Conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state 13C NMR

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Abstract

Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local α-helical form is present around Gly10, whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local β-sheet form as viewed from Gly10 and Phe22, and both β-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from α-helix to β-sheet, and from random coil to β-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local β-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic α-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel β-sheets at pH 7.5 and a mixture of antiparallel and parallel β-sheets at pH 3.3.

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