Efficient Protein and Protein-Ligand complex characterization via SEC-MALS

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Research programs in the laboratories of the four applicants (Drs. Stetefeld, McKenna, Khajehpour and O'Neil) are focused on investigating a variety of interesting biological systems including integral membrane proteins with catalytic activities and integral membrane protein channels, a chlorophyll biosynthesis enzyme, monoclonal antibodies from llama, viral nucleic acid recognition proteins, a regulatory bacterial kinase and a transcription activator. The common feature that links each of these research groups is the structural and functional characterization of specific protein samples and their potential ligands to investigate their role in regulating the biological systems of interest. Rapid advances in the field of structural biology have allowed the research groups included in this proposal to determine high-resolution three-dimensional images of protein-ligand complexes, and begin to characterize their biochemical function and mechanism in terms of key molecular features that dictate the interaction. As an integral component of this strategy, light scattering approaches in combination with size exclusion chromatography that allow the absolute determination of molecular features of a protein in solution and the relevant protein-ligand complex have emerged as a central feature of protein structure/function research laboratories. The requested Size Exclusion Chromatography-Multiple Angle Light Scattering instrument (SEC-MALS) provides a valuable tool (i) to explore the absolute molecular mass information at every point of the size-separated sample, (ii) to identify protein and relevant protein-ligand complexes and their association state, (iii) to detect higher-order aggregates and protein conjugates, and (iv) to detect the molecular mass of the conjugates to rapidly and unambiguously determine stoichiometry. When combined with the already existing infrastructure accessible to our research groups, SEC-MALS provides a powerful means to analyze biomolecular interactions and to study molecular details of protein binding and protein stability. No SEC-MALS instrument is currently available in the Province of Manitoba. Therefore there is a clear and urgent need for the acquisition of this crucial piece of infrastructure.