The role of the
peptide hormone calcitonin in skeletal protection has led to its use as a therapeutic for
osteoporosis. However,
calcitonin aggregation into
amyloid fibrils limits its therapeutic efficacy, necessitating a modification of
calcitonin's aggregation kinetics. Here, we report a direct relationship between human
calcitonin (hCT) concentration and aggregation lag time. This kinetic trend was contrary to the conventional understanding of
amyloid aggregation and persisted over a range of aggregation conditions, as confirmed by
thioflavin-T kinetics assays, CD spectroscopy, and transmission EM. Dynamic light scattering, 1H NMR experiments, and seeded
thioflavin-T assay results indicated that differences in initial
peptide species contribute to this trend more than variations in the primary nucleus formation rate. On the basis of kinetics modeling results, we propose a mechanism whereby a structural conversion of hCT monomers is needed before incorporation into the fibril. Our kinetic mechanism recapitulates the experimentally observed relationship between
peptide concentration and lag time and represents a novel mechanism in
amyloid aggregation. Interestingly, hCT at low pH and
salmon calcitonin (sCT) exhibited the canonical inverse relationship between concentration and lag time. Comparative studies of hCT and sCT with molecular dynamics simulations and CD indicated an increased α-helical structure in sCT and low-pH hCT monomers compared with neutral-pH hCT, suggesting that α-helical monomers represent a growth-competent species, whereas unstructured random coil monomers represent a growth-incompetent species. Our finding that initial monomer concentration is positively correlated with lag time in hCT aggregation could help inform future efforts for improving therapeutic applications of CT.