Kinetic role of nonnative species in the folding of bovine pancreatic trypsin inhibitor.
Kinetic role of nonnative species in the folding of bovine pancreatic trypsin inhibitor.
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非本地物种在牛胰腺胰蛋白酶抑制剂折叠中的动力学作用。
DOI：
10.1073/pnas.89.20.9900
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发表时间：
1992
影响因子：
11.1
通讯作者：
Kim,PS
中科院分区：
文献类型：
--
作者：
Weissman,JS;Kim,PS
我们以前已经证明，在牛胰蛋白酶抑制剂的氧化折叠过程中，只有带有天然二硫键的中间体被很好地填充。然而，这些研究也证实了较早的结论[Creighton，T.E.(1977)J.Mol.比奥尔。113,275-293]，动力学上首选的折叠路线中的限速转变涉及分子内二硫键重排。因此，具有非天然二硫键的中间体必须在折叠过程中瞬时形成。在牛胰蛋白酶抑制剂的动力学折叠实验中，可以在低水平检测到两个特定的非本地物种，标记为[30-51；5-14]和[30-51；5-38]，其中数字表示参与二硫键的残基。通过与纯化的可逆捕获的中间体合作，这两个外来物种的作用已经被直接检验。这些物种之间的交换相对较快，并且与最初形成的天然二硫键中间体[30-51；14-38]交换较快。因此，在动力学折叠实验中检测到的两种非本地物种的低丰度主要反映了与这种天然中间体相比，它们的热力学稳定性较低。在很小的程度上，这些外来物种形成了生产性的天然中间体[30-51；5-55]，它是天然蛋白质的直接前体。然而，也会产生等量的[5-55；14-38]，这是一种非生产性的死胡同中间体。因此，在牛胰酶抑制剂折叠过程中检测到的外来物种并不致力于形成生产性的天然中间体，也不能专门用于指导折叠朝着生产性路线进行。
We have shown previously that during the oxidative folding of bovine pancreatic trypsin inhibitor only intermediates with native disulfide bonds are well populated. Nevertheless, these studies also confirmed the earlier conclusion [Creighton, T. E. (1977) J. Mol. Biol. 113, 275-293] that the rate-limiting transition in the kinetically preferred route for folding involves intramolecular disulfide bond rearrangements. Consequently, intermediates with nonnative disulfide bonds must form transiently during folding. Two specific nonnative species, denoted [30-51; 5-14] and [30-51; 5-38], in which numbers indicate residues participating in a disulfide bond, can be detected at low levels in kinetic folding experiments with bovine pancreatic trypsin inhibitor. By working with purified reversibly trapped intermediates, the role of these two nonnative species has been examined directly. These species are found to be in relatively rapid exchange with each other and with an initially formed native two-disulfide intermediate [30-51; 14-38]. Thus, the low abundance of the two nonnative species detected in kinetic folding experiments reflects primarily their low thermodynamic stability as compared to this native intermediate. To a small extent, these nonnative species form the productive native intermediate [30-51; 5-55], which is the immediate precursor to the native protein. However, an equal amount of [5-55; 14-38], a nonproductive dead-end intermediate, is also produced. Thus, the nonnative species detected during the folding of bovine pancreatic trypsin inhibitor are not committed to forming the productive native intermediate, nor do they serve to direct folding specifically toward a productive route.