Lee Andrew L, Sapienza Paul J
Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
Front Mol Biosci. 2018 May 25;5:47. doi: 10.3389/fmolb.2018.00047. eCollection 2018.
Thymidylate synthase (TS) is a homodimeric enzyme with evidence for negative regulation of one protomer while the other protomer acts on substrate, so called half-the-sites reactivity. The mechanisms by which multisubunit allosteric proteins communicate between protomers is not well understood, and the simplicity of dimeric systems has advantages for observing conformational and dynamic processes that functionally connect distance-separated active sites. This review considers progress in overcoming the inherent challenges of accurate thermodynamic and atomic-resolution characterization of interprotomer communication mechanisms in symmetric protein dimers, with TS used as an example. Isothermal titration calorimetry (ITC) is used to measure ligand binding cooperativity, even in cases where the two binding enthalpies are similar, and NMR spectroscopy is used to detect site-specific changes occurring in the two protomers. The NMR approach makes use of mixed-labeled dimers, enabling protomer-specific detection of signals in the singly ligated state. The rich informational content of the NMR signals from the singly ligated state, relative to the apo and saturated states, requires new considerations that do not arise in simple cases of 1:1 protein-ligand interactions.
胸苷酸合成酶（TS）是一种同二聚体酶，有证据表明一个亚基存在负调控，而另一个亚基作用于底物，即所谓的半位点反应性。多亚基别构蛋白在亚基之间进行通讯的机制尚未完全了解，二聚体系统的简单性有利于观察功能上连接远距离活性位点的构象和动态过程。本综述以TS为例，探讨了在克服对称蛋白质二聚体中亚基间通讯机制的精确热力学和原子分辨率表征所面临的固有挑战方面取得的进展。等温滴定量热法（ITC）用于测量配体结合协同性，即使在两种结合焓相似的情况下也是如此，核磁共振波谱法用于检测两个亚基中发生的位点特异性变化。核磁共振方法利用混合标记的二聚体，能够在单配体状态下对亚基特异性信号进行检测。相对于无配体和饱和状态，单配体状态下核磁共振信号丰富的信息内容需要新的考量，而这在简单的1:1蛋白质-配体相互作用情况中不会出现。
