Structural diversity of amyloid fibril formed in human calcitonin as revealed by site‐directed 13C solid‐state NMR spectroscopy
Structural diversity of amyloid fibril formed in human calcitonin as revealed by site‐directed 13C solid‐state NMR spectroscopy
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DOI：
10.1002/mrc.1323
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发表时间：
2004-02
影响因子：
2
通讯作者：
A. Naito;M. Kamihira;R. Inoue;H. Saitô
中科院分区：
文献类型：
--
作者：
A. Naito;M. Kamihira;R. Inoue;H. Saitô
通过定点13 C固态NMR光谱法检查了pH 4.1水溶液中人降钙素（hCT）的原纤维形成，并与pH 3.3和7.5下对应于三种不同净电荷的原纤维形成进行了比较。值得注意的是，所观察到的13 C化学位移和线形的13 C CP/MAS光谱之间的原纤维在不同的pH值制备的显着不同。发现在pH 7.5和4.1时，在中心核心区域形成反平行β折叠结构。在C末端区域，在pH 7.5和4.1下均形成无规卷曲，尽管pH 4.1下的无规卷曲区域大于pH 7.5下的无规卷曲区域。通过两步自催化反应机制分析的原纤化动力学表明，分别测定原纤形成的成核和成熟反应的速率常数k1和k2，并且该值与Lys 18和His 20的净正电荷而不是Asp 15的负电荷的存在良好相关。此外，尝试通过13 C REDOR测量，通过考虑先前提出的3自旋系统分析的偶极相互作用，评估13 C，15 N标记的hCT和模型五肽的相邻链的酰胺氮和羰基碳之间的原子间距离。反平行β折叠的独特链堆积被认为是主要的原纤维结构，尽管不能排除由垂直于原纤维方向的滑动的一个或两个残基组成的链堆积的贡献的可能性。此外，Phe 16的苯环似乎在β折叠的同一侧对齐，并通过在β链之间形成π-π相互作用使β折叠稳定。版权所有© 2004年约翰威利父子有限公司。
Fibril formation in human calcitonin (hCT) from aqueous solution at pH 4.1 was examined and compared with those at pH 3.3 and 7.5 corresponding to three different net charges by means of site‐directed 13C solid‐state NMR spectroscopy. Notably, the observed 13C chemical shifts and lineshapes of the 13C CP/MAS spectra differed substantially among fibrils prepared at different pHs. It was found that antiparallel β‐sheet structures were formed at pH 7.5 and 4.1 in the central core regions. In the C‐terminal region, random coils were formed at both pH 7.5 and 4.1, although the random coil region at pH 4.1 was larger than that at pH 7.5. Fibrillation kinetics analyzed by a two‐step autocatalytic reaction mechanism showed that the rate constants k1 and k2 for nucleation and maturation reactions of the fibril formation, respectively, were separately determined and the values correlated well with the net positive charges of Lys18 and His20 rather than the existence of a negative charge of Asp15. Further, an attempt was made to assess interatomic distances between amide nitrogen and carbonyl carbon of neighboring chains of 13C, 15N‐labeled hCT and a model pentapeptide by 13C REDOR measurements by taking into account its dipolar interaction analyzed by the 3 spin system proposed previously. A unique chain packing of the antiparallel β‐sheets was proposed as a dominant fibril structure, although the possibility of a contribution of chain packing consisting of sliding one or two residues perpendicular to the fibril direction cannot be ruled out. In addition, it appears that the phenyl rings of Phe16 are aligned on the same side of the β‐sheet and make the β‐sheet stable by forming π–π interactions between the β‐strands. Copyright © 2004 John Wiley & Sons, Ltd.