Characterization of human calcitonin fibrillation in aqueous urea solution by 1H NMR spectroscopy.
The inhibitory effects of urea on the normally rapid fibrillation of human calcitonin (hCT) were investigated by 1H NMR. From subtle differences in the chemical shift of hCT in the presence and absence of urea, the occurrence of weak interactions between urea and hCT was confirmed. The chemical shifts on the NH and C alpha protons of residues in the C-terminal (Gln24-Pro32) region were unaffected by the urea interactions, while the chemical shifts of the N-terminal (Cys1-Cys7) and central (Met8-Pro23) residues were observed to move significantly downfield with increasing urea concentrations. These findings suggest that urea serves to stabilize the monomeric form of hCT and to promote the concentration of the extended hCT conformer. However, it was also found that even by storing hCT in urea the fibrillation process cannot be circumvented, as the gradual carbamylation of the N-terminus takes place. The time course of 1D and 2D spectra of carbamylated hCT showed that cross peaks of residues in the N-terminal and central regions disappear faster than those in the C-terminal regions, indicating that the fibrillation of carbamylated hCT is initiated in the N-terminal and central regions. It is postulated that carbamylation increases the hydrophobicity of the N-terminal region and hence fosters fibrillation, even in urea solution.
Kanaori
K
K
International Research Laboratories, Ciba-Geigy Japan Ltd., Takarazuka, Japan.
Nosaka
A Y
AY
D002116:Calcitonin / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D011487:Protein Conformation; D014508:Urea / Q000737:chemistry*
D014508:Urea; D002116:Calcitonin