THERMAL UNFOLDING OF DODECAMERIC GLUTAMINE-SYNTHETASE - INHIBITION OFAGGREGATION BY UREA

Thermal unfolding of dodecameric manganese.glutamine synthetase (622,0 00 M-r) at pH 7 and similar to 0.02 ionic strength occurs in two obser vable steps: a small reversible transition (T-m similar to 42 degrees C; Delta H congruent to 0.9 J/g) followed by a large irreversible tran sition (T-m similar to 81 degrees C; Delta H congruent to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Second ary structure, hydrophobicity, and oligomeric structure of the equilib rium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M:) destabilizes the dode camer (with a tertiary structure similar to that without urea at 55 de grees C) and inhibits aggregation accompanying un folding at less than or equal to 0.2 mg protein/ml. With increasing temperature (30-70 deg rees C) or incubation times at 25 degrees C: (5-35h) in 3 Pn urea, onl y dodecamer and unfolded monomer are detected. In addition, the loss i n enzyme secondary structure is pseudo-first-order (t(1/2) = 1,030 s a t 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (T-max similar to 64 degree s C; Delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isotherma l calorimetry (Delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Gi nsburg A, 1995, Protein Sci 4:1544-1552), after correcting for the bin ding of urea to protein sites exposed during unfolding (-42 J/g). Refo lding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.