Structural diversity of amyloid fibril formed in human calcitonin as revealed by site-directed 13C solid-state NMR spectroscopy
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.