Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability.
The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 --> Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.
Doucette
Peter A
PA
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Whitson
Lisa J
LJ
Cao
Xiaohang
X
Schirf
Virgil
V
Demeler
Borries
B
Valentine
Joan Selverstone
JS
Hansen
Jeffrey C
JC
Hart
P John
PJ
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003460:Crystallization; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects*; D019791:Guanidine / Q000494:pharmacology*; D006801:Humans; D007202:Indicators and Reagents; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010720:Phosphines / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics*; D000072105:Superoxide Dismutase-1; D014461:Ultracentrifugation
D004220:Disulfides; D019791:Guanidine; D007202:Indicators and Reagents; D010720:Phosphines; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C000606290:SOD1 protein, human; C080938:tris(2-carboxyethyl)phosphine