Salamanca Silvia, Villegas Virtudes, Vendrell Josep, Li Li, Aviles Francesc X, Chang Jui-Yoa
Institut de Biotecnologia i Biomedicina i Departament de Bioquimica i Biologia Molecular, Universitat Autònoma de Barcelona 08193 Bellaterra, Spain.
J Biol Chem. 2002 May 17;277(20):17538-43. doi: 10.1074/jbc.M200040200. Epub 2002 Mar 13.
The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.
利用二硫键重排技术阐明了水蛭羧肽酶抑制剂（LCI）的解折叠和变性曲线。在硫醇引发剂和变性剂存在的情况下，天然LCI通过重排其天然二硫键而变性，并转化为一组重排异构体的混合物。LCI的104种可能的重排异构体中有9种可以区分，其总量占变性LCI总量的90%。绘制随着变性剂浓度增加，天然LCI转化为重排异构体的比例的变性曲线表明，达到50%变性所需的硫氰酸胍和盐酸胍的浓度分别为2.4 m和3.6 m。相比之下，即使在高浓度（8 m）下，天然LCI也抗尿素变性。LCI的解折叠途径是根据变性过程中LCI重排异构体相对浓度的变化来定义的。两组重排异构体在解折叠途径中呈现不同变化。一组在强变性条件下作为中间体积累，对应于开放或松弛结构，其中发现了珠状异构体。另一组的相对丰度与变性条件呈负相关，应该具有另一种比未折叠状态更紧凑的非天然结构。与其他含二硫键的蛋白质相比，LCI的解折叠速率常数较低。总体而言，本研究结果表明，具有潜在生物技术应用价值的分子LCI具有缓慢的解折叠动力学且高度稳定。
