Effect of electrostatic interaction on fibril formation of human calcitonin as studied by high resolution solid state 13C NMR.
Fibrillation of a human calcitonin mutant (hCT) at the position of Asp(15) (D15N-hCT) was examined to reveal the effect of the electrostatic interaction of Asp(15) with charged side chains. The secondary structures of fibrils and soluble monomers in the site-specific (13)C-labeled D15N-hCTs were determined using (13)C cross-polarization magic angle spinning and dipolar decoupled magic angle spinning NMR approaches, sensitive to detect (13)C signals from the fibril and the soluble monomer, respectively. The local conformations and structures of D15N-hCT fibrils at pH 7.5 and 3.2 were found to be similar to each other and those of hCT at pH 3.3 and were interpreted as a mixture of antiparallel and parallel beta-sheets, whereas they were different from the hCT fibril at pH 7.5 whose structure is proposed to be antiparallel beta-sheets. Thus the negatively charged Asp(15) in the hCT molecule turned out to play an essential role in determining the structures and orientations of the hCT molecules. Fibrillation kinetics of D15N-hCT was analyzed using a two-step autocatalytic reaction mechanism. The results indicated that the replacement of Asp(15) with Asn(15) did not reduce the rate constants of the fibril formation but rather increased the rate constants at neutral pH.
Kamihira
Miya
M
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Kamigori, Hyogo 678-1297, Japan.
Oshiro
Yuki
Y
Tuzi
Satoru
S
Nosaka
Atsuko Y
AY
Saitô
Hazime
H
Naito
Akira
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D002116:Calcitonin / Q000737:chemistry*; D002247:Carbon Isotopes; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D020894:Microfibrils / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D055672:Static Electricity
D002247:Carbon Isotopes; D011994:Recombinant Proteins; D001224:Aspartic Acid; D002116:Calcitonin