Abstract
Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M~r~) at pH 7 and βΌ0.02 ionic strength occurs in two observable steps: a small reversible transition (T~m~ βΌ 42Β°C; Ξ__H__ β
0.9 J/g) followed by a large irreversible transition (T~m~ βΌ 81Β°C; Ξ__Hβ
__ 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 55Β°C) and inhibits aggregation accompanying unfolding at β€50.2 mg protein/mL. With increasing temperature (30β70Β°C) or incubation times at 25Β°C (5β35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudoβfirstβorder (t~1/2~ = 1,030s at 20.0Β°C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (T~max~ βΌ 64Β°C; Ξ__H__ = 17 Β± 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (Ξ__H__ = 57 Β± 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4:144β1552), after correcting for the binding of urea to protein sites exposed during unfolding (β42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.