Differential Scanning Calorimetry Study of Reversible, Partial Unfolding Transitions in Dodecameric Glutamine Synthetase from Escherichia coli

Abstract

Partial unfolding of dodecameric glutamine synthetase (GS) from Escherichia coli has been studied by differential scanning calorimetry (DSC). A single endotherm (tm = 51.6 ± 0.1 °C and ΔHcal = 211 ± 4 kcal/mol of enzyme) was observed in DSC experiments with Mn«GS in the presence of 1.0 mM free Mn2+ and 100 mM KCl at pH 7. The dodecameric structure of Mn·GS was retained throughout heating cycles, and thermal transitions were reversible as shown by rescans [with 6–18 mg of GS (Mr 622 000) from 15 to 68 °C at 20–60 °C/h] and by >93% recovery of activity. A cooperative ratio ΔHcal/ΔH∨H of 1.6 ± 0.1 and deconvolution analysis show two cooperative units (two-state transitions): t1 = 50.4 and t2 = 51.7 °C; the ratio of the relative sizes of thermally labile domains is ~ 1:2 as judged by ΔH2/ΔH1 ≅ 2. However, the thermally induced overall enthalpy change (0.34 cal/g) for GS dodecamer is only 5-10% of that for thermal unfolding of small globular proteins at 50 °C. The t1 and t2 values from deconvolutions of DSC data agree with t0.5 values previously calculated from spectral measurements of temperature-induced exposures of ~0.7 of 2 Trp and ~2 of 17 Tyr per subunit, respectively [Shrake et al. (1989) Biochemistry 28, 6281-6294], over a 14 °C temperature range using both stabilizing and destabilizing conditions for Mn·GS. No uncoupling of Trp and Tyr exposures or of cooperative units in DSC experiments with Mn·GS occurred in the presence of either 150 mM Gln (tm = 58.6 °C) or 10 mM free [Mn2+] (tm = 43.9 °C). Thus, cooperative interactions apparently link partial unfolding reactions of all subunits within the GS dodecamer so that only two two-state transitions are observed. © 1991, American Chemical Society. All rights reserved.