The rate-limiting step in the formation of the native dimeric state of human Cu, Zn superoxide dismutase (SOD1) is a very slow monomer folding reaction that governs the lifetime of its unfolded state. Mutations at dozens of sites in SOD1 are known to cause a fatal motor neuron disease, amyotrophic lateral sclerosis, and recent experiments implicate the unfolded state as a source of soluble oligomers and histologically observable aggregates thought to be responsible for toxicity. To determine the thermodynamic properties of the transition state ensemble (TSE) limiting the folding of this high contact order β-sandwich motif, a combined thermal/urea denaturation thermodynamic/kinetic analysis was performed. The barriers to folding and unfolding are dominated by the activation enthalpy at 298 K and neutral pH; the activation entropy is favorable and reduces the barrier height for both reactions. The absence of secondary structure formation or large-scale chain collapse prior to crossing the barrier for folding led to the conclusion that dehydration of nonpolar surfaces in the TSE is responsible for the large and positive activation enthalpy. Although the activation entropy favors the folding reaction, the transition from the unfolded state to the native state is entropically disfavored at 298 K. The opposing entropic contributions to the free energies of the TSE and the native state during folding provide insights into structural properties of the TSE. The results also imply a crucial role for water in governing the productive folding reaction and enhancing the propensity for the aggregation of SOD1.

Dozens of mutations throughout the sequence of the gene encoding superoxide dismutase 1 (SOD1) have been linked to toxic protein aggregation in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A parsimonious explanation for numerous genotypes resulting in a common phenotype would be mutation-induced perturbation of the folding freeenergy surface that increases the populations of high-energy states prone to aggregation. The absence of intermediates in the folding of monomeric SOD1 suggests that the unfolded ensemble is a potential source of aggregation. To test this hypothesis, here we dissected SOD1 into a set of peptides end-labeled with FRET probes to model the local behavior of the corresponding sequences in the unfolded ensemble. Using timeresolved FRET, we observed that the peptide corresponding to the loop VII-β8 sequence at the SOD1 C-terminus was uniquely sensitive to denaturant. Utilizing a two-dimensional form of maximum entropy modeling, we demonstrate that the sensitivity to denaturant is the surprising result of a two-state-like transition from a compact to an expanded state. Variations of the peptide sequence revealed that the compact state involves a nonnative interaction between the disordered Nterminus and the hydrophobic C-terminus of the peptide. This nonnative intramolecular structure could serve as a precursor for intermolecular association and result in aggregation associated with ALS. We propose that this precursor would provide a common molecular target for therapeutic intervention in the dozens of ALS-linked SOD1 mutations.

The folding reaction of a stable monomeric variant of Cu/Zn superoxide dismutase (mSOD1), an enzyme responsible for the conversion of superoxide free radicals into hydrogen peroxide and oxygen, is known to be among the slowest folding processes that adhere to two-state behavior. The long lifetime, $10 s, of the unfolded state presents ample opportunities for the polypeptide chain to transiently sample nonnative structures before the formation of the productive folding transition state. We recently observed the formation of a nonnative structure in a peptide model of the C-terminus of SOD1, a sequence that might serve as a potential source of internal chain friction-limited folding. To test for friction-limited folding, we performed a comprehensive thermodynamic and kinetic analysis of the folding mechanism of mSOD1 in the presence of the viscogens glycerol and glucose. Using a, to our knowledge, novel analysis of the folding reactions, we found the disulfide-reduced form of the protein that exposes the C-terminal sequence, but not its disulfide-oxidized counterpart that protects it, experiences internal chain friction during folding. The sensitivity of the internal friction to the disulfide bond status suggests that one or both of the crosslinked regions play a critical role in driving the friction-limited folding. We speculate that the molecular mechanisms giving rise to the internal friction of disulfide-reduced mSOD1 might play a role in the amyotrophic lateral sclerosis-linked aggregation of SOD1.

Aggregates of Cu, Zn Superoxide Dismutase (SOD1) in the spinal cord are a hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Mutations throughout the SOD1 sequence have been found to cause ALS, suggesting a general gain‐of‐function mechanism for the toxicity of this disease. We have previously argued that the aggregation of the unfolded chain, following synthesis on the ribosome and prior to maturation, provides a parsimonious explanation for the common phenotype. To determine whether segments of unfolded SOD1 are capable of adopting structure prior to the exceedingly slow (>10 sec) productive folding reaction, we dissected SOD1 into peptides that were labeled with a donor/acceptor FRET pair at or near their N/C‐termini. Time‐resolved FRET (tr‐FRET) analysis revealed that the peptide corresponding the C‐terminal region of SOD1 forms urea sensitive structure. Utilizing 2‐dimensional Maximum Entropy Modeling (MEM), we found that this peptide undergoes a discrete transition between a compact state and a random coil‐like structure. Mutations show this collapse results from a non‐native intramolecular interaction involving a hydrophobic sequence at the C‐terminus as well as a polar/charged sequence at the N‐terminus. Ongoing studies are examining the behavior of these segments in the full‐length protein with tr‐FRET. These results are consistent with the results of proteolysis and crystallographic studies from other labs and together suggest that this region of SOD1 may present a potential target for novel therapeutics to treat ALS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

the WD40 binding domain of Cdc4. The highly-cooperative switch-like dependence on the number of phosphorylated sites on Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. Further experimental attention is necessary to determine the physicochemical/mechanistic basis of its highly cooperative binding. We used single molecule fluorescence techniques to study the dimensions and dynamics of Sic1's N-terminal targeting region (residues 1-90, henceforth Sic1), phosphorylated Sic1 (pSic1), and the pSic1-WD40 dynamic complex. Previous single molecule Frster Resonance Energy Transfer (smFRET) measurements [Liu, 2014] observed end-to-end reconfiguration on timescales larger than~1ms; resulting in FRET histograms with multiple conformational sub-ensembles. Sic1, pSic1, and the pSic1-WD40 complex are examined using smFRET to study the dynamics and dimensions of the various sub-ensembles. In a refinement to the conventional approaches for inferring dimensions from smFRET experiments, we use distance distributions from Monte Carlo simulations which extensively sample coarse-grained protein conformations. The application of polymer physics theory/simulation towards smFRET data interpretation, and towards IDP binding, contributes to the growing toolkit for understanding the diverse behaviours of IDPs.