Structure of the ADRP domain of SARC-CoV nsp3. FSPS studies confirmed its phophatase activity |
Biomedical structural proteomics at FSPS
Sample Production
An expression-ready clone library has been created and maintained containing all known and predicted coding regions for most available strains. The library has been constructed using experimentally validated clones for E. coli, baculovirus and mammalian expression systems and is updated with publicly released information. Sequencing of clone products ensures quality assurance. Clones from the FSPS library are used for protein production through primary expression strategies and predefined alternate pathways. Sufficient amounts of soluble folded proteins, domains, and other constructs are expressed and purified for functional and structural analysis. Analytical and biophysical characterization approaches are used to ensure high quality products.
Structural Proteomics
The structures and substructures of SARS-CoV proteins and complexes are being determined by using either NMR or X-ray crystallography. The method used is based on size and protein behavior. Rapid optimization strategies through variation in clones, expression systems, and crystallization conditions enhance the likelihood of successful structure determination. In addition, a set of pathways outside the traditional automated structure determination pipelines have been developed for more difficult structures, such as glycosylated and membrane-bound proteins. All structures are fully validated, annotated, and released according to established standards of the NIH Protein Structure Initiative. To date FSPS has solved structures for six SARS-CoV protein constructs and has assigned fold and/or function to the six Open Reading Frames.
Functional Proteomics
Several approaches are used to probe the function of each protein and its interactions, such as the characterization of the life cycle and host response; identification and characterization of protein-protein interactions, and identification and characterization of ligands. Cryo-electron microscopy and iterative single-particle image reconstruction is used to produce 2-D and 3-D maps of viral coat protein complexes from formalin-inactivated native and fusion-activated virus preparations. The effects on the viral lifecycle and intracellular host response for each SARS-CoV protein will be defined using a combination of viral cDNA cloning, site-directed mutagenesis, antisense functional mapping and microarray-based functional mapping using live virus produced from cDNA knockouts. In addition, SARS-CoV cellular receptors and the entry process are being identified and characterized using ligand "fishing" techniques (e.g. TAP-TAG approaches).
Computational Biology
FSPS continues to collect and integrate existing data to derive hypotheses that are being experimentally tested utilizing the methods in sample production, functional proteomics, and structural proteomics. These bioinformatics services support the pipeline of activities from design of primers to structural analysis and are done through sequence analysis that facilitates the in silico discovery phase of functional proteins, protein domains and substrates/cofactors/inhibitors, and large scale virtual docking to identify possible inhibitors of solved structures. Bioinformatics will additionally be used to reconstruct essential pathways and important networks involving SARS-CoV proteins, to produce a prioritized list of possible drug/vaccine targets, and to interact with the public data resources to optimize dissemination of data to the public.
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Funded by NIH, National Institute of Allergy and Infectious Diseases 