Investigations of biomolecular interactions by isothermal titration calorimetry

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Several researchers in the Department of Biochemistry of the Université de Montréal are investigating important macromolecular complexes and characterizing their binding affinity and specificity. These complexes include protein/protein, protein/RNA, protein/DNA, metal/protein, RNA/RNA and protein/ligand complexes. Formation of these complexes is important for a wide variety of molecular processes such as transcriptional or translational regulation, RNA processing, glycolysis, viral infection, and cancer. Specific studies include 1) Transcriptional control of plant defense responses; 2) messenger RNA transport and localization in mammals; 3) Structural investigation of reaction complexes in Schiff-base forming aldolases; 4) Macromolecular interactions regulating the phosphorylation of the carboxyl-terminal domain of RNA polymerase II; 5) Structural investigations of large RNAs 6) Structure function studies of the bacterial mercury detoxification pathway. In order to fully understand the role of specific functional groups within important macromolecular complexes, it is crucial to be able to thoroughly characterize both the binding affinity and the specificity of the complex formation.  Over the last ten years, there have been a number of significant technical advancements in measuring the binding affinities of macromolecular complexes in the micromolar to nanomolar range.  There are now several techniques that enable researchers to accurately and reproducibly determine binding constants in these ranges such as fluorescence anisotropy (FA), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC).  ITC is particularly interesting as it is the only technique that allows direct measurement of true binding affinity since it does not require any modification of the molecules of interest and the measurements can be done with all of the molecules free and in solution. We are requesting a microcalorimeter so that we can fulfill a pressing need for recording ITC experiments with our macromolecular complexes in our own department.  These ITC experiments are crucial to further our understanding of the structure function studies ongoing within our laboratories.