The requested low volume isothermal titration calorimeter (ITC) will be primarily used by NIH funded faculty investigators in the University of Pittsburgh School of Medicine, who are broadly focused on understanding the functions of biological macromolecules at the molecular level. The only ITC instrument within the School of Medicine is not broadly accessible and requires 2.0 milliliters to fill the sample cell. The large volumes required are often prohibitive for samples that are difficult to express or costly to produce, especially when the interactions are weak and high concentrations are required in both the sample cell and syringe. The requested instrument, which has been redesigned to have improved heat sensing signal-to-noise, requires only about 0.28 milliliters to fill the sample cell, thus lowering the sample requirements by about seven-fold. These improvements will allow investigators to perform a greater number of measurements with the same or less amount of material, which are often available in only limited amounts and are time- and resource-intensive to prepare. This, along with other improvements in the instrument relative to its predecessor, such as a roughly two-fold improvement in the response time and a nearly fully automated procedure for washing the sample cell and syringe, greatly expands the applicability of this instrument to a much broader range of biomolecular systems. The requested instrument will be housed in the Department of Structural Biology and its use, operation, and maintenance will be overseen by a junior investigator, who is well-versed in the application of ITC to diverse macromolecular systems. The costs for operating and maintaining the instrument will be borne by the users, although the University will cover any shortfalls in the unlikely event this is necessary. The major and minor users of the instrument are focused on a) applying the instrument as a complement to NMR spectroscopy for studying ligand binding in solution, b) investigating protein-drug interactions, c) validating the integrity of recombinant proteins for structural studies, and d) investigating the binding properties of signaling receptors or engineered signaling receptors with multiple domains. These studies are providing either the groundwork or directly contributing to efforts that will aid and assist in the development of new and more effective therapies for viral diseases, such as AIDS, and other diseases, such as cancer and neurological disorders. The instrument will be made available for use by all NIH funded investigators both within the School of Medicine and throughout the University of Pittsburgh, and is expected to have a broad impact in diverse areas of biomedical science.
The requested low volume isothermal titration calorimeter will enable measurements of the overall thermodynamics and stoichiometry of binding for biological macromolecules to their various partners, which includes small molecule co-factors, substrates, and drugs, as well as proteins, RNA, and DNA. The ability to detect and accurately quantify these interactions is essential for many purposes, such as validating binding thermodynamics and stoichiometry predicted from structural, computational, or drug screening studies and assessing how this binding is altered as the molecules or solution conditions vary.