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South African Journal of Science

Print version ISSN 0038-2353

Abstract

KUHNERT, Diane C.; GILDENHUYS, Samantha  and  DIRR, Heini W.. Effect of macromolecular crowding on the stability of monomeric glutaredoxin 2 and dimeric glutathione transferase A1-1. S. Afr. j. sci. [online]. 2008, vol.104, n.1-2, pp. 76-80. ISSN 0038-2353.

The effect of macromolecular crowding on the structure and stability of monomeric glutaredoxin 2 (Grx2) and its homodimeric structural homologue human glutathione transferase A1-1 (hGST A1-1) was investigated using dextran 70 as crowding agent. Far-UV circular dichroism and fluorescence spectroscopic data indicated that repulsive steric interactions between the proteins and dextran (50-300 mg/ml) had little effect on the global structures of the native proteins. Urea-induced unfolding of both proteins was reversible (recoveries of >80%) at low dextran concentrations (<100 mg/ml) but resulted in significant losses in refolding recoveries at higher levels of dextran, due to aggregation. The two-state global unfolding processes of Grx2 and hGST A1-1, as well as their m-values (unfolding cooperativity parameter), were unaffected by 100 mg/ml dextran, demonstrating the absence of specific intermolecular interactions between protein and crowder. Dextran at 100 mg/ml enhanced the stability of Grx2 and hGST A1-1 by 1.1 kcal/mol and 2.2 kcal/mol, respectively. Compaction of the unfolded states of both proteins is indicated by an increase in alpha-helical content and in the decreased solvent exposure of their tryptophan residues. The dextran-induced formation of compact states of urea-denatured Grx2 and hGST A1-1 is ascribed to steric excluded volume effects, which induce an entropic destabilization of expanded unfolded states, thereby shifting the equilibrium between native and unfolded states towards the native state. Quantitatively, however, the extent of stabilization of Grx2 is lower than that predicted by the equivalent hard particle model for the excluded volume effect of dextran on protein stability.

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