A protein caught in a kinetic trap: Structures and stabilities of insulin disulfide isomers
A protein caught in a kinetic trap: Structures and stabilities of insulin disulfide isomers
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DOI：
10.1021/bi0202981
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发表时间：
2002-12-17
期刊：
影响因子：
2.9
通讯作者：
Weiss, MA
中科院分区：
文献类型：
--
作者：
Hua, QX;Jia, WH;Weiss, MA
胰岛素原含有六个半胱氨酸，其特定配对（A6-A11、A7-B7 和 A20-B19）是胰岛素折叠的定义特征。胰岛素链重组研究表明，配对信息包含在 A 和 B 结构域内。预先通过直接化学合成制备的两种含有非天然二硫桥的胰岛素异构体（[A7-A11，A6-B7，A20-B19]和[A6-A7，A11-B7，A20-B19]）具有亚稳态和生物活性。值得注意的是，相同的两种异构体优先由天然胰岛素或胰岛素原在盐酸胍中二硫键重配后形成。不存在其他二硫键异构体表明观察到的物种表现出更大的相对稳定性和/或动力学可达性。第一个异构体（[A7-A11,A6-B7,A20-B19]，胰岛素交换）的结构已被描述[Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat.结构。生物。 2、129-138]。在这里，我们证明了第二种异构体（胰岛素交换2）的有序性低于第一种异构体。 B 链中保留了类似天然的结构元素，而 A 链很大程度上是无序的。胍变性的热力学研究证明了异构体相对于天然胰岛素的不稳定性 (DeltaDeltaG(u) > 3 kcal/mol)。相比之下，胰岛素样生长因子 I (IGF-I) 和相应的异构体 IGF-swap 作为分叉折叠途径的替代产物形成，表现出类似的协同展开转变。胰岛素异构体在结构和稳定性上与二硫键类似物相似，其部分折叠提供了氧化折叠中间体的模型。每个都表现出类似原生的 B 链和有序性较低的 A 链。这种普遍的不对称性与分级二硫键途径一致，其中 B 链中的新生结构为 A 链的折叠提供了模板。亚稳态二硫化物异构体的结构为能源景观的地形提供了探针。
Proinsulin contains six cysteines whose specific pairing (A6-A11, A7-B7, and A20-B19) is a defining feature of the insulin fold. Pairing information is contained within A and B domains as demonstrated by studies of insulin chain recombination. Two insulin isomers containing non-native disulfide bridges ([A7-A11,A6-B7,A20-B19] and [A6-A7,A11-B7,A20-B19]), previously prepared by directed chemical synthesis, are metastable and biologically active. Remarkably, the same two isomers are preferentially formed from native insulin or proinsulin following disulfide reassortment in guanidine hydrochloride. The absence of other disulfide isomers suggests that the observed species exhibit greater relative stability and/or kinetic accessibility. The structure of the first isomer ([A7-A11,A6-B7,A20-B19], insulin-swap) has been described [Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat. Struct. Biol. 2, 129-138]. Here, we demonstrate that the second isomer (insulin-swap2) is less ordered than the first. Nativelike elements of structure are retained in the B chain, whereas the A chain is largely disordered. Thermodynamic studies of guanidine denaturation demonstrate the instability of the isomers relative to native insulin (DeltaDeltaG(u) > 3 kcal/mol). In contrast, insulin-like growth factor I (IGF-I) and the corresponding isomer IGF-swap, formed as alternative products of a bifurcating folding pathway, exhibit similar cooperative unfolding transitions. The insulin isomers are similar in structure and stability to two-disulfide analogues whose partial folds provide models of oxidative folding intermediates. Each exhibits a nativelike B chain and less-ordered A chain. This general asymmetry is consistent with a hierarchical disulfide pathway in which nascent structure in the B chain provides a template for folding of the A chain. Structures of metastable disulfide isomers provide probes of the topography of an energy landscape.