Fig 1.
Fibrillization and fibril morphology of the Aβ variants.
(A) Fibrillization kinetics of Aβ40 variants monitored by ThT assay in 10 mM phosphate buffer, pH 7.4. The peptide concentration was 25 μM. The signal was normalized to the plateau signal of WT. (B) TEM of the end-point products of Aβ variants. The scale bars are 200 nm.
Fig 2.
Native PAGE, AUC analysis, far-UV CD spectra, and FTIR spectra of the Aβ variants.
(A) Native PAGE of freshly prepared WT, A2T, and A2V variants. (B) SV experiments of AUC of freshly prepared WT (black), A2T (blue), and A2V (red) variants. (C) Far-UV CD spectra of freshly prepared WT (black), A2T (blue), and A2V (red) variants. (D) FTIR spectra of fibrils of WT (black), A2T (blue), and A2V (red) variants. The peaks were indicated.
Fig 3.
The effects of full-length and N-terminal A2T and A2V variants on Aβ fibrillization.
Fibrillization kinetics of WT Aβ40 co-incubated with different ratios of (A) full-length of A2T or A2V and (B) NTF of A2T or A2V from residue 1 to 10. ThT assay was performed in 10 mM phosphate buffer, pH 7.4. WT Aβ40 was 25 μM. The WT to full-length A2T/A2V ratio were 1 to 1 and 1 to 2. The WT to A2T/A2V NTF ratio were 1 to 1 and 1 to 6. The signal was normalized to the plateau signal of WT.
Fig 4.
HSQC spectra of 15N labeled Aβ40 co-incubated with full-length WT, A2T, or A2V Aβ40.
(A) 15N labeled Aβ40 co-incubated with two molar equivalents of full-length WT (blue) or A2T (red). (B) 15N labeled Aβ40 co-incubated with two molar equivalents of full-length WT (blue) or A2V (red).
Fig 5.
NMR CSP and cross peak intensity differences in Aβ40 co-incubated with full-length of WT (control), A2T, and A2V variants.
(A) The CSPs between control and A2T (Blue) or A2V (Red). (B) The differential reduction of cross peak intensity between control and A2T (Blue) or A2V (Red).
Table 1.
Amino acid Sequence of N-terminal A2T peptides.
Fig 6.
Fibrillization and cytotoxicity of WT Aβ40 co-incubated with A2T NTFs.
(A) Fibrillization of Aβ40 in the absence and presence of A2T NTFs. Aβ40 WT co-incubated with A2T NTFs, monitored by ThT assay in 10 mM phosphate buffer, pH 7.4. Final Aβ concentration was 25 μM and A2T NTFs concentration were 125 μM. The signal was normalized to the plateau signal of WT. (B) Cell viability of Aβ in the absence and presence of A2T NTFs. The end-point products of ThT assay were treated to neuroblastoma SH-SY5Y cells with final concentration of 10 μM. After 1 day incubation, cell viability was measured by MTT assay. The data were normalized with buffer control. The effect of NTFs were compared with the cells treated WT Aβ alone using one-way ANOVA (*, p< 0.05). (C) Cytotoxicity of Aβ in the absence and presence of A2T NTFs. Cytotoxicity was examined by LDH assay. The end-point products of ThT assay were treated to neuroblastoma SH-SY5Y cells with final concentration of 10 μM. After 1 day incubation, cytotoxicity was measured by LDH assay. Triton X-100 was used as a positive control for 100% cytotoxicity.
Fig 7.
TEM images of the end-point products of Aβ incubated with various A2T NTFs.
A2T NTFs ranged from residue 1-x, denoted as Nx (x = 4–10). The amino acid sequences are listed in Table 1. The TEM images of WT Aβ incubated with different A2T NTFs after 4 days in quiescence in 10 mM phosphate buffer, pH 7.4. Aβ40 WT concentration was 25 μM and A2T NTFs were 125 μM. The scale bars are 100 nm.