Microcalorimetric analysis of biomolecular interactions

马尼托巴大学的四位申请人（McKenna、Stetefeld、Mark和O 'Neil博士）的实验室研究项目集中于研究各种有趣的生物系统，包括病毒核酸识别、叶绿素生物合成、抗菌抗性、卷曲螺旋稳定性和病毒复制调控。连接这些研究小组的共同特征是蛋白质-配体相互作用在调节感兴趣的生物系统中发挥的核心作用。因此，定义控制蛋白质及其配体（包括其他蛋白质，核酸，小分子或离子）之间相互作用的特征是完全理解给定蛋白质如何执行其生物学功能所需的关键信息。结构生物学领域的快速发展使得包括本提案中的研究小组在内的研究小组能够确定蛋白质-配体复合物的高分辨率三维图像，并开始根据决定相互作用的关键分子特征来表征其生化功能和机制。然而，仅仅了解静态蛋白质结构并不能确保准确预测功能和生物活性，因此必须与许多技术相结合，以整合从蛋白质结构测定到生物化学和生物物理表征的步骤。作为这一战略的一个组成部分，量热法，允许确定的热力学特征的蛋白质-配体相互作用已经成为蛋白质结构/功能研究实验室的一个中心特征。所要求的等温滴定量热仪（ITC）可提供有关驱动生物分子缔合的能量力的快速、定量和准确的信息。当与我们的研究小组现有的基础设施相结合时，ITC提供了一种强大的手段来分析生物分子相互作用的热力学参数，以研究蛋白质结合和蛋白质稳定性的分子细节。马尼托巴大学目前没有国际贸易中心的仪器。因此，显然需要迅速购置这一关键的基础设施。

Research programs in the laboratories of the four applicants (Drs. McKenna, Stetefeld, Mark, and O'Neil) at the University of Manitoba are focused on investigating a variety of interesting biological systems including viral nucleic acid recognition, chlorophyll biosynthesis, antibacterial resistance, coiled-coil stability, and regulation of viral replication. The common feature that links each of these research groups is the central role that protein-ligand interactions play in regulating the biological system of interest. Therefore, defining the features that govern the interaction between proteins and their ligands (including other proteins, nucleic acids, small molecules, or ions) is a crucial piece of information required to fully understand how a given protein performs it biological function. Rapid advances in the field of structural biology have allowed research groups, including those in this proposal, to determine high-resolution three-dimensional images of protein-ligand complexes, and begin to characterize their biochemical function and mechanism in terms of key molecular features that dictate the interaction. However, knowledge of static protein structure alone does not ensure accurate prediction of function and biological activity, and must therefore be combined with a number of technologies to integrate the steps from protein structure determination to biochemical and biophysical characterization. As an integral component of this strategy, calorimetric approaches that allow determination of thermodynamic features of a protein-ligand interaction have emerged as a central feature of protein structure/function research laboratories. The requested Isothermal Titration Calorimeter (ITC) provides rapid, quantitative, and accurate information on the energetic forces that drive biomolecular associations. When combined with the already existing infrastructure accessible to our research groups, ITC provides a powerful means to analyze thermodynamic parameters of biomolecular interactions to study molecular details of protein binding and protein stability. No ITC instrument is currently available at the University of Manitoba. Therefore there is a clear and rapid need for the acquisition of this crucial piece of infrastructure.