Figure 3. Isolation and characterization of G37C oligomers.
A: Toxic Aß forms DTT resistant oligomers during purification.
After dialysis, different species were eluted from a Superdex-75 column in PBS at 4 °C. Toxic AßG37C (red) and harmless AßL34T (green) were found in one or two peaks detected at 215 nm. The presence of DTT (5 mM) to the purified oligomeric fraction do not change its elution profile from a Superdex-75 column equilibrated in PBS + DTT (blue),
B: Elution profile of Standards on the Superdex-75 column in PBS at 4 °C.
C: Oligomeric urea-resistant AßG37C (oAßG37CUR) is not stabilized by S-S bonds.
Equal quantities of oAßG37CUR (0.5 moles) were analyzed by Tricine-SDS-PAGE (15 %) under reducing (50 mM DTT or 50 mM TCEP or 2.5 % ß mercaptotethanol) or non-reducing (-) conditions. The same analysis was done for AßG37C with DTT and ß mercaptoethanol as reducing agent.
D: Fibrillation of oAßG37CUR monitored by ThT fluorescence.
80 µM oAßG37CUR was incubated with 20 μM ThT at 30 °C.
E: Morphology of oAßG37CUR fibers in TEM.
After 7 days of fibrillation at 30 °C, oAßG37CUR was stained with 2 % uranyl acetate and observed by TEM. Scale bar: 200 nm.
F: oAßG37CUR exhibits rich beta-parallel sheet structure in ATR-FTIR.
ATR-FTIR spectrum of oAßG37CUR at t0 (black) and after 15 days at 30 °C (red).
G: Conformational changes in oAßG37CUR during fibrillation in bis-ANS.
50 µM oAßG37CUR was loaded onto a Superdex-75 column in PBS. Left, the absorbance at 215 nm (red) and fluorescence in the presence of 1 µM bis-ANS (black) of oAßG37CUR loaded at t0. At different times during incubation at 30 °C in PBS + 5mM DTT, aliquots were taken for bis-ANS fluorescence analysis (right).
H: Toxicity of oAßG37CUR in PC12 cells.
The cytotoxicity of oligomeric oAßG37CUR in PC12 cells was determined with MTT assay. At time 0 (white) and after 24 hours of incubation in PBS (+ 5 mM DTT for oAβG37CUR) (black), 0.5 μM of AβG37C or oAβG37CUR was added to PC12 cells. After 16 hours, cell viability was measured with MTT assays. The cell viability of cell treated with PBS was set as 100%. The values shown indicate the mean+S.D. of three experiments. ***p<0.001