Limits...
Tetramerization reinforces the dimer interface of MnSOD.

Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS - PLoS ONE (2013)

Bottom Line: Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure.Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD.We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

Show MeSH

Related in: MedlinePlus

Thermostability of WT and RP-mutant ScMnSOD and CaMnSODc.The S. cerevisiae enzymes in (A) are: (a) as-isolated ScMnSOD, (b) oxidized ScMnSOD and (c) as-isolated K182R, A183P ScMnSOD. The C. albicans enzymes in (B) are: (a) as-isolated CaMnSODc; (b) reduced CaMnSODc; (c) oxidized CaMnSODc and (d) as-isolated K184R, L185P CaMnSODc. Unfolding transitions are shown in black lines. The components (gray) were deconvoluted using a two-state irreversible model for WT ScMnSOD and a non-two-state reversible model for RP-mutant ScMnSOD, and WT and RP-mutant CaMnSODc. Reduced or oxidized enzymes were prepared by adding sodium hyposulfite or potassium permanganate to the sample solution prior to the DSC scan.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646814&req=5

pone-0062446-g008: Thermostability of WT and RP-mutant ScMnSOD and CaMnSODc.The S. cerevisiae enzymes in (A) are: (a) as-isolated ScMnSOD, (b) oxidized ScMnSOD and (c) as-isolated K182R, A183P ScMnSOD. The C. albicans enzymes in (B) are: (a) as-isolated CaMnSODc; (b) reduced CaMnSODc; (c) oxidized CaMnSODc and (d) as-isolated K184R, L185P CaMnSODc. Unfolding transitions are shown in black lines. The components (gray) were deconvoluted using a two-state irreversible model for WT ScMnSOD and a non-two-state reversible model for RP-mutant ScMnSOD, and WT and RP-mutant CaMnSODc. Reduced or oxidized enzymes were prepared by adding sodium hyposulfite or potassium permanganate to the sample solution prior to the DSC scan.

Mentions: To investigate the impact of the quaternary structure and the residue substitutions at dimer interfaces on MnSOD thermostability, we monitored the unfolding transitions of WT and RP-mutant yeast MnSODs by DSC (Materials and Methods). Although the heat treatment of all WT and RP-mutant yeast MnSODs led to irreversible aggregation of the proteins, the DSC profiles were fitted using either a two-state irreversible model or a non-two-state reversible model, depending on which model yielded a better fitting. WT and mutant proteins are both partially loaded with Mn (Table S1). WT and RP-mutant ScMnSOD are loaded with 0.70 and 0.71 Mn per subunit, respectively. WT and RP-mutant CaMnSODc are loaded with 0.59 and 0.43 Mn per subunit, respectively. All four rest predominantly in the reduced (2+) state. The DSC profile of as-isolated ScMnSOD showed a single transition at 91°C, corresponding to one irreversible process (Figure 8A–a). This suggests cooperativity in the aggregation of apo and metallated subunits in ScMnSOD.


Tetramerization reinforces the dimer interface of MnSOD.

Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS - PLoS ONE (2013)

Thermostability of WT and RP-mutant ScMnSOD and CaMnSODc.The S. cerevisiae enzymes in (A) are: (a) as-isolated ScMnSOD, (b) oxidized ScMnSOD and (c) as-isolated K182R, A183P ScMnSOD. The C. albicans enzymes in (B) are: (a) as-isolated CaMnSODc; (b) reduced CaMnSODc; (c) oxidized CaMnSODc and (d) as-isolated K184R, L185P CaMnSODc. Unfolding transitions are shown in black lines. The components (gray) were deconvoluted using a two-state irreversible model for WT ScMnSOD and a non-two-state reversible model for RP-mutant ScMnSOD, and WT and RP-mutant CaMnSODc. Reduced or oxidized enzymes were prepared by adding sodium hyposulfite or potassium permanganate to the sample solution prior to the DSC scan.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646814&req=5

pone-0062446-g008: Thermostability of WT and RP-mutant ScMnSOD and CaMnSODc.The S. cerevisiae enzymes in (A) are: (a) as-isolated ScMnSOD, (b) oxidized ScMnSOD and (c) as-isolated K182R, A183P ScMnSOD. The C. albicans enzymes in (B) are: (a) as-isolated CaMnSODc; (b) reduced CaMnSODc; (c) oxidized CaMnSODc and (d) as-isolated K184R, L185P CaMnSODc. Unfolding transitions are shown in black lines. The components (gray) were deconvoluted using a two-state irreversible model for WT ScMnSOD and a non-two-state reversible model for RP-mutant ScMnSOD, and WT and RP-mutant CaMnSODc. Reduced or oxidized enzymes were prepared by adding sodium hyposulfite or potassium permanganate to the sample solution prior to the DSC scan.
Mentions: To investigate the impact of the quaternary structure and the residue substitutions at dimer interfaces on MnSOD thermostability, we monitored the unfolding transitions of WT and RP-mutant yeast MnSODs by DSC (Materials and Methods). Although the heat treatment of all WT and RP-mutant yeast MnSODs led to irreversible aggregation of the proteins, the DSC profiles were fitted using either a two-state irreversible model or a non-two-state reversible model, depending on which model yielded a better fitting. WT and mutant proteins are both partially loaded with Mn (Table S1). WT and RP-mutant ScMnSOD are loaded with 0.70 and 0.71 Mn per subunit, respectively. WT and RP-mutant CaMnSODc are loaded with 0.59 and 0.43 Mn per subunit, respectively. All four rest predominantly in the reduced (2+) state. The DSC profile of as-isolated ScMnSOD showed a single transition at 91°C, corresponding to one irreversible process (Figure 8A–a). This suggests cooperativity in the aggregation of apo and metallated subunits in ScMnSOD.

Bottom Line: Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure.Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD.We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

Show MeSH
Related in: MedlinePlus