title: Structural diversity of amyloid fibril formed in human calcitonin as revealed by site-directed 13C solid-state NMR spectroscopy.
abstract: 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 (13)C solid-state NMR spectroscopy. Notably, the observed (13)C chemical shifts and lineshapes of the (13)C CP/MAS spectra differed substantially among fibrils prepared at different pHs. It was found that antiparallel beta-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 k(1) and k(2) for nucleation and maturation reactions of the fibril formation, respectively, were separately determined and the values correlated well with the net positive charges of Lys(18) and His(20) rather than the existence of a negative charge of Asp(15). Further, an attempt was made to assess interatomic distances between amide nitrogen and carbonyl carbon of neighboring chains of (13)C, (15)N-labeled hCT and a model pentapeptide by (13)C REDOR measurements by taking into account its dipolar interaction analyzed by the 3 spin system proposed previously. A unique chain packing of the antiparallel beta-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 Phe(16) are aligned on the same side of the beta-sheet and make the beta-sheet stable by forming pi-pi interactions between the beta-strands.
authors.affiliation: Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Naito
authors.forename: Miya
authors.initials: M
authors.lastname: Kamihira
authors.forename: Ryozo
authors.initials: R
authors.lastname: Inoue
authors.forename: Hazime
authors.initials: H
authors.lastname: Saitô
mesh_terms: D000595:Amino Acid Sequence; D002116:Calcitonin / Q000737:chemistry*; D002247:Carbon Isotopes; D014644:Genetic Variation; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D009842:Oligopeptides; D002116:Calcitonin