A Kinetic Analysis of the Folding and Unfolding of OmpA in Urea and Guanidinium Chloride: Single and Parallel Pathways
A Kinetic Analysis of the Folding and Unfolding of OmpA in Urea and Guanidinium Chloride: Single and Parallel Pathways
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DOI：
10.1021/bi300974y
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发表时间：
2012-10-23
期刊：
影响因子：
2.9
通讯作者：
Otzen, Daniel E.
中科院分区：
文献类型：
--
作者：
Andersen, Kell K.;Wang, Huabing;Otzen, Daniel E.
大肠杆菌外膜蛋白OmpA可以从尿素变性状态折叠成脂囊泡和表面活性剂胶束。然而，一个完整的动力学描述的OmpA的折叠和去折叠，这可以提供基础的蛋白质工程研究的蛋白质的折叠途径，是缺乏的。在这里，我们使用两种不同的变性剂来探测的展开机制的OmpA在表面活性剂辛基麦芽糖苷（OM）的存在下。展开的OmpA胶束的存在下，实现与有效的变性剂氯化胍（GdmCl），导致单相展开。相比之下，OmpA在尿素中仅在OM的临界胶束浓度以下展开，并且这发生在不同的相中，我们将其归因于结合了不同量的表面活性剂的状态的存在，从完全“裸露”到部分被表面活性剂覆盖。多个平行的重折叠阶段归因于折叠前不同程度的折叠。动力学结果用于推导OmpA在表面活性剂中的稳定性，使用尿素或GdmCl作为变性剂，得到相当的结果，并表明一个最低限度的三态折叠方案，涉及变性状态D，折叠中间体I，和天然状态N。N和I相对于D分别稳定15.6和2.6千卡/摩尔。OmpA的周质结构域对表面活性剂胶束的稳定性没有贡献。然而，与OmpA相比，BBP（具有缩短的环的OmpA的最小跨膜β-桶形式）被类似于10 kcal/mol去稳定化，突出了环对OmpA稳定性的贡献。
The outer membrane protein OmpA from Escherichia coli can fold into lipid vesicles and surfactant micelles from the urea denatured state. However, a complete kinetic description of the folding and unfolding of OmpA, which can provide the basis for subsequent protein engineering studies of the protein's folding pathway, is lacking. Here we use two different denaturants to probe the unfolding mechanism of OmpA in the presence of the surfactant octyl maltoside (OM). Unfolding of OmpA in the presence of micelles, achieved with the potent denaturant guanidinium chloride (GdmCl), leads to single-phase unfolding. In contrast, OmpA unfolds in urea only below OM's critical micelle concentration, and this occurs in different phases, which we attribute to the existence of states that have bound different amounts of surfactant, from completely "naked" to partly covered by surfactant. Multiple parallel refolding phases are attributed to different levels of collapse prior to folding. Kinetic results used to derive the stability of OmpA in surfactant, using either urea or GdmCl as the denaturing agent, give comparable results and indicate a minimalist three state folding scheme involving denatured state D, folding intermediate I, and native state N. N and I are stabilized by 15.6 and 2.6 kcal/mol, respectively, relative to D. The periplasmic domain of OmpA does not contribute to stability in surfactant micelles. However, BBP, a minimalist transmembrane beta-barrel version of OmpA with shortened loops, is destabilized by, similar to 10 kcal/mol compared to OmpA, highlighting loop contributions to OmpA stability.