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. 1995 Aug;4(8):1544–1552. doi: 10.1002/pro.5560040812

Urea-induced dissociation and unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric and spectral studies.

M Zolkiewski 1, N J Nosworthy 1, A Ginsburg 1
PMCID: PMC2143178  PMID: 8520480

Abstract

Urea-induced dissociation and unfolding of manganese.glutamine synthetase (Mn.GS) have been studied at 37 degrees C (pH 7) by spectroscopic and calorimetric methods. In 0 to approximately 2 M urea, Mn.GS retains its dodecameric structure and full catalytic activity. Mn.GS is dissociated into subunits in 6 M urea, as evidenced by a 12-fold decrease in 90 degrees light scattering and a monomer molecular weight of 51,800 in sedimentation equilibrium studies. The light scattering decrease in 4 M urea parallels the time course of Trp exposure but occurs more rapidly than changes in secondary structure and Tyr exposure. Early and late kinetic steps appear to involve predominantly disruption of intra-ring and inter-ring subunit contacts, respectively, in the layered hexagonal structure of Mn.GS. The enthalpies for transferring Mn.GS into urea solutions have been measured by titration calorimetry. After correcting for the enthalpy of binding urea to the protein, the enthalpy of dissociation and unfolding of Mn.GS is 14 +/- 4 cal/g. A net proton uptake of approximately 50 H+/dodecamer accompanies unfolding reactions. The calorimetric data are consistent with urea binding to multiple, independent sites in Mn.GS and the number of binding sites increasing approximately 9-fold during the protein unfolding.

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Selected References

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