Differential scanning calorimetry study of reversible, partial unfolding transitions in dodecameric glutamine synthetase from Escherichia coli.
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 degrees C and delta Hcal = 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 degrees C at 20-60 degrees C/h] and by greater than 93% recovery of activity. A cooperative ratio delta Hcal/delta HvH of 1.6 +/- 0.1 and deconvolution analysis show two cooperative units (two-state transitions): t1 = 50.4 and t2 = 51.7 degrees C; the ratio of the relative sizes of thermally labile domains is approximately 1:2 as judged by delta H2/delta H1 approximately equal to 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 degrees 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 approximately 0.7 of 2 Trp and approximately 2 of 17 Tyr per subunit, respectively [Shrake et al. (1989) Biochemistry 28, 6281-6294], over a 14 degrees C temperature range using both stabilizing and destabilizing conditions for Mn.GS.(ABSTRACT TRUNCATED AT 250 WORDS)
Section on Protein Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
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Ginsburg
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Zolkiewski
D002152:Calorimetry, Differential Scanning; D002712:Chlorides; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry*; D046911:Macromolecular Substances; D008345:Manganese / Q000737:chemistry; D017895:Manganese Compounds; D011489:Protein Denaturation; D013816:Thermodynamics
D002712:Chlorides; D046911:Macromolecular Substances; D017895:Manganese Compounds; D008345:Manganese; D005974:Glutamate-Ammonia Ligase; C025340:manganese chloride