Riek R, Hornemann S, Wider G, Glockshuber R, Wüthrich K
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
FEBS Lett. 1997 Aug 18;413(2):282-8. doi: 10.1016/s0014-5793(97)00920-4.
The recombinant murine prion protein, mPrP(23-231), was expressed in E. coli with uniform 15N-labeling. NMR experiments showed that the previously determined globular three-dimensional structure of the C-terminal domain mPrP(121-231) is preserved in the intact protein, and that the N-terminal polypeptide segment 23-120 is flexibly disordered. This structural information is based on nearly complete sequence-specific assignments for the backbone amide nitrogens, amide protons and alpha-protols of the polypeptide segment of residues 121-231 in mPrP(23-231). Coincidence of corresponding sequential and medium-range nuclear Overhauser effects (NOE) showed that the helical secondary structures previously identified in mPrP(121-231) are also present in mPrP(23-231), and near-identity of corresponding amide nitrogen and amide proton chemical shifts indicates that the three-dimensional fold of mPrP(121-231) is also preserved in the intact protein. The linewidths in heteronuclear 1H-15N correlation spectra and 15N[1H]-NOEs showed that the well structured residues 126-230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23-231) outside of this domain.
重组鼠朊病毒蛋白mPrP(23 - 231)在大肠杆菌中表达并进行了均匀的15N标记。核磁共振实验表明，先前确定的C端结构域mPrP(121 - 231)的球状三维结构在完整蛋白中得以保留，而N端多肽片段23 - 120则呈灵活无序状态。该结构信息基于对mPrP(23 - 231)中121 - 231位残基多肽片段的主链酰胺氮、酰胺质子和α - 质子的几乎完整的序列特异性归属。相应的顺序和中程核Overhauser效应(NOE)的一致性表明，先前在mPrP(121 - 231)中鉴定出的螺旋二级结构在mPrP(23 - 231)中也存在，并且相应酰胺氮和酰胺质子化学位移的近乎一致表明mPrP(121 - 231)的三维折叠在完整蛋白中也得以保留。异核1H - 15N相关谱和15N[1H] - NOE中的线宽表明，结构良好的126 - 230位残基具有几纳秒的相关时间，这是小球状蛋白的典型特征，而在此结构域之外的mPrP(23 - 231)所有残基的相关时间均短于1纳秒。
