Equilibrium thermodynamic analysis of amyotrophic lateral sclerosis-associated mutant apo Cu,Zn superoxide dismutases.
The folding and thermodynamic properties of metal free (apo) superoxide dismutases (SODs) are systematically analyzed using equilibrium guanidinium chloride (GdmCl) curves and differential scanning calorimetry (DSC). Chemically and structurally diverse amyotrophic lateral sclerosis (ALS)-associated mutations (G85R, G93R, E100G, I113T) are introduced into a pseudo-wild-type background that has no free cysteines, resulting in highly reversible unfolding. Analysis of the protein concentration dependence of GdmCl curves reveals formation of a monomer intermediate in equilibrium with native dimer and unfolded monomer. Global fitting of the data enables quantitative measurement of free energy changes for both dimer dissociation and monomer intermediate stability. All the mutations decrease protein stability, mainly by destabilizing the monomer intermediate, but also by tending to weaken dimerization, even for mutations far from the dimer interface. Thus, the effects of mutations seem to propagate through the apo protein, and result in increased population of both intermediate and unfolded monomers. This may underlie increased formation of toxic aggregates by mutants in ALS. Analysis of DSC data for apo SODs is consistent with stability measurements from GdmCl curves and provides further evidence for increased aggregation by mutant proteins through increased ratios of van't Hoff to calorimetric enthalpies of unfolding.
Vassall
Kenrick A
KA
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry and Department of Chemistry and Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Stathopulos
Peter B
PB
Rumfeldt
Jessica A O
JA
Lepock
James R
JR
Meiering
Elizabeth M
EM
D002152:Calorimetry, Differential Scanning; D009154:Mutation; D010957:Plasmids; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013816:Thermodynamics
D011994:Recombinant Proteins; D013482:Superoxide Dismutase