The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.

Nicolai, E., DI VENERE, A., Rosato, N., Rossi, A., FINAZZI AGRO', A., Mei, G. (2006). Physico-chemical properties of molten dimer ascorbate oxidase. THE FEBS JOURNAL, 273(22), 5194-5204 [10.1111/j.1742-4658.2006.05515.x].

Physico-chemical properties of molten dimer ascorbate oxidase

NICOLAI, ELEONORA;DI VENERE, ALMERINDA;ROSATO, NICOLA;ROSSI, ANTONELLO;FINAZZI AGRO', ALESSANDRO;MEI, GIAMPIERO
2006-11-01

Abstract

The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.
nov-2006
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10 - BIOCHIMICA
English
Con Impact Factor ISI
Trypsin; Models, Molecular; Dimerization; Protein Denaturation; Copper; Plant Proteins; Protein Binding; Ascorbate Oxidase; Protein Structure, Quaternary; Hydrostatic Pressure; Protein Folding; Protein Structure, Tertiary; Protein Conformation
Nicolai, E., DI VENERE, A., Rosato, N., Rossi, A., FINAZZI AGRO', A., Mei, G. (2006). Physico-chemical properties of molten dimer ascorbate oxidase. THE FEBS JOURNAL, 273(22), 5194-5204 [10.1111/j.1742-4658.2006.05515.x].
Nicolai, E; DI VENERE, A; Rosato, N; Rossi, A; FINAZZI AGRO', A; Mei, G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/26317
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