Amyloid formation in denatured single-mutant lysozymes where residual structures are modulated

Abstract

Reduced hen lysozyme has a residual structure involving long-range interaction. It has been demonstrated that a single mutation (A9G, W62G, W111G, or W123G) in the residual structure differently modulates the long-range interactions of reduced lysozyme. To examine whether such variations in the residual structure affect amyloid formation, reduced and alkylated mutant lysozymes were incubated under the amyloid-fibrillation condition. From the analyses of CD spectra and thioflavine T fluorescences, it was suggested that variation in residual structure led to different amyloid formation. Interestingly, the extent of amyloid formation did not always correlate with the extent to which the residual structure was maintained, resulting in the involvement of a hydrophobic cluster normally contained in W111 in the reduced lysozyme.

Figure 1.

Transverse relaxation rates (R2) in the reduced and alkylated HELs at pH 2.0. Six clusters are detected in the wild-type lysozyme. This figure was schematically redrawn based on our former results (Wirmer et al. 2004).

Figure 2.

Time dependence of CD spectra of CAM HELs after 0 d or 28 d of incubation in 50 mM sodium malate at pH 2.0.

Figure 3.

The fluorescence emission at 485 nm of ThT in the presence of CAM HELs during the incubation at pH 2.0.

Figure 4.

Electron micrographs of solutions of the CAM wild-type HEL (A), CAM A9G HEL (B), CAM W62G HEL (C), CAM W111G HEL (D), and CAM W123G HEL (E) in 50 mM sodium malate at pH 2.0 after incubation at 30°C for 4 mo and a protein concentration of 8 mg/mL. The scale bars represent 250 nm.