Wiegand Thomas, Cadalbert Riccardo, von Schroetter Christine, Allain Frédéric H-T, Meier Beat H
Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland.
J Biomol NMR. 2018 Aug;71(4):237-245. doi: 10.1007/s10858-018-0196-z. Epub 2018 Jun 12.
Segmental isotope labelling enables the NMR study of an individual domain within a multidomain protein, but still in the context of the entire full-length protein. Compared to the fully labelled protein, spectral overlap can be greatly reduced. We here describe segmental labelling of the (double-) hexameric DnaB helicase from Helicobacter pylori using a ligation approach. Solid-state spectra demonstrate that the ligated protein has the same structure and structural order as the directly expressed full-length protein. We uniformly C/N labeled the N-terminal domain (147 residues) of the protein, while the C-terminal domain (311 residues) remained in natural abundance. The reduced signal overlap in solid-state NMR spectra allowed to identify structural "hotspots" for which the structure of the N-terminal domain in the context of the oligomeric full-length protein differs from the one in the isolated form. They are located near the linker between the two domains, in an α-helical hairpin.
片段同位素标记能够对多结构域蛋白中的单个结构域进行核磁共振（NMR）研究，且仍处于完整全长蛋白的背景下。与完全标记的蛋白相比，谱线重叠可大大减少。我们在此描述了使用连接方法对幽门螺杆菌的（双）六聚体DnaB解旋酶进行片段标记。固态光谱表明，连接后的蛋白与直接表达的全长蛋白具有相同的结构和结构有序性。我们对该蛋白的N端结构域（147个残基）进行了均匀的碳/氮标记，而C端结构域（311个残基）保持自然丰度。固态NMR光谱中信号重叠的减少使得能够识别出结构“热点”，在寡聚全长蛋白背景下N端结构域的结构与分离形式下的结构不同。它们位于两个结构域之间的连接子附近，处于一个α螺旋发夹结构中。
