Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201, USA.
J Biomol NMR. 2011 Aug;50(4):403-10. doi: 10.1007/s10858-011-9526-0. Epub 2011 Jul 6.
NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domain-domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domain-domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domain-domain interactions in human apolipoprotein E (apoE), a multi-domain, 299-residues α-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples in which the N-terminal (NT-) domain was (2)H(99%)/(15)N-labeled whereas the C-terminal (CT-) domain was either (15)N- or (15)N/(13)C-labeled. 3-D (15)N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT- and the CT-domains and determination of the NMR structure of apoE.
由于与特定的域-域相互作用的明确识别特别困难，因此对大型多结构域蛋白质进行 NMR 结构测定是一项具有挑战性的任务。分段标记是一种 NMR 策略，允许对一个结构域进行同位素标记，而使另一个结构域未标记。这大大简化了光谱重叠，并允许快速识别结构域-域相互作用。这里提出了一种新的分段标记策略，用于检测结构域间 NOE。为了鉴定人载脂蛋白 E（apoE）中的结构域-结构域相互作用，apoE 是一种多结构域、299 个残基的α螺旋蛋白质，利用柱上表达蛋白连接生成分段标记的 apoE 样品，其中 N 端（NT-）结构域是（2）H（99％）/（15）N 标记，而 C 端（CT-）结构域是（15）N-或（15）N/（13）C 标记。这些分段标记的 apoE 样品的 3D（15）N 编辑 NOESY 谱允许直接观察 NT-结构域的骨架酰胺质子与 CT-结构域的脂肪质子之间的结构域间 NOE。这种简单的方法可以明确识别 78 个结构域间 NOE，从而准确定义 NT-和 CT-结构域的相对位置，并确定 apoE 的 NMR 结构。
