Differential scanning calorimetry study of reversible, partial unfolding transitions in dodecameric glutamine synthetase from Escherichia coli. 1991

A Ginsburg, and M Zolkiewski
Section on Protein Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.

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)

UI MeSH Term Description Entries
D008345 Manganese A trace element with atomic symbol Mn, atomic number 25, and atomic weight 54.94. It is concentrated in cell mitochondria, mostly in the pituitary gland, liver, pancreas, kidney, and bone, influences the synthesis of mucopolysaccharides, stimulates hepatic synthesis of cholesterol and fatty acids, and is a cofactor in many enzymes, including arginase and alkaline phosphatase in the liver. (From AMA Drug Evaluations Annual 1992, p2035)
D011489 Protein Denaturation Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein. Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D002152 Calorimetry, Differential Scanning Differential thermal analysis in which the sample compartment of the apparatus is a differential calorimeter, allowing an exact measure of the heat of transition independent of the specific heat, thermal conductivity, and other variables of the sample. Differential Thermal Analysis, Calorimetric,Calorimetric Differential Thermal Analysis,Differential Scanning Calorimetry,Scanning Calorimetry, Differential
D002712 Chlorides Inorganic compounds derived from hydrochloric acid that contain the Cl- ion. Chloride,Chloride Ion Level,Ion Level, Chloride,Level, Chloride Ion
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005974 Glutamate-Ammonia Ligase An enzyme that catalyzes the conversion of ATP, L-glutamate, and NH3 to ADP, orthophosphate, and L-glutamine. It also acts more slowly on 4-methylene-L-glutamate. (From Enzyme Nomenclature, 1992) EC 6.3.1.2. Glutamine Synthetase,Glutamate Ammonia Ligase (ADP),Glutamate Ammonia Ligase,Ligase, Glutamate-Ammonia,Synthetase, Glutamine
D013816 Thermodynamics A rigorously mathematical analysis of energy relationships (heat, work, temperature, and equilibrium). It describes systems whose states are determined by thermal parameters, such as temperature, in addition to mechanical and electromagnetic parameters. (From Hawley's Condensed Chemical Dictionary, 12th ed) Thermodynamic
D046911 Macromolecular Substances Compounds and molecular complexes that consist of very large numbers of atoms and are generally over 500 kDa in size. In biological systems macromolecular substances usually can be visualized using ELECTRON MICROSCOPY and are distinguished from ORGANELLES by the lack of a membrane structure. Macromolecular Complexes,Macromolecular Compounds,Macromolecular Compounds and Complexes,Complexes, Macromolecular,Compounds, Macromolecular,Substances, Macromolecular
D017895 Manganese Compounds Inorganic chemicals that contain manganese as an integral part of the molecule. Compounds, Manganese

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