For biophysical studies using heteronuclear NMR analysis of amyloid beta peptide, construction of an efficient and high yield expression system of uniformly isotopic labeled amyloid beta peptide is desirable. Here we succeeded in obtaining (15)N-labeled amyloid beta 1-40 expressed by attachment to hen egg white lysozyme as a fusion protein.
Amyloid fibril formation of amyloid beta peptide 1-40 (Abeta 1-40) was reported to be retarded in the presence of 150mM phosphate buffer at pH 7 [Monji, Ustumi, Ueda, Imoto, Yoshida, Hashioka, Tashiro and Tashiro, J. Neurochemistry, 77, 1425-1432 (2007)]. In order to elucidate the reason why phosphate ion retards the amyloid fibril formation, we examined the preferential binding sites of phosphate ion to Abeta 1-40 using chemical shift perturbation analysis of heteronuclear NMR. In titration analysis of (15)N-labeled Abeta1-40 in the presence of 150 mM phosphate ion or 150 mM chloride ion, we identified the residues affected by these ions in Abeta 1-40. As a result, we found the tendency that phosphate ion preferentially bound to some residues located on the C-terminus region where the region was reported to be the potential beta-strand region in Abeta1-40. Therefore, we suggested that phosphate ions interacted with the potential beta-strand region in Abeta1-40 to be hard to form beta-sheet in Abeta 1-40, resulting in retardation of the amyloid fibril formation.
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