Microcalorimetric analysis of biomolecular interactions

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Research programs in the laboratories of the four applicants (Drs. McKenna, Stetefeld, Mark, and O'Neil) at the University of Manitoba are focused on investigating a variety of interesting biological systems including viral nucleic acid recognition, chlorophyll biosynthesis, antibacterial resistance, coiled-coil stability, and regulation of viral replication. The common feature that links each of these research groups is the central role that protein-ligand interactions play in regulating the biological system of interest. Therefore, defining the features that govern the interaction between proteins and their ligands (including other proteins, nucleic acids, small molecules, or ions) is a crucial piece of information required to fully understand how a given protein performs it biological function. Rapid advances in the field of structural biology have allowed research groups, including those 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. However, knowledge of static protein structure alone does not ensure accurate prediction of function and biological activity, and must therefore be combined with a number of technologies to integrate the steps from protein structure determination to biochemical and biophysical characterization. As an integral component of this strategy, calorimetric approaches that allow determination of thermodynamic features of a protein-ligand interaction have emerged as a central feature of protein structure/function research laboratories. The requested Isothermal Titration Calorimeter (ITC) provides rapid, quantitative, and accurate information on the energetic forces that drive biomolecular associations. When combined with the already existing infrastructure accessible to our research groups, ITC provides a powerful means to analyze thermodynamic parameters of biomolecular interactions to study molecular details of protein binding and protein stability. No ITC instrument is currently available at the University of Manitoba. Therefore there is a clear and rapid need for the acquisition of this crucial piece of infrastructure.