Unit: Biochemistry and Biophysics of Macromolecules
Director: Deshmukh N. GOPAUL
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 constants as well as the thermodynamic parameters are determined by fluorescence spectroscopy and microcalorimetry. We are also interested in solving the structures and understanding the biochemistry of proteins involved in host invasion of Plasmodium falciparum, and also in the signaling pathway of NF-KB.
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.
We have been able to produce soluble proteins for both Int1 and Int4 in E. coli and are in the process of screening crystallization conditions using robotics. 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. We have also been able to produce one of the proteins involved in the invasion of the host by Plasmodium falciparum and are now trying to solubilize the protein in order to proceed with crystallization trials. We have also been successful in characterizing crystalline forms of the protein involved in the NF-KB pathway, but we need to improve the quality of the data.
Reaction pathway for site specific recombination catalyzed by Cre from Phage P1
(i) Binding synapse of DNA substrates with Cre enzyme (ii) Cleavage by conserved Try324 (iii) Holliday junction intermediate formation (iv) Isomerization of the Holliday junction (v) Second strand cleavage by previously inactive Try324 (vi) Recombined products
Keywords: Site-specific recombinase, integase, X-ray crystallography, structure, biochemistry