Sunday, May 18, 2025
5/18/2025
Isothermal Titration Calorimeter - We request the Affinity Isothermal Titration Calorimeter (ITC) from TA Instruments (Waters Corporation) which is robust (user error is unlikely to harm the instrument) and sensitive. ITC provides accurate, label-free, and direct measurement of affinity between two molecules, number of binding sites, enthalpy and entropy of binding, and change of heat capacity upon binding. ITC will increase the rigor of binding measurement performed at the University of Montana (UM) as other direct binding measurement methods currently available at UM require surface attachment or labeling of one of the binding partners and are less sensitive for similar sized binding partners (e.g., surface plasmon resonance and fluorescence-based methods). UM-available indirect binding methods require calculation of affinity using assumptions that often cannot be verified (e.g., competitive fluorescence anisotropy). ITC will advance the research objectives of the UM research community including six minor user projects, and the 3 major user NIH-funded projects described below: 1) Measurement of affinity between a family of transcription factors (nuclear receptors) and their cofactors. Such data will aid in design of the next generation of nuclear receptor drugs that cause less adverse effects. This is important because nuclear receptors bind about 1 out of every 6 approved drugs and many cause treatment-limiting adverse effects. ITC will provide a rigorous check of the currently used indirect affinity measurement (competitive fluorescence anisotropy). Such a check is critical because accurate affinity measurement is fundamental to testing the central hypothesis of this work. 2) Work to accurately predict how mutations affect protein stability and the most prevalent protein conformations. Such prediction is fundamental to many areas, including understanding the impact of genetic information on health. ITC provides a key measure of the algorithm’s accuracy for prediction of a mutation's effect on protein conformation prevalence. The percentage of a model protein found in one of these conformations directly impacts its affinity for a ligand, allowing determination of the portion of the protein in that conformation, and thus the prediction accuracy of the algorithm. In addition, because ITC measurements provide both the change in enthalpy and entropy, ITC data permits extrapolation of binding constants obtained at one temperature to any other temperature for efficient comparison with other data. 3) Work to measure binding affinity between the protein PlzA from the bacteria that causes Lyme disease (Borrelia burgdorferi) and its putative and known endogenous ligands (c-di-GMP and RNA). PlzA is essential for infectivity in mice, however PlzA’s ability to bind c-di-GMP is not, pointing to the probable importance of RNA binding. ITC and PlzA mutants will be used to identify PlzA residues and determinants that mediate RNA binding. In addition, ITC will be used to evaluate the binding of different RNA ligands, including recently identified native B. burgdorferi RNAs.
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