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The completion and near completion of the sequencing phase of the genome projects has ushered in the age of proteomics, the study of all gene products in an organism. This flood of sequence information coupled with recent advances in molecular and structural biology have led to the concept of 'structural genomics', the determination of protein three-dimentionsl structures on a genome-wide scale in order to obtain insight on protein function.
Our team is part of the Ontario Centre for Structural Proteomics (OCSP)- a Canadian and NIH-funded Center affiliated with the Midwest Center for Structural Genomics (MCSG). Our main goal is to determine three-dimentional structures, using X-ray crystallography, of protein families that can not be predicted from existing protein structure data (PDB). This approach should eventually lead to completion of the protein folding space and elucidation of the function of these proteins.
Since 1998 we focus on the development of technology and methodology for large-scale, high throughput protein sample preparation and structure solving. Our technological aims are:
- Effective valid protein target identification in sequenced genomes
- Development of highly parallel and cost-effective methods to clone, express and purify proteins on a scale required for structural studies
- Improvement of protein crystallization process by creating more effective crystallization screens
- Acceleration of 3D protein structures solving process
- Development of effective database
Our group selects protein targets from all three kingdoms of life (Bacteria, Archaea and Eukarya), with an emphasis on previously unknown folds and on proteins from disease-causing organisms that are more challenging for structural studies. One of the main criteria for selection is the absence of strong sequence similarity (less that 30% ID) to proteins with known 3D structure deposited into PDB. Our project has already covered more than 3000 structurally uncharacterized proteins from Escherichia coli, Thermotoga maritima, Pseudomonas aeruginosa, Thermoplasma acidophilum, Helicobacter pillory, Archeoglobus fulgidus, Methanobacterium thermoautothrophicum, Saccharomyces cerevisiae and other completed genomes.
We developed and standardized the procedure for high throughput protein purification by affinity chromatography, which permits us to purify 10-12 different proteins in parallel. We have also developed a pipeline for screening for crystallization conditions, which allows us to obtain initial crystallization conditions for 30-40% of purified proteins. Although not completely automated, this strategy remains one of the most efficient in the field of structural proteomics.
Currently our pipeline produces more that 60 structures of novel protein families representatives per year. This information is used for accurate structure prediction/modeling of several hundred homologues of these proteins.
Our group benefits from collaborations with all of the investigators of the NESG and MCSG as well as many structural and functional biology labs in Canada and around the world. Coupled with this effort is a program in enzyme genomics, focusing on characterizing enzymatic activities for proteins purified for structural proteomics.
An important use of three-dimentionsl structural information of proteins is to uncover clues as to a protein's function that are not detectable from sequence analysis. The long term goal is to determine experimental structures for all proteins, becuase subtle differences in protein structure contribute to the diversity and complexity of life.
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