Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry
Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry
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DOI：
10.3791/57806
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发表时间：
2018-06-01
影响因子：
1.2
通讯作者：
Reichmann, Dana
中科院分区：
文献类型：
--
作者：
Fassler, Rosi;Edinger, Nufar;Reichmann, Dana
生物体在其生命周期中经常需要应对波动的环境，包括温度、pH值、活性氧积累等的变化。这些波动可导致广泛的蛋白质展开、聚集和细胞死亡。因此，细胞已经进化出一个动态的和应激特异性的分子伴侣网络，在应激条件下维持一个“健康”的蛋白质组。不依赖atp的伴侣蛋白是一类主要的分子伴侣蛋白，作为一线防御分子，以应激依赖的方式保护蛋白质不聚集。这些伴侣蛋白的一个共同特征是它们能够利用结构可塑性进行应力特异性激活、识别和释放错误折叠的来访者。在本文中，我们重点研究了一种内在无序伴侣蛋白的功能和结构分析，即细菌氧化还原调节的Hsp33，它在氧化应激过程中保护蛋白质免受聚集。在这里，我们提出了多种技术的工具箱，用于研究氧化还原调节的伴侣活性，以及绘制伴侣活性的构象变化。具体来说，我们描述了一个工作流程，其中包括制备完全还原和完全氧化的蛋白质，然后使用光散射分析体外伴侣抗聚集活性，重点是抗聚集活性的程度及其动力学。为了克服聚集分析期间积累的频繁异常值，我们描述了Kfits的使用，Kfits是一种新颖的图形工具，可以轻松处理动力学测量。该工具可以很容易地应用于其他类型的动力学测量，以去除异常值和拟合动力学参数。为了将功能与蛋白质结构联系起来，我们描述了一种结构质谱技术的设置和工作流程，氢氘交换质谱技术允许在Hsp33活性的不同阶段对伴侣和底物的构象变化进行映射。同样的方法可以应用于其他蛋白质-蛋白质和蛋白质-配体的相互作用。
Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client.In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogendeuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.