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  Director : Deshmukh N. Gopaul (gopauld@pasteur.fr)


  abstract

 

The Macromolecular Biochemistry and Biophysics lab is engaged in the characterization of protein/DNA and protein/protein complexes by the determination of their three dimensional structures using x-ray crystallography. The binding aonstants as well as the thermodynamic paratmeters are determined by fluorescence spectroscopy and microcalorimetry.



  report

cale

The Lab was started in early 2002. The main research axis is the biochemical and structural characterization of macromolecular complexes, namely recombinases from the family of Tyrosine site-specific recombinases, as complexes with their DNA substrates. The methodology we use for the structural characterization requires the crystallization and x-ray diffraction of the protein/DNA complexes. We have started other collaborative projects with local labs including the study of the protein/protein interactions involved in cell motility/recognition in the case of the invasion of Plasmodium falciparum, the causative agent of Malaria. We have additionally taken on the characterization of some components involved in the regulation of some signal transduction pathways.

Structural and biochemical characterization of IntI1 and IntI4 integrases.

The integrons have been identified as being part of the strategy for bacteria to acquire resistance against antibiotics. This system involves the use of an integrase IntI and of variable DNA sequences known as attI or attC, when they are proximal or distal to the open reading frame carrying the resistance gene, respectively. We plan on tackling the three dimensional structures of IntI1 from Pseudomonas aeruginosa and Int4 from Vibrio cholera in the presence or absence of DNA substrates by x-ray crystallography. We also want to establish the biochemical properties of these enzymes, namely their binding and kinetic constants by fluorescence anisotropy, calorimetry and Surface Plasmon Resonance (Biacore) methods. As substrates we will use recombination intermediates including 4-way junctions as well as linear suicide substrates. The binary approach of establishing the structure and the biochemical parameters will give a deeper understanding of the reaction mechanism, isomerization of intermediates and the formation of products. This information will in turn be used for the generation of activators or inhibitors of the integrases, which hopefully will be adequate for preventing or reversing the acquisition of resistance genes.

Keywords: Site-specific recombinase, integase, x-ray crystallography, structure, biochemistry



  web site

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  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Fejt, Francoise, (ffejt@pasteur.fr) Gopaul, Deshmukh N., IP, (Group Leader,gopauld@pasteur.fr) MacDonald, Douglas, Pasteur Foundation fellow, (dmacdona@pasteur.fr) Miras, Isabelle, IP, Technician (imiras@pasteur.fr)

Activity Reports 2002 - Institut Pasteur
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