Mechanism and thermodynamics of guanidinium chloride-induced denaturation of ALS-associated mutant Cu,Zn superoxide dismutases.


Mutations in human copper zinc superoxide dismutase (hSOD) that are associated with amyotrophic lateral sclerosis (ALS) have been proposed to destabilize the protein and thereby enhance toxic protein aggregation. In previous studies, denaturation of metallated (holo) hSODs was found to be irreversible, and complicated by the formation of intermolecular disulfide bonds. Here, ALS-associated mutations (E100G, G93A, G85R and A4V) are introduced into a pseudo wild-type background containing no free cysteine residues. The guanidinium chloride-induced denaturation of the holo proteins is generally found to be highly reversible (except for A4V, which tended to aggregate), enabling quantitative analysis of the effects of the mutations on protein stability. Denaturation and renaturation curves were monitored by tryptophan fluorescence, circular dichroism, enzyme activity, chemical cross-linking and analytical sedimentation, as a function of equilibration time and protein concentration. There is strong kinetic hysteresis, with curves requiring exceptionally long times (many days for pseudo wild-type) to reach equilibrium, and evidence for the formation of kinetic and equilibrium intermediate(s), which are more highly populated at lower protein concentrations. The effects of metal dissociation were included in the data fitting. The full protein concentration dependence is best described using a three-state model involving metallated native dimer, metallated monomeric intermediate and unfolded monomers with no bound metals; however, at high protein concentrations the unfolding approaches a two-state transition with metal binding to both the native dimers and unfolded monomers. We show that the E100G, G93A and G85R mutations decrease overall protein stability, largely by decreasing monomer stability with little effect on dimer dissociation. Comparison of the chemical denaturation data with ALS disease characteristics suggests that aggregation of some mutant hSOD may occur through increased population of partially folded states that are less stable than the monomeric intermediate and accessed from the destabilized holo protein. Study holds ProTherm entries: 18920, 18921, 18922, 18923, 18924, 18925, 18926, 18927, 18928, 18929, 18930, 18931, 18932, 18933, 18934, 18935, 18936, 18937, 18938 Extra Details: dG_H2O data are in units of kcal/mol.dimer; m in kcal/mol.dimer/M superoxide dismutase; amyotrophic lateral sclerosis; protein stability and folding; dimeric protein; guanidine hydrochloride denaturation

Submission Details

ID: YvZdZji63

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Rumfeldt JA;Stathopulos PB;Chakrabarrty A;Lepock JR;Meiering EM,J. Mol. Biol. (2006) Mechanism and thermodynamics of guanidinium chloride-induced denaturation of ALS-associated mutant Cu,Zn superoxide dismutases. PMID:16307756
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Superoxide dismutase [Cu-Zn] P00441 SODC_HUMAN
100.0 Superoxide dismutase [Cu-Zn] P60052 SODC_PANTR
95.5 Superoxide dismutase [Cu-Zn] Q8HXQ4 SODC_PONPY
94.2 Superoxide dismutase [Cu-Zn] Q8HXQ3 SODC_HYLLA
93.5 Superoxide dismutase [Cu-Zn] Q8HXP9 SODC_SAPAP
92.2 Superoxide dismutase [Cu-Zn] Q8HXP8 SODC_CALJA
91.6 Superoxide dismutase [Cu-Zn] Q8HXQ1 SODC_MACFA
91.6 Superoxide dismutase [Cu-Zn] Q8HXQ2 SODC_MACFU
91.6 Superoxide dismutase [Cu-Zn] Q8HXQ0 SODC_MACMU