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

用于生物化学、结构生物学、免疫学、工程和化学生物学的蛋白质-配体相互作用的无标记测量。

• 批准号: RTI-2022-00597
• 负责人: Trant, John
• 金额: $ 10.93万
• 依托单位: University of Windsor
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743045
• 关键词: Label; free; measurement; protein; ligand

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.

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。虽然许多工具可以在复杂的生物环境中使用替代测量（如荧光位移和酶联测定）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（如微尺度热电泳）或固定（如表面等离子体共振）。前者通常是无标记的，但由于差异蛋白表达、化合物隔离和其他间接因素导致显著错误而引起并发症；后者精度很高，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方进行任何修改，而直接提供由两组分结合产生的热力学参数。对于标准ITC，定量蛋白质相互作用需要大量（毫克）的分离蛋白质，但Malvern MicroCal PEAQ-ITC，一种纳米ITC （nITC）可以使用低微克量的蛋白质，与我们实验室蛋白质生产中现成的量一致。这一工具在温莎大学、距离我们2小时的近邻西部大学、或我们的跨境合作伙伴韦恩州立大学和奥克兰大学都是缺乏的，将被申请人和经常使用的用户广泛使用，以研究各种分子，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，如纳米粒子、病毒、培养我们多样化和跨学科的下一代药物化学和生物化学研究人员。这也吸引了这些上市机构的合作伙伴对未来合作的兴趣。nITC是由nserc资助的11名研究人员（2名申请者和9名经常使用者）组成的团队申请的，其中包括7名早期职业研究人员，横跨3个科学和工程部门，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领域。研究人员进行了突破性的创新科学，但是缺乏能够研究广泛生物分子相互作用的最先进的仪器阻碍了该团队的研究进展。这种能力的进一步延迟将对HQP训练产生严重影响，并将对NSERC研究计划产生长期的负面影响。该设备对至少80名HQP的培训至关重要。所请求的工具对于若干多学科合作团队赠款和工业合同的进展至关重要，其中包括药物发现、生物技术、细胞生物学和当前抗击COVID-19大流行等领域的国家和国际伙伴关系。