title: Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.
abstract: Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.
authors.affiliation: Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Nosworthy
authors.forename: A
authors.initials: A
authors.lastname: Ginsburg
mesh_terms: D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology*
chemical_list: D046911:Macromolecular Substances; D014508:Urea; D005974:Glutamate-Ammonia Ligase