Probing Substrate-Loaded Carrier Proteins by Nuclear Magnetic Resonance.
Probing Substrate-Loaded Carrier Proteins by Nuclear Magnetic Resonance.
复制标题
通过核磁共振探测负载底物的载体蛋白。
DOI：
10.1007/978-1-0716-3214-7_12
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Frueh,DominiqueP
中科院分区：
文献类型：
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作者：
Arya,Neeru;Marincin,KennethA;Frueh,DominiqueP
载体蛋白 (CP) 是非核糖体肽合成酶 (NRPS) 的核心角色，因为它们与所有催化结构域相互作用，并且它们共价地固定产生最终产物的底物和中间体。因此，CP 及其合作伙伴域如何根据 CP 货物的功能相互识别和互动对于理解和设计 NRPS 至关重要。然而，持有底物的不稳定硫酯键的快速水解对确定结构域识别的分子机制的分子和生物物理研究提出了挑战。在本章中，我们描述了一种抵消水解的方案，并通过核磁共振（NMR）光谱在原子水平上研究负载的载体蛋白。该方法依靠 NMR 管中的腺苷酸化域原位加载 CP，以达到稳定状态的底物负载 CP。我们描述了评估样品完整性和维持 CP 负载所需的对照和实验读数。我们的方法为进行后续 NMR 实验提供了基础，并获得单独或存在其他域的底物负载 CP 的动力学、热力学、动态和结构参数。
Carrier proteins (CPs) are central actors in nonribosomal peptide synthetases (NRPSs) as they interact with all catalytic domains, and because they covalently hold the substrates and intermediates leading to the final product. Thus, how CPs and their partner domains recognize and engage with each other as a function of CP cargos is paramount to understanding and engineering NRPSs. However, rapid hydrolysis of the labile thioester bonds holding substrates challenges molecular and biophysical studies to determine the molecular mechanisms of domain recognition. In this chapter, we describe a protocol to counteract hydrolysis and study loaded carrier proteins at the atomic level with nuclear magnetic resonance (NMR) spectroscopy. The method relies on loading CPs in situ, with adenylation domains in the NMR tube, to reach substrate-loaded CPs at steady state. We describe controls and experimental readouts necessary to assess the integrity of the sample and maintain loading on CPs. Our approach provides a basis to conduct subsequent NMR experiments and obtain kinetic, thermodynamic, dynamic, and structural parameters of substrate-loaded CPs alone or in the presence of other domains.