|Structural Dynamics of Macromolecules - URA 2185|
|HEAD||DELARUE Marc / email@example.com|
|MEMBERS||BARBOSA Isabelle, GOUGE Jerome, Dr. POGGI Luisa, ROMAIN Felix
The aim of the group is to understand at the molecular level the structure and energetics of ligand binding in validated drug targets of various pathogens, using X-Ray crystallography and computational structural biology. In addition, we have embarked in a larger project aimed at understanding the nature of intermediates along the reaction pathway of a catalytic cycle for fundamental objects of molecular biology, such as DNA polymerases.
a) Mutant of Taq polymerase (coll. J.L. Jestin, I.P.)
The structure of a Taq polymerase mutant originally isolated by in vitro directed evolution was solved and refined at 2.5 Angstrom, in a crystal form different from that of the wild-type enzyme. We have also recently obtained crystals of the wild-type in the mutant crystallisation conditions (and vice-versa) and will be refining models with this data in order to assess the significance and biological relevance of the structural difference seen thus far.
b) Structural studies of human pol μ (coll. F. Rougeon, I.P.)
Pol mu is a recently discovered DNA polymerase involved in Non-Homologous End-Joining repair system of DNA double-strand breaks. We have shown that pol mu is much more sensitive to protease limited digestion than TdT and used this property to probe conformational change in the presence of various substrates. In addition, the activity of 10 different single point mutants in the active site of TdT is being studied. Two other mutants were found to display pol mu phenotype and will also be crystallized and structurally characterized. We will extend our studies of the pol X family to bacterial ones.
c) Structural studies of a bacterial homolog of the nicotinic receptor (coll. P.J. Corringer, I.P.)
We have solved the structure of the extra-cellular domain of one of the bacterial analogs of the nicotinic receptor ; this protein is a proton-sensitive cation channel. The refinement is nearly complete using 2.4 Angstrom data, as well as the structure solution of another crystal form grown above the activation pH. Low resolution data have also been collected for the entire protein.
i) NOMAD-Ref We have put on-line and regularly updated a variety of refinement tools that use Normal Modes as a reduced set of degrees of freedom to conduct refinement of models with either X-Ray data, cryo-electron microscopy data, or just packing energy when given a preliminary solution of the docking problem. ii) PDB_Hydro We have updated and accelerated a new method to calculate electrostatics properties of macromolecules and their complexes in a dipolar solvent of variable density thereby generalizing the Poisson-Boltzmann Equation method (Azuara et al., JMB, submitted). iii) Conformational transitions (Coll. S. Doniach, Stanford). We have pursued our work aiming at producing physically exact trajectories along the reaction pathway between two known structural forms of the same macromolecule and have put it on-line, together with extensive comparisons with existing methods using adenylate kinase as an example: http://lorentz.dynstr.pasteur.fr/joel/index.php. We also have extended this work to make it work with full atomic representation and to detect which residues contribute most to the height of the transition state energy barrier. This work has been selected for and awarded (2007) an ARTS grant by Apple, Inc.
Keywords: Crystallography, DNA polymerases, DNA repair, structural transition, drug design
Delarue M.Dealing with structural variability in molecular replacement and crystallographic refinement through normal-mode analysis.Acta Cryst. D. 2008 64:40-8.
Franklin J, Koehl P, Doniach S, Delarue M.MinActionPath: maximum likelihood trajectory for large-scale structural transitions in a coarse-grained locally harmonic energy landscape. Nucleic Acids Res. 2007 35:W477-82.
Delarue M, Duclert-Savatier N, Miclet E, Haouz A, Giganti D, Ouazzani J, Lopez P, Nilges M, Stoven V.Three dimensional structure and implications for the catalytic mechanism of 6-phosphogluconolactonase from Trypanosoma brucei.J Mol Biol. 2007 366:868-81.
Delarue M.An asymmetric underlying rule in the assignment of codons: possible clue to a quick early evolution of the genetic code via successive binary choices. RNA. 2007 13:161-9.
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Activity Reports 2007 - Institut Pasteur
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