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Mapping the Core of the B2-Microglobulin Amyloid Fibril by H/D Exchange


H/D exchange of amyloid fibrils
Uniformly 15N- and 15N/13C-labeled recombinant human ß2-m was expressed in methyltrophic yeast Pichia pastoris (6, 7). The 1H-15N HSQC spectrum of ß2-m monomer in the presence of 95% DMSO and 5% H2O at pH 5.0 and 25°C showed that all the backbone resonances were located in the limited range of HN chemical shifts from 7.6 to 8.6 ppm, suggesting that the protein was highly denatured (Fig. 3a). We unambiguously assigned 89 among 97 potentially visible residues in the HSQC spectrum.

We next examined whether dissolution of lyophilized amyloid fibrils in 95% DMSO and 5% D2O actually quenches the H/D exchange reaction. Most of the backbone amide protons remained unchanged, indicating the negligible exchange reaction after dissolution in 95% DMSO and 5% D2O (Fig. 3b). The 1H-15N-HSQC spectra of the samples in which H/D exchange were performed for various periods in deuterated buffer at pD* 2.5 and 25°C showed that the H/D exchange kinetics of many amide protons were extremely slow. Even after the longest exchange period examined (8 days), many peaks showed intensities as strong as ~80% of those in the reference spectrum (Fig. 3c). However, it was also evident that several peaks showed considerable decrease in intensity.

The degree of exchange of the amyloid fibrils calculated for each residue from the peak intensities with and without an 8 day-incubation in D2O was plotted against the residue number (Fig. 4a). The plot showed a characteristic trapezoidal shape, with a relatively flat part in the middle of the sequence and slopes decreasing in intensity at both the N- and C-termini. The pattern of the protected residues was contrary to that in the native state, in which only the ß-stands are protected (circles in Fig. 4a).

Hydrogen bond network in amyloid fibrils

The significance of the present results can be more clearly seen by visualizing the protected residues in the native structure of ß2-m (Fig. 4b). In the amyloid fibrils, in addition to the regions corresponding to the core regions of the native structure, many residues in the native loops become similarly protected from exchange. On the other hand, the N- and C-terminal ends and ßA-strand were not protected. Persistent secondary structures are the most likely reason for the marked protection from H/D exchange. The trapezoidal shape of the protection suggested that, in addition to the native ß-strands except ßA, the native loops were transformed to ß-sheets in amyloid fibrils.

The present results may explain why amyloid fibrils are very rigid and stable, with a needle-like morphology. It is likely that the extensive hydrogen bond network of ß-structure, more than that of the native globular state, confers rigidity on the amyloid fibrils and resistance to protease digestion.

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