Tsutsui Yuko, Wintrode Patrick L
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106, USA.
J Mol Biol. 2007 Aug 3;371(1):245-55. doi: 10.1016/j.jmb.2007.05.039. Epub 2007 May 18.
Alpha-1 antitrypsin (alpha(1)-AT) is a member of the serpin class of protease inhibitors, and folds to a metastable state rather than its thermodynamically most stable native state. Upon cleavage by a target protease, alpha(1)-AT undergoes a dramatic conformational change to a stable form, translocating the bound protease more than 70 A to form an inhibitory protease-serpin complex. Numerous mutagenesis studies on serpins have demonstrated the trade-off between the stability of the metastable state on the one hand and the inhibitory efficiency on the other. Studies of the equilibrium unfolding of serpins provide insight into this connection between structural plasticity and metastability. We studied equilibrium unfolding of wild-type alpha(1)-AT using hydrogen-deuterium/exchange mass spectrometry to characterize the structure and the stability of an equilibrium intermediate that was observed in low concentrations of denaturant in earlier studies. Our results show that the intermediate observed at low concentrations of denaturant has no protection from hydrogen-deuterium exchange, indicating a lack of stable structure. Further, differential scanning calorimetry of alpha(1)-AT at low concentrations of denaturant shows no heat capacity peak during thermal denaturation, indicating that the transition from the intermediate to the unfolded state is not a cooperative first-order-like phase transition.. Our results show that the unfolding of alpha(1)-AT involves a cooperative transition to a molten globule form, followed by a non-cooperative transition to a random-coil form as more guanidine is added. Thus, the entire alpha(1)-AT molecule consists of one cooperative structural unit rather than multiple structural domains with different stabilities. Furthermore, our results together with previous mutagenesis studies suggest a possible link between an equilibrium molten globule and a functional intermediate that may be populated during the protease inhibition.
α-1抗胰蛋白酶（α(1)-AT）是丝氨酸蛋白酶抑制剂家族（serpin）的成员之一，它折叠成亚稳态而非热力学上最稳定的天然状态。在被靶蛋白酶切割后，α(1)-AT会发生剧烈的构象变化，转变为稳定形式，将结合的蛋白酶移位超过70埃，形成抑制性蛋白酶-丝氨酸蛋白酶抑制剂复合物。众多关于丝氨酸蛋白酶抑制剂的诱变研究表明，一方面是亚稳态的稳定性，另一方面是抑制效率，两者之间存在权衡。对丝氨酸蛋白酶抑制剂平衡去折叠的研究有助于深入了解结构可塑性与亚稳态之间的这种联系。我们使用氢-氘/交换质谱法研究了野生型α(1)-AT的平衡去折叠，以表征在早期研究中在低浓度变性剂中观察到的平衡中间体的结构和稳定性。我们的结果表明，在低浓度变性剂中观察到的中间体对氢-氘交换没有保护作用，这表明缺乏稳定结构。此外，低浓度变性剂条件下α(1)-AT的差示扫描量热法显示，热变性过程中没有热容峰，这表明从中间体到未折叠状态的转变不是协同的一级相变。我们的结果表明，α(1)-AT的去折叠涉及到向熔融球状体形式的协同转变，随后随着更多胍的加入，向无规卷曲形式进行非协同转变。因此，整个α(1)-AT分子由一个协同结构单元组成，而不是由具有不同稳定性的多个结构域组成。此外，我们的结果与先前的诱变研究共同表明，平衡熔融球状体与蛋白酶抑制过程中可能出现的功能中间体之间可能存在联系。
