Correlation between disulfide reduction and conformational unfolding in bovine pancreatic trypsin inhibitor.
Correlation between disulfide reduction and conformational unfolding in bovine pancreatic trypsin inhibitor.
复制标题
牛胰蛋白酶抑制剂中二硫键还原与构象去折叠之间的相关性。
DOI：
10.1021/bi962310t
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Anderson,S
中科院分区：
文献类型：
--
作者：
Ma,LC;Anderson,S
牛胰蛋白酶抑制剂 (BPTI) 的天然双二硫键中间体，Cys14 和 Cys38 之间的二硫键被还原，在 BPTI 的二硫键偶联折叠途径中起着特别重要的作用，因为它参与反应的速率决定步骤 [Creighton & Goldenberg (1984)J.Mol.生物。 179、497−526； Weissman & Kim (1991)《科学》253, 1386−1393]。为了直接研究构象稳定性和还原性展开动力学之间的关系，并深入了解 BPTI 硫醇/二硫键介导的折叠/解折叠反应中的限速过渡态，对基于该中间体的类似天然双二硫键类似物的 BPTI 变体进行了检查。引入的氨基酸取代使其热力学稳定性较差。这些变体中二硫化物相对于二硫苏糖醇还原的动力学稳定性也因取代而降低。从化学变性测量获得的稳定自由能（ΔG）和从这一系列变体的还原解折叠反应获得的构象转变的活化能（ΔG⧧conf）是高度相关的。观察到的相关性意味着二硫键还原与这组蛋白质变体中的构象稳定性直接耦合。它还强烈表明还原性解折叠反应限速步骤中的过渡态涉及蛋白质的高度解折叠构象。这些数据与构象耦合氧化还原折叠路径一致，涉及以未折叠 (30−51) 和未折叠 (5−55) 作为反应物种的两条平行路径。此外，这些结果为观察到的野生型 BPTI 重折叠反应中的一二硫键 (30−51) 中间体形成的 5−55 个二硫键形成速率相对于 14−38 个键形成速率降低了 1000 倍提供了理论解释。这些数据符合蛋白质折叠过程中蛋白质二硫键形成的一般范例，其中折叠中间体中的天然样结构加速了溶剂暴露的二硫键的形成，但抑制了核心二硫键的形成。该模型预测，在折叠反应后期形成埋藏二硫键的“重排”机制（即非天然二硫键参与限速步骤）可能是高稳定性表面含二硫键蛋白质氧化还原折叠途径的共同特征。
The native-like two-disulfide intermediate of bovine pancreatic trypsin inhibitor (BPTI), with the disulfide between Cys14 and Cys38 reduced, plays a particularly important role in the disulfide-coupled folding pathway of BPTI because of its participation in the rate-determining step of the reaction [Creighton & Goldenberg (1984)J.Mol. Biol. 179, 497−526; Weissman & Kim (1991)Science 253, 1386−1393]. In order to study directly the relationship between conformational stability and reductive unfolding kinetics, and to gain insight concerning the rate-limiting transition state in the thiol/disulfide-mediated folding/unfolding reaction of BPTI, BPTI variants based on a native-like two-disulfide analog of this intermediate, , were examined. The amino acid replacements introduced into rendered it thermodynamically less stable. The kinetic stability, with respect to reduction by dithiothreitol, of the disulfides in these variants was also decreased by the substitutions. The stabilization free energy (ΔG), obtained from chemical denaturation measurements, and the activation energy of the conformational transition (ΔG⧧conf), from the reductive unfolding reaction for this series of variants, were highly correlated. The observed correlation implies a direct coupling of disulfide reduction to conformational stability in this set of protein variants. It also strongly suggests that the transition state in the rate-limiting step of the reductive unfolding reaction involves a highly unfolded conformation of the protein. These data are consistent with a conformation-coupled redox folding pathway for involving two parallel paths with unfolded (30−51) and unfolded (5−55) as the reactive species. Furthermore, the results provide a theoretical explanation for the observed 1000-fold diminution in the rate of 5−55 disulfide bond formation, relative to that of 14−38 bond formation, from the one-disulfide (30−51) intermediate in the wild-type BPTI refolding reaction. The data fit a general paradigm for protein disulfide formation during protein folding whereby native-like structure in folding intermediates accelerates formation of solvent-exposed disulfides but inhibits formation of core disulfides. This model predicts that a “rearrangement” mechanism (i.e., with non-native disulfides involved in the rate-limiting step) to form buried disulfides at a late stage in the folding reaction may be a common feature of redox folding pathways for surface disulfide-containing proteins of high stability.