Monitoring Biomolecular Interactions using Isothermal Titration Calorimetry

蛋白质的正常功能与其在正确的时间以正确的亲和力识别和结合特定目标分子的能力密切相关。申请人的研究兴趣各不相同，但仍然集中在蛋白质的表征及其与靶分子（如离子、金属辅助因子、小分子和其他蛋白质）在健康和疾病中的相互作用。为了更全面地了解这些相互作用，我们需要详细了解相关的大分子参数，如强度、所涉及的分子数量和各种能量系数。等温滴定量热法（ITC）是一种可靠的生物物理方法，可以直接测量溶液中的这些参数。此外，ITC很有吸引力，因为它不需要对所研究的分子进行修饰，而修饰可能会破坏相互作用的程度。ITC设计的技术进步大大减少了样品体积，允许使用微量热计直接测量溶液中的蛋白质-配体相互作用。我们需要这样的设备来满足女王大学探索新型蛋白质相互作用的结构、生物物理和化学基础的迫切需要。从这些实验中获得的数据将使人们更好地了解它们在细胞功能和调节中的作用。本提案的资助将为高素质人员（HQP）提供持续的机会，以获得现代蛋白质结构和功能研究中最广泛使用的方法之一的有价值的培训。这项工作不仅将扩大HQP在这一研究领域的培训和合作工作，而且还将使申请人在绿色化学、细胞调节和农业领域做出具有更广泛意义的关键发现。

The proper functioning of a protein is intimately associated with its ability to recognize and bind to specific target molecules at the right time and with the right affinity. The applicants' research interests are diverse, but nonetheless converge on to the characterization of proteins and their interactions with target molecules such as ions, metal co-factors, small molecules and other proteins in health and disease. To gain a more comprehensive understanding of these interactions one requires detailed knowledge about the associated macromolecular parameters such as the strength, the number of molecules involved, and various energetic coefficients. Isothermal titration calorimetry (ITC) is a proven and robust biophysical methodology that can directly measure these parameters in solution. Moreover, ITC is attractive because it does not require modification of the molecules under investigation, which can potentially disrupt the extent of interaction. Technical advances in ITC design have allowed for significantly reduced sample volumes, allowing for direct measurement of protein-ligand interactions in solution using a microcalorimeter. We are requesting such a device to fulfill a pressing need to explore the structural, biophysical and chemical basis of novel protein interactions at Queen's University. Data derived from these experiments will lead to an improved understanding of their role in cellular function and regulation. Funding of this proposal will provide a continuing opportunity for the high-qualified personnel (HQP) to gain valuable training with one of the most widely used approaches in modern protein structure and function studies. This work will not only expand HQP training and collaborative work in this area of research, but will also position the applicants in making key discoveries that have broader implications in the fields of green chemistry, cellular regulation, and agriculture.