Fig 1.
Characterization of monomer and oligomer properties for human and rabbit prion proteins.
Oligomerization of human prion protein (A) and rabbit prion protein (B) in the buffer (20 mM NaOAc, 150 mM NaCl, pH 4.0) was monitored by gel filtration chromatography. The prion protein oligomers were prepared with a buffer (20 mM NaOAc, 150 mM NaCl, pH 4.0) at 47°C. Particle sizes of both human prion protein (C) and rabbit prion protein (D) were analyzed by DLS spectroscopy. Secondary structures of human prion protein (E) and rabbit prion protein (F) were detected by Far-UV CD spectroscopy. Both DLS and CD experiments were performed at 25°C. The buffer used for prion monomers contained 20 mM NaOAc, pH 5.5.
Fig 2.
NaCl concentration-dependent oligomerization of recHuPrPC and recRaPrPC monitored by gel filtration chromatography.
The oligomerization experiments of prion proteins were conducted in the buffer (20 mM NaOAc, 50–200 mM NaCl, pH 4.0) at 57°C (n = 3; Error bars, S.D.).
Fig 3.
Urea-induced unfolding transitions of recRaPrPC and recHuPrPC proteins analyzed at 25°C by Far-UV CD spectroscopy.
The buffer contained 20 mM NaOAc, pH 5.5. The unfolded fraction calculated from Δε at 222 nm is plotted as a function of the urea concentration. Insert: ΔG (kJ/mol) versus the urea concentration.
Fig 4.
Thermal-induced unfolding transitions of recRaPrPC and recHuPrPC proteins analyzed by Far-UV CD spectroscopy.
The buffer contained 20 mM NaOAc, 0–200 mM NaCl, pH 5.5. The unfolded fraction calculated from Δε at 222 nm is plotted as a function of temperature.
Table 1.
Apparent thermodynamic parameters associated with urea-induced unfolding transitions of recHuPrPC and recRaPrPC at 25°C.
The buffer contained 20 mM NaOAc, pH 5.5. ΔGH2ON→U is designated as the apparent free energy of unfolding extrapolated to zero concentration of denaturant, mN→U is the cooperativity of the unfolding transition, and Cm is the concentration of urea required to denature 50% of the protein. The CD spectrum was an average of three consecutive scans. Each experiment was repeated in triplicate for each sample.
Table 2.
Apparent thermodynamic parameters associated with thermal-induced unfolding transitions of recHuPrPC and recRaPrPC.
The buffer contained 20 mM NaOAc, 0–200 mM NaCl, pH 5.5. ΔG0°CN→U is designated as the apparent free energy of unfolding extrapolated to 0°C, mN→U is the cooperativity of the unfolding transition, and Tm is the temperature at the midpoint of unfolding. The CD spectrum was an average of three consecutive scans. One experiment was conducted for each sample.
Fig 5.
Incubation temperature-dependent oligomerization of recHuPrPC and recRaPrPC monitored by gel filtration chromatography.
The oligomerization experiments of prion proteins were conducted in a buffer (20 mM NaOAc, 150 mM NaCl, pH 4.0) at 37–67°C (n = 3; Error bars, S.D.).
Fig 6.
15% SDS-PAGE analysis of protease K digestion of recHuPrPO and recRaPrPO proteins.
The oligomeric proteins (40 mM) were digested by protease K (2 μg/ml) for 0–80 min, in a buffer (10 mM Tris-HCl, 2 mM CaCl2, pH 7.4) at 37°C.
Fig 7.
Comparison of recHuPrPO-induced and recRaPrPO-induced toxicities on human glioblastoma cell lines U87.
Cells were incubated with oligomeric PrPO proteins at different concentrations for 48 h (37°C). Cytotoxicity was quanitified as a function of cell viability by the MTS assay (n = 3, mean±SD; *, p<0.01; ***, p<0.001; by Multiple Comparison Test).