Isotopically discriminated NMR spectroscopy: A tool for investigating complex protein interactions in vitro
Isotopically discriminated NMR spectroscopy: A tool for investigating complex protein interactions in vitro
复制标题
DOI：
10.1021/ja070505q
复制
发表时间：
2007-05-23
影响因子：
15
通讯作者：
Bermel, Wolfgang
中科院分区：
文献类型：
--
作者：
Golovanov, Alexander P.;Blankley, Richard T.;Bermel, Wolfgang
提出了一种新的NMR方法来观察体外多组分蛋白质-蛋白质-配体相互作用，这将有助于了解蛋白质相互作用的细胞网络如何在分子水平上运作以及如何用药物控制它们。该方法独特地允许在单个样品中同时密切监测混合物的至少两种多肽组分，而不会增加信号重叠，并且可用于研究由两个多肽独立见证的复杂（例如，顺序、竞争、合作、变构、诱导等）结合事件。一种多肽用N-15均匀标记，另一种用N-15和C-13均匀标记。分别记录了这些分子的H-1-N-15相关光谱，并根据与酰胺基氮相连的C-13′/C-12′原子的类型进行了区分。两种不同同位素标记的分子状态的任何变化都将通过酰胺基团的指纹信号单独报告，例如，添加未标记的配体。据我们所知，目前还没有其他技术能够以类似的细节监测复杂的绑定事件。该方法可以很容易地与传统的蛋白质核磁共振技术相结合，并纳入各种应用。
A new NMR approach is presented for observing in vitro multicomponent protein-protein-ligand(s) interactions, which should help to understand how cellular networks of protein interactions operate on a molecular level and how they can be controlled with drugs. The method uniquely allows at least two polypeptide components of the mixture to be simultaneously closely monitored in a single sample, without increased signal overlap, and can be used to study complex (e.g., sequential, competitive, cooperative, allosteric, induced, etc.) binding events, witnessed by two polypeptides independently. One polypeptide is uniformly labeled with N-15 and another with N-15 and C-13. The H-1-N-15 correlation spectra are recorded for each of these molecules separately, discriminated on the basis of the type of C-13'/C-12' atom attached to the amide group nitrogen. Any changes to the state of the two differently isotopically labeled molecules will be reported individually by fingerprint signals from amide groups, e.g., as unlabeled ligands are added. To our knowledge, no other technique currently exists which can monitor complex binding events in similar detail. The proposed method can be combined easily with traditional protein NMR techniques and incorporated in a variety of applications.