Investigations of biomolecular interactions by isothermal titration calorimetry

蒙特利尔大学生物化学系的几位研究人员正在研究重要的大分子复合物，并表征其结合亲和力和特异性。这些复合物包括蛋白质/蛋白质、蛋白质/RNA、蛋白质/DNA、金属/蛋白质、RNA/RNA和蛋白质/配体复合物。这些复合物的形成对于多种分子过程如转录或翻译调节、RNA加工、糖酵解、病毒感染和癌症是重要的。具体研究包括：1）植物防御反应的转录调控; 2）信使RNA在哺乳动物中的转运和定位; 3）形成醛缩酶的席夫碱反应复合物的结构研究; 4）调节RNA聚合酶II羧基端结构域磷酸化的大分子相互作用; 5）大RNA的结构研究6）细菌汞解毒途径的结构功能研究。为了充分了解重要大分子复合物中特定官能团的作用，关键是能够彻底表征复合物形成的结合亲和力和特异性。 在过去的十年中，在测量微摩尔至纳摩尔范围内的大分子复合物的结合亲和力方面取得了许多重大的技术进步。 现在有几种技术，使研究人员能够准确和可重复地确定这些范围内的结合常数，如荧光各向异性（FA），表面等离子体共振（SPR）和等温滴定量热法（ITC）。 ITC是特别令人感兴趣的，因为它是允许直接测量真实结合亲和力的唯一技术，因为它不需要对感兴趣的分子进行任何修饰，并且可以在所有分子游离和在溶液中的情况下进行测量。我们需要一台微量热计，以便我们能够满足记录ITC实验的迫切需要，这些实验是在我们自己的部门用我们的大分子复合物进行的。 这些ITC实验对于进一步理解我们实验室内正在进行的结构功能研究至关重要。

Several researchers in the Department of Biochemistry of the Université de Montréal are investigating important macromolecular complexes and characterizing their binding affinity and specificity. These complexes include protein/protein, protein/RNA, protein/DNA, metal/protein, RNA/RNA and protein/ligand complexes. Formation of these complexes is important for a wide variety of molecular processes such as transcriptional or translational regulation, RNA processing, glycolysis, viral infection, and cancer. Specific studies include 1) Transcriptional control of plant defense responses; 2) messenger RNA transport and localization in mammals; 3) Structural investigation of reaction complexes in Schiff-base forming aldolases; 4) Macromolecular interactions regulating the phosphorylation of the carboxyl-terminal domain of RNA polymerase II; 5) Structural investigations of large RNAs 6) Structure function studies of the bacterial mercury detoxification pathway. In order to fully understand the role of specific functional groups within important macromolecular complexes, it is crucial to be able to thoroughly characterize both the binding affinity and the specificity of the complex formation.  Over the last ten years, there have been a number of significant technical advancements in measuring the binding affinities of macromolecular complexes in the micromolar to nanomolar range.  There are now several techniques that enable researchers to accurately and reproducibly determine binding constants in these ranges such as fluorescence anisotropy (FA), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC).  ITC is particularly interesting as it is the only technique that allows direct measurement of true binding affinity since it does not require any modification of the molecules of interest and the measurements can be done with all of the molecules free and in solution. We are requesting a microcalorimeter so that we can fulfill a pressing need for recording ITC experiments with our macromolecular complexes in our own department.  These ITC experiments are crucial to further our understanding of the structure function studies ongoing within our laboratories.