Nosworthy N J, Ginsburg A
Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Protein Sci. 1997 Dec;6(12):2617-23. doi: 10.1002/pro.5560061213.
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.
在pH 7和离子强度约为0.02的条件下，十二聚体锰谷氨酰胺合成酶（分子量622,000）的热解折叠过程可观察到两个步骤：一个小的可逆转变（熔解温度约42℃；焓变约等于0.9 J/g），随后是一个大的不可逆转变（熔解温度约81℃；焓变约等于23.4 J/g），在此过程中二级结构丧失且形成可溶性聚集体。平衡中间体的二级结构、疏水性和寡聚结构与天然蛋白质相同，然而一些芳香族残基更加暴露。尿素（3 M）使十二聚体不稳定（其三级结构类似于55℃时无尿素的情况），并在蛋白质浓度≤0.2 mg/mL时抑制解折叠过程中伴随的聚集。随着温度升高（30 - 70℃）或在3 M尿素中于25℃孵育时间延长（5 - 35小时），仅检测到十二聚体和解折叠的单体。此外，酶二级结构的丧失为准一级反应（在4.5 M尿素中于20.0℃时半衰期t1/2 = 1,030秒）。对3 M尿素中的酶进行差示扫描量热法显示一个吸热峰（最高温度约64℃；焓变 = 17 ± 2 J/g）。解离和解折叠的焓变与通过等温量热法进行尿素滴定测定的值一致（焓变 = 57 ± 15 J/g；佐尔基耶夫斯基M、诺兹沃西NJ、金斯堡A，1995年，《蛋白质科学》4：1544 - 1552），在校正尿素与解折叠过程中暴露的蛋白质位点的结合（-42 J/g）之后。热解折叠后尿素稀释时可重新折叠并组装成有活性的酶。
