Biophysical and spectroscopical insights into structural modulation of species in the aggregation pathway of superoxide dismutase 1

Fig. 1. Time-dependent SEC results of SOD1 aggregation. a Size exclusion chromatogram of EDTA and DTT-treated SOD1(30 μM) at different times (t = 10, 15, and 30 min), b fitting of decrease in dimer area with for first order (Eq. 6), c the time evolution of soluble species observed between 6 and 18 ml, d at lower elution volume (6–13 ml).

Fig. 2. Results of the transition from monomer to non-native dimer. a–h Time evolution of soluble species at higher elution volume (13–18 ml), i Size exclusion chromatogram of EDTA and DTT-treated SOD1 (30 μM) at different times, j fitting of ratio of monomer area at t = 0 over t = 0–6 with second-order equation (Eq. 7).

Fig. 3. SERS spectra of protein and oligomer. a Represents the SOD1 (PDB ID: IHL5100) positive charge residues responsible for the electrostatic attractions with the silver nanoparticle and surface charge (blue for positive, red for negative) give an idea about possible attachment orientation of SOD1 protein, (R = Arginine, K = Lysine, H = Histidine) b give idea about possible attachment orientation of oligomer with the silver nanoparticle, c SERS spectra of protein (black), oligomer (red) and oligomer formed in presence of quercetin (blue)65,85,90,91,101,102.

Fig. 4. Different concentrations of aggregation SOD1 for the kinetics analysis. a Aggregation of different concentrations of SOD1 in 2 mM EDTA and 80 mM DTT at 37 °C, 25 mM phosphate buffer pH 7.4, b EDTA and DTT induced fibrillation of SOD1 alone and in the presence of seeds. c log-log plot of aggregation rate vs. SOD1 concentration. d–h Different concentrations of SOD1 aggregation early time rise of the ThT fluorescence intensity fits well with the relation Δ = A(cosh(bt)-1) referring to the presence of secondary pathways. i Calculation of the secondary nucleus size from the parameter κ.

Fig. 5. Global fitting of SOD1 aggregation kinetics data to various kinetic models. a nucleation elongation, b secondary nucleation dominant, c fragmentation dominant, d fragmentation and secondary nucleation, e multi-step secondary nucleation dominant, and f saturating elongation models.

Fig. 6. Effect of quercetin on ANS fluorescence spectra of DTT and EDTA-treated SOD1. a ANS fluorescence spectra of SOD1 30 μM monitored at different time points after adding EDTA and DTT. b ANS fluorescence spectra of SOD1 30 μM aggregation at different times in the presence of quercetin (50 µM).

Fig. 7. SEC results of SOD1 aggregation in the presence of quercetin. a Size exclusion chromatogram of EDTA and DTT-treated SOD1 30 μM in the presence of 50 µM quercetin at different times, b fitting of decrease in dimer area with first-order equation, c SEC at different times to monitor the formation of different soluble species formed during the SOD1 aggregation in the presence of quercetin (50 μM).

Fig. 8. Inhibitory effect of quercetin on SOD1 aggregation kinetics. a Aggregation kinetics of 30 μM SOD1 in the presence of different concentrations of quercetin (10, 30, 40, and 50 μM), b plot of lag time and kapp versus quercetin concentration, c Aggregation of different concentrations (20, 30, 40, 50 µM) SOD1 protein in the presence of 25 µM quercetin, d log-log plot of half time versus protein concentration.

Fig. 9. PCA results of protein and oligomer. PCA scatter plot for the first two principal components (PC1 and PC2) for (a) protein and oligomer, (b) oligomer and oligomer in presence of quercetin. Each dot represents one spectrum (black for protein, red for oligomer, green for oligomer in presence of quercetin), (c) PC loading plot for PC1 and PC2 for protein and oligomer, (d) PC loading plot for PC1 and PC2 for oligomer and oligomer formed in presence of quercetin.

Fig. 10. Representation of steps in the aggregation mechanism of SOD1. After removal of metal ions and disulfide bond from native protein dimer rapidly converts into monomer which is first-order reaction. Then monomer takes extended form which has tendency to form non-native dimers which act as nucleus. Once the critical concentration of nucleus is reached, oligomerization process starts expeditiously. Then larger oligomers fragmented and act as seeds for further aggregation. Overall, SOD1 aggregation follows a fragmentation-dominant mechanism in which the formation of non-native dimer serves as rate-limiting step.