein implicated in the SAR of Arabidopsis thaliana, associated with its signal peptide (embedded in the central hydrophobic cavity). Right: The LTP1 Oxylipin from barley, a new case of a covalent linkage with its lipid (-Keto 9 hydroxy 10 oxo 12(Z)-octadecenoic acid). These two examples describe the different binding modes in the LTP field.
2- Studies on gas-protein interactions (from 1 to 50 Bars).
Analysis and elucidation of the general anesthesia mechanism. Using neutral gas like Ar, Xe, Kr, N2 or reactive species like O2, CO, N2O.
Xenon and krypton are known to be inert in chemistry but they easily insert in buried hydrophobic cavities in proteins. Protein-rare gas were investigated in he phasing of electron density maps, either by MIR (xenon) or MAD (krypton) techniques. The usefulness of the technique made this method now routinely employed with all synchrotron places.
X-ray view of the active site of urate oxidase (from Aspergillus flavus). This particular oxidase is able to use directly dioxygen from air, without needs of any co-factor to activate O2. Two sites in the protein: one for the substrate (uric acid, here 8-azaxanthin as inhibitor) and a second for the reactive species (O2, H2O and H2O2). Left: in normal conditions, a water molecule usually occupies this latter site. Under dioxygen pressure (> 20 bars) a molecule of dioxygen is clearly visible.
On the other hand, xenon as well as N2O possesses strong anesthetic properties making them useful in surgery. However their mechanisms of action still need to be elucidated. X-ray diffraction was employed using model globular proteins like urate oxidase, elastase acetylcholine esterase or anexins.
Left: urate oxidase tetramer showing the xénon sites (together with the 8-azaxanthin inhibitor). Right: Elastase xenon site located in the active site cleft, close to the surface.
3- Analyses of protein and nucleic acids 3D structures under very high hydrostatic pressures (from 1 to 20 kBars).
How life has emerged on earth remains a mystery. Molecules with backbones forming stable double helices held together by specific pairings have an exceptional importance in this saga, as they combine the encoding of genetic information with (in some cases) catalytic activity. Many scenarios involve extreme conditions, and this motivated structural investigations on this particular architecture under very high pressure. This study – the first report on the application of high-pressure macromolecular crystallography (HPMX) to nucleic acids – is part of a broader program where high pressure is used to investigate physicochemical properties of DNA and RNA molecules. The pressure range extended from ambient pressure to up 2 GPa (20,000 atm):
Left: a crystal of the octamer d(GGTATACC)2 is selected, mounted in a diamond anvil pressure cell. Middle: The diffraction diagrams at ambient pressure and at 1,8 GPa (18.000 atm). One clearly observes the displacement of the strong meridian reflections, an indication about the stacking compression of the double helix. Right: The X-ray structure of the A DNA double helix at ambient pressure (green) and 1.8 GPa (red). The deformation is very anisotropic, the Watson-Crick base-pairs are only slightly affected while the base stacking is reduced from 2.9 Å to 2.6 Å.
Topic # 1:
– “The 1.45 Å resolution structure of the cryptogein cholesterol complex. A close up view of a sterol carrier protein (SCP) active site.” M.B. Lascombe, M. Ponchet, P. Venard, M. L. Milat, J. P. Blein and Thierry Prangé. Acta Crystallogr. (2002) D58, 1442-1447.
– “The defective in induced resistance protein of Arabidopsis thaliana, DIR1, is a new type of lipid transfer protein.” M.B. Lascombe, B. Bakan, N. Buhot, D. Marion, J.P. Blein, C. Lamb and T. Prangé Protein Sci. (2008) 17, 1522-1530.
– “The crystal structure of oxylipin-conjugated barley LTP1 highlights the unique plasticity of the hydrophobic cavity of these plant lipid binding proteins”. B. Bakan, M. Hamberg, V. Larue, T. Prangé, D. Marion and M.B. Lascombe. Biochem. Biophys. Res. Commun. (2009) 390, 780-785.
Topic # 2:
– “Exploring hydrophobic sites in proteins with xenon and krypton noble gas.” T. Prangé, L. Pernot, N. Colloc’h, S. Longhi, W. Bourguet, R. Fourme. and M. Schiltz. Protein Struct. and Funct. (1998) 30(1), 61-73.
– “Use of noble gases xenon and krypton as heavy atoms in protein structure determination.” M. Schiltz, R. Fourme and T. Prangé. Methods in Enzymology, (2004). 374, 83-119.
– “Protein crystallography under xenon and nitrous oxide pressure: Comparisons with in vivo pharmacology studies and implications in the mechanism of inhaled anesthetic action”. N. Colloc’h, J. Sopkova de Oliveira Santos, P. Retailleau, J. J. Risso, M. Lemaire, T. Prangé and J. H. Abraini. Biophys. J. (2007) 92, 217-224.
– “Oxygen pressurized crystallography: Probing the dioxygen binding site in cofactorless urate oxidase and implication for its catalytic mechanism.”. N. Colloc’h, L. Gabison, J. Sopkova-de Oliveira Santos, M. El Hajji, B. Castro, J. H. Abraini and T. Prangé. Biophys J. (2008) 95, 2415-2422.
– “Comment la bio-cristallographie permet de proposer un mécanisme d’action des gaz anesthésiques”. N. Colloc’h, J. Abraini et T. Prangé. Reflet de la physique (2010). 19, 10-13.
– “A pressure-response crystallographic study of urate oxidase with xenon and nitrous oxide”. G. Marassio, T. Prangé, H.N. David, J. Sopkova-de Olivera-Santos, L. Gabison, N. delcroix, J.H. Abraini and N. Colloc’h. FASEB. J. (2011) 25, 2266-2275.
Topic # 3:
– “Adaptation of base-paired double helix molecular architecture to extreme hydrostatic pressure”. E. Girard, T. Prangé, A. C. Dhaussy, E. Migianu-Griffoni, M. Lecouvey, J. C. Chervin, M. Mezouar, R. Kahn and R. Fourme. Nucleic Acid. Res. (2007) 35, 4800-4808.
– “Structure-function perturbation and dissociation of tetrameric urate oxydase by high hydrostatic pressure”. E. Girard, S. Maréchal, J. Perez, S. Finet, R. Kahn, R. Fourme, G. Marassio, A.C. D’Haussy, T. Prangé, M. Giffard, F. Dulin, F. Bonneté, R. Lange, J. Abraini, M. Mézouar and N. Colloc’h. Biophys. J. (2010) 98, 2365-2373.
– “A new paradigm for MX beamlines derived for HPMX methodology and results”. R. Fourme, E. Girard, A.C. Dhaussy, T. Prangé, I. Ascone, M. Mezouar and R. Kahn. J. synchr. Rad. (2011) 18, 31-36.