Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state

Abstract

Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

Network Hamiltonian models reveal pathways to amyloid fibril formation

“…The progression of epochs that emerges from network Hamiltonian simulations - from Condensation to Local Ordering to Nucleation to Fibril Growth and finally Maturation - is in agreement with prior works that indicate a similar progression in unseeded fibril formation dynamics: from a lag phase in ThT fluorescence with prolific nucleation 33 , to the formation of on-pathway oligomers 12 (compare with the nucleation epoch in the Dendrite Consolidation model), to other events, such as fragmentation with secondary nucleation (compare with defect breakage and defect to end connections), and elongation (end to end connections) 34 – 36 . Furthermore, the appearance of a large, pre-fibrillar component during the condensation phase of 1-ribbon formation via the condensate annealing mechanism is consistent with the transparent hydrogels observed during the fibrillization of superoxide dismutase 37 and -lactoglobulin 38 . Such similarities are further addressed in the “ Discussion ” section.…”

Effects of Protein Crowders and Charge on the Folding of Superoxide Dismutase 1 Variants: A Computational Study

“…In this paper, we studied the effect of self-crowding to gain a better insight into the self-assembly of proteins . We analyzed the effect of crowding in two ways: by looking at how crowding affects the stability of one protein variant in the crowded ensemble in comparison to the stability of other protein variants in the crowded case or by comparing the thermal stability of the protein variants between the single and the crowded ensembles.…”

“…In this paper, we studied the effect of self-crowding to gain a better insight into the self-assembly of proteins. 66 We analyzed the effect of crowding in two ways: by looking at how crowding affects the stability of one protein variant in the crowded ensemble in comparison to the stability of other protein variants in the crowded case or by comparing the thermal stability of the protein variants between the single and the crowded ensembles. Comparing the wild type (WT c ) and Mutant within the single ensemble, we see from Figure 3 (top panel) that the free energy minima for the folded configurations (with high Q) are approximately the same at 157 K (which is very close to their folding temperature at approximately 158 K), while the unfolded basin is deeper for the Mutant than WT c .…”

Transient Diffusive Interactions with a Protein Crowder Affect Aggregation Processes of Superoxide Dismutase 1 β-Barrel

“…In addition to crowding effects, other factors may contribute to protein aggregation inside living cells, including chemical (e.g., decreased pH , ) and mechanical (e.g., shear or stretch − ) factors. Indeed, aggregation of SOD1 has a strong dependence on pH, and we previously observed aggregate formation of SOD1 under dilute experimental conditions applying shear flow . To further understand the real conformational behavior of SOD1 inside living cells, R 2 relaxation dispersion measurements in living cells should be used to acquire information on the off-pathway transitions of SOD1 in situ .…”