Kinetics of Amyloid Fibril Formation of Human Calcitonin

Abstract

Amyloid fibril formation is one of the common phenomena associated with many serious diseases such as Alzheimer’s disease, Parkinson’s, bovine spongiform encephalopathy (BSE), scrapie, and so on. Independent of the constituent polypeptides, the amyloid fibrils exhibit highly organized filamentous structures which are typically ∼100 A in diameter, as revealed by electron microscopy and atomic force microscopy. Mechanism of the amyloid fibril formation has been extensively studied by various spectroscopic techniques, related to misfolding of proteins. Especially, solid-state NMR spectroscopy has made a great contribution to determine the structures of the fibrils from several peptides/proteins at the molecular level. For example, Alzheimer’s β-amyloid peptides, which consist of 40–42 amino acid residues, have gained insights into the three-dimensional (3D) structures in the fibrils as a double-layered cross-β structure with parallel β-sheets by accumulating the local and spatial conformational restraints [1–3]. Also, an 11-residue fragment of human transthyretin (TTR) in its fibrillar form which in vivo is allied with familial amyloid polyneuropathy and senile systemic amyloidosis, was revealed the complete 3D structures of the extended β-strand conformation, by establishing dihedral angles of the backbone and 13C– 15N distances [4,5]. These results indicate that solid-state NMR spectroscopy is a powerful tool to determine the non-crystal, non-soluble, fibrillar structures. In this chapter, a solid-state NMR application on the kinetics analyses of the amyloid fibril formation is described. Human calcitonin (hCT) is a thyroid hormone which regulates the mineral metabolism in the bones [6– 8]. hCT contains 32 amino acid residues and its sequence is CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPNH2 with a disulfide bond between Cys1 and Cys7 and a C-terminus amide. In a high concentrated solution, however, it is known to form the amyloid fibrils, which are typically 80 A in diameter [9,10].