Isothermal Titration Calorimetry for Studying Macromolecular Interactions

我们NSERC dg资助的研究项目的统一主题是建立生物分子相互作用（蛋白质与核酸的相互作用，小分子与蛋白质和核酸的相互作用等）在生物体（如病毒，细菌，酵母和人类）的细胞过程中的作用。我们在推进研究和培训活动方面面临的关键挑战是缺乏研究生物分子相互作用所需的关键基础设施。没有这一点，我们就无法进行跨学科的研究来培养UofL的学生和研究人员。结构生物学方法的最新进展扩大了我们对生物分子如何相互交流和功能的理解。然而，为了研究生物分子复合物的高分辨率结构，亲和力和化学计量学的信息是必不可少的。因此，我们建议安装等温滴定量热法（ITC）。这种广泛使用和可靠的仪器有助于直接测量由于生物分子相互作用而产生或吸收的热量。ITC可以直接、快速、准确地测定结合亲和力，并且可以研究各种天然状态下未改变的生物分子的相互作用。在单个实验中，ITC可以精确地确定大范围的结合常数、反应化学计量以及焓和熵等热力学参数。除了结合亲和力外，ITC还可以帮助阐明分子相互作用的机制。UofL没有ITC工具；因此，迫切需要这种关键的基础设施。结合UofL现有的基础设施，ITC的安装将加速我们实验室和我们合作伙伴实验室的HQP培训，并显著推进研究和培训活动。

The unifying theme of our NSERC DG-funded research programs is to establish the role of biomolecular interactions (interactions of proteins with nucleic acids, small molecule interactions with proteins and nucleic acids, etc.,) in cellular processes in organisms such as viruses, bacteria, yeast, and humans. The pivotal challenge we face in advancing our research and training activities is the lack of critical infrastructure we require to study biomolecular interactions. Without this, we cannot perform interdisciplinary research to train students and researchers at the UofL. Recent advances in structural biology methods have expanded our understanding of how biomolecules communicate with each other and function. However, to study the high-resolution structures of biomolecular complexes, information on affinity and stoichiometry is essential. Therefore, we propose to install isothermal titration calorimetry (ITC). This widely used and reliable instrument aids a direct measure of the heat generated or absorbed as a result of biomolecular interaction. ITC allows the direct, rapid, and accurate determination of binding affinities and it is possible to study interactions of a wide range of unaltered biomolecules in their native state. In a single experiment, ITC accurately determines a wide range of binding constants, reaction stoichiometry, as well as thermodynamic parameters such as enthalpy and entropy. In addition to binding affinities, ITC can help elucidate the mechanisms underpinning molecular interactions. The UofL does not have an ITC instrument; hence, there is an urgent need for this critical infrastructure. In conjunction with the existing infrastructure at the UofL, the installation of ITC will accelerate HQP training in our laboratories, and in our collaborator's laboratories, and significantly advance research and training activities.