Label-free measurement of protein-ligand interactions for biochemistry, structural biology, immunology, engineering, and chemical biology.

分子相互作用的直接测量对于理解蛋白质及其配体是必不可少的。虽然许多工具可以使用替代测量（例如荧光置换和酶联测定）在复杂的生物环境中提供估计，但其他工具可以测量分离的蛋白质-配体相互作用，但需要标记（例如微尺度热泳）或固定（例如表面等离子体共振）。前者通常是无标记的，但遭受由差异蛋白质表达、化合物螯合和其他间接因素引起的并发症，这些因素导致显著的误差;后者是高度准确的，但分子的修饰会影响测量的有效性。等温滴定量热法（ITC）的独特之处在于不需要对任何一种组分进行任何修饰，而直接提供由两种组分结合产生的热力学参数。定量蛋白质相互作用需要大量（mg）分离蛋白质用于标准ITC，但马尔文MicroCal PEAQ-ITC，一种nanoITC（nITC）可用于低µg量的蛋白质，与我们实验室蛋白质生产中容易获得的量一致。该工具在UWindsor，在我们的2小时距离最近的邻居，西方，或在我们的跨境合作伙伴韦恩州立大学和奥克兰大学缺乏，并将被申请人和频繁用户广泛使用，以研究从生物活性离子，核酸，碳水化合物，肽和蛋白质到超分子组装体，如纳米颗粒，病毒，和洞穴，并培养我们的多元化和跨学科的下一代药物化学和生物化学研究人员。这也引起了这些上市机构合作伙伴对未来合作的兴趣。nITC是由11名NSERC资助的研究人员（两名申请人和九名经常使用者）组成的团队提出的，其中包括科学和工程三个部门的七名早期职业调查人员，涵盖蛋白质科学，细胞生物学，免疫学，药物发现和新型生物材料等领域。研究人员进行了突破性的创新科学，但缺乏能够研究广泛生物分子相互作用的最先进仪器阻碍了团队的研究进展。进一步延迟获得这种能力对HQP培训有严重影响，并将对NSERC研究计划产生长期的负面影响。 该设备对于培训至少80名现任HQP至关重要。所要求的文书对几个合作性、多学科团队赠款和工业合同的进展至关重要，这些合同包括药物发现、生物技术、细胞生物学和当前抗击COVID-19大流行病领域的国家和国际伙伴关系。

The direct measurement of molecular interactions is essential for understanding proteins and their ligands. Although many tools can provide an estimate in a complex biological milieu using a surrogate measurement (e.g. fluorescent displacement and enzyme-linked assays), others can measure isolated protein-ligand interactions but require labelling (e.g. microscale thermophoresis) or immobilization (e.g. surface plasmon resonance). The former are often label-free but suffer from complications arising from differential protein expression, compound sequestration, and other indirect factors that lead to significant error; the latter are highly accurate, but the modifications of the molecules can affect the validity of the measurement. Isothermal titration calorimetry (ITC) is unique in not requiring any modification of either partner, while directly providing the thermodynamic parameters arising from the binding of two components. Quantifying protein interactions requires significant amounts (mgs) of isolated protein for standard ITC, but the Malvern MicroCal PEAQ-ITC, a nanoITC (nITC) can work with low µg amounts of protein, consistent with the amounts readily available from protein production from our labs. This tool is lacking at UWindsor, at our 2-hours distant closest neighbour, Western, or at our cross-border partners at Wayne State and Oakland University, and will be extensively used by the applicants and frequent users to study a wide range of molecules from biologically active ions, nucleic acids, carbohydrates, peptides and proteins, to supramolecular assemblies such as nanoparticles, viruses, and cavitands and to train our diverse and interdisciplinary next generation of medicinal chemistry and biochemistry researchers. This has also drawn interest from partners at those listed institutions for future collaboration. The nITC is requested by a team of eleven NSERC-funded researchers (two applicants and nine frequent users), including seven early-career investigators across three departments in both science and engineering, spanning the fields of protein science, cellular biology, immunology, drug discovery and novel biomaterials. The researchers conduct ground-breaking innovative science, but the lack of a state-of-the-art instrument capable of studying a wide range of biomolecular interactions hampers the team's research progress. Further delay in the acquisition of this capability has severe implications for HQP training, and will have a long-lasting negative impact on NSERC research programs. This equipment is critical for the training of at least 80 current HQP. The requested instrument is central to the progress of several collaborative, multidisciplinary team grants and industrial contracts that include national and international partnerships in the fields of drug discovery, biotechnology, cellular biology, and the current fight against the COVID-19 pandemic.