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

RP-mutant CaMnSODc is more subject to GdHCl-induced unfolding than the wild type.The molar CD at 224 nm was used to monitor changes in α-helical structure content as a function of [GdHCl]. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The sample solutions contained 0.2 mg/mL (monomer concentration) MnSOD in 25 mM potassium phosphate (pH 7.4).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646814&req=5

pone-0062446-g007: RP-mutant CaMnSODc is more subject to GdHCl-induced unfolding than the wild type.The molar CD at 224 nm was used to monitor changes in α-helical structure content as a function of [GdHCl]. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The sample solutions contained 0.2 mg/mL (monomer concentration) MnSOD in 25 mM potassium phosphate (pH 7.4).

Mentions: In order to understand the impact of the quaternary structure and the dimer interface on MnSOD stability, we used CD spectroscopy to monitor the denaturant-induced unfolding transitions of WT and RP-mutant yeast MnSODs (Figure S4). The molar CD at 224 nm was used to monitor changes in α-helical structure content as a function of [GdHCl] (Figure 7). Under these experimental conditions, ScMnSOD is a tetramer while CaMnSODc is a dimer or “loose tetramer”. The sharp decrease in helical structure content occurred at ∼3.4 M GdHCl in ScMnSOD and ∼1.6 M GdHCl in CaMnSODc (Figure 7). The unfolding of RP-mutant CaMnSODc occurred at a lower concentration (∼1.2 M) of GdHCl than that of WT CaMnSODc (Figure 7). By contrast, the unfolding profiles of WT and RP-mutant ScMnSOD are comparable to each other (Figure 7).


Tetramerization reinforces the dimer interface of MnSOD.

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

RP-mutant CaMnSODc is more subject to GdHCl-induced unfolding than the wild type.The molar CD at 224 nm was used to monitor changes in α-helical structure content as a function of [GdHCl]. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The sample solutions contained 0.2 mg/mL (monomer concentration) MnSOD in 25 mM potassium phosphate (pH 7.4).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062446-g007: RP-mutant CaMnSODc is more subject to GdHCl-induced unfolding than the wild type.The molar CD at 224 nm was used to monitor changes in α-helical structure content as a function of [GdHCl]. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The sample solutions contained 0.2 mg/mL (monomer concentration) MnSOD in 25 mM potassium phosphate (pH 7.4).
Mentions: In order to understand the impact of the quaternary structure and the dimer interface on MnSOD stability, we used CD spectroscopy to monitor the denaturant-induced unfolding transitions of WT and RP-mutant yeast MnSODs (Figure S4). The molar CD at 224 nm was used to monitor changes in α-helical structure content as a function of [GdHCl] (Figure 7). Under these experimental conditions, ScMnSOD is a tetramer while CaMnSODc is a dimer or “loose tetramer”. The sharp decrease in helical structure content occurred at ∼3.4 M GdHCl in ScMnSOD and ∼1.6 M GdHCl in CaMnSODc (Figure 7). The unfolding of RP-mutant CaMnSODc occurred at a lower concentration (∼1.2 M) of GdHCl than that of WT CaMnSODc (Figure 7). By contrast, the unfolding profiles of WT and RP-mutant ScMnSOD are comparable to each other (Figure 7).

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