Figure 1.
Protein-concentration dependence of high-order oligomerization.
(A) 12.5% SDS-PAGE. (B) 10% native-PAGE. (C) Activity staining of the bands in native-PAGE. M is the protein marker, TT is tetramer and O is high-order oligomers. The proteins were dissolved in 20 mM PBS buffer, pH 7.4.
Figure 2.
Effects of solution conditions on the dynamic equilibrium between tetramer and high-order oligomers characterized by 10% native-PAGE.
(A) Salt-dependence. (B) pH-dependence. (C) Heat treatment at 80°C. (D) Heat treatment at 95°C. The proteins were dissolved in 20 mM PBS buffer with a protein concentration of 1 mg/ml. TT is tetramer and O is high-order oligomers. BSA was used as a molecular weight reference and a loading control of the native-PAGE. For clarity, the BSA band was not shown in panels B, C and D.
Figure 3.
Stability of purified tetramers and high-order oligomers separated by ion-exchanging.
Most of the high-order oligomers were quickly dissociated into tetramers after collection. The purified fractions were concentrated to a protein concentration of 0.3 mg/ml and heated at 80°C or 95°C for a given time. After heat treatment, the samples were analyzed by 10% native-PAGE (upper panel) followed by activity staining (lower panel). A weak band from octomer can be observed when the tetramers were heated at 80°C for 90 min or 95°C for 60 min. TT is tetramer and O is high-order oligomers. The oligomeric size was characterized by the position of the native tetramer as well as the BSA control (data not shown).
Figure 4.
Effect of mutations on the high-order oligomerization and stability of tcSOD.
(A) Native-PAGE. (B) Activity staining. (C) Stability at 95°C. The protein concentration was 1 mg/ml for the native-PAGE and activity staining analysis and 0.33 mg/ml for the stability experiments. The presented data were calculated from three repetitions.
Figure 5.
SDS-PAGE and SEC analysis of the proteins cross-linked by glutaraldehyde.
(A) SDS-PAGE analysis of the WT and M202 samples with or without cross-linking after heating at 25°C or 80°C for 50 min. The cross-linking was performed at the incubation temperature for 1 min or 5 min. after heat treatment. The right panel shows the SDS-PAGE of BSA treated at the same conditions as a control. M is monomer, D is dimer and TT is tetramer. (B) SEC analysis of the WT TcSOD and M202 cross-linked at 25°C or 80°C. The SEC samples were cross-linked for 5 min after heating at the given temperature for 50 min with a final protein concentration of 0.27 mg/ml. 1–6 represent the WT protein without cross-linking, WT protein cross-linked at 25°C, WT protein cross-linked at 80°C, M202 without cross-linking, M202 in cross-linked at 25°C and M202 cross-linked at 805°C, receptivity. The arrows indicate the peaks from the off-pathway aggregates.
Figure 6.
Native-PAGE and activity staining of the proteins cross-linked by glutaraldehyde.
(A) 10% Native-PAGE. (B) Activity staining. The proteins were heated for a given time (0, 5 or 50 min) at a given temperature ranging from 25°C to 80°C, and then cross-linked by glutaraldehyde for 10 min at the incubation temperature. TT1 and TT2 indicate two different forms of cross-linked tetramers. TT1 is active and TT2 is inactive. The protein concentration was 1 mg/ml. The sample cross-linked at 80°C contained insoluble aggregates (data not shown).
Figure 7.
Cross-linking of tcSOD by DSP.
(A) SDS-PAGE. (B) Native-PAGE (upper) and activity staining (lower). The samples were incubated at the given temperature for 50 min followed by 10 min cross-linking by DSP. M is monomer, D is dimer, TT is tetramer and O is high-order oligomers.
Figure 8.
Crystal structure of ApSOD (PDB ID: 1COJ).
The four subunits are distinguished in different colors. The coordinated Fe2+ ions are shown in the sphere model. The last 10 residues at the C-terminus are highlighted in red.
Figure 9.
A proposed model for the role of active high-order oligomers in tcSOD hyperthermostability against off-pathway aggregation.
The formation of active high-order oligomers is a reversible process, while the aggregation pathway is irreversible. The high-order oligomerization confers an additional energy gap between the native state and aggregates. The removals of the oligomerization ability by the M202 mutation facilitate the proteins to aggregate under extreme conditions.