Atomic Force Microscopy of Proteasome Assemblies
Atomic Force Microscopy of Proteasome Assemblies
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DOI：
10.1007/978-1-61779-105-5_9
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发表时间：
2011-01-01
期刊：
影响因子：
--
通讯作者：
Osmulski, Pawel A.
中科院分区：
文献类型：
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作者：
Gaczynska, Maria;Osmulski, Pawel A.
蛋白酶体是所有真核生物必需的主要蛋白酶。这种大的、多亚基的、模块化的和多功能的酶负责大多数受调节的细胞内蛋白质降解。它是多酶泛素-蛋白酶体途径的一部分，广泛参与蛋白质的识别、标记和切割。“蛋白酶体”这个名字是指几种类型的蛋白质组装体共享一个共同的催化核心颗粒。额外的蛋白质模块附着在核心上，调节其活动，并扩大其功能。蛋白酶体的结构已经用多种方法进行了广泛的研究。解决了几个物种的核心颗粒的晶体结构。然而，仅确定了用PA 26活化剂修饰的核颗粒的单一结构。核磁共振光谱学被成功地应用于探测一种更简单的，古细菌类型的核心颗粒。反过来，电子显微镜在探索许多类别的组装体的空间排列方面非常有效。然而，高阶复合物的构成还没有很好地建立。此外，晶体结构提供的蛋白酶体分子动力学的信息非常有限。原子力显微镜（AFM）是解决其他方法无法回答的问题的理想技术。例如，AFM非常适合研究蛋白酶体的变构调节，蛋白质动力学在酶催化中的作用，以及组件中模块和亚基的空间组织。在这里，我们提出了一种方法，探针的构象多样性和动态的酵母核心颗粒使用振荡模式原子力显微镜在液体中。我们正在利用观察到的管状核心颗粒配备有通向催化室的摆动门。我们演示了如何识别不同的门在AFM图像中的构象，以及如何表征配体控制和突变干扰的门动力学。
The proteasome is the essential prime protease in all cukaryotes. The large, multisubunit, modular, and multifunctional enzyme is responsible for the majority of regulated intracellular protein degradation. It constitutes a part of the multienzyme ubiquitin-proteasome pathway, which is broadly implicated in recognition, tagging, and cleavage of proteins. The name "proteasome" refers to several types of protein assemblies sharing a common catalytic core particle. Additional protein modules attach to the core, regulate its activities, and broaden its functional capabilities. The structure of proteasomes has been studied extensively with multiple methods. The crystal structure of the core particle was solved for several species. However, only a single structure of the core particle decorated with PA26 activator has been determined. NMR spectroscopy was successfully applied to probe a much simpler, archeabacterial type of the core particle. In turn, electron microscopy was very effective in exploring the spatial arrangement of many classes of assemblies. Still, the makeup of higher-order complexes is not well established. Besides, the crystal structure provided very limited information on proteasome molecular dynamics. Atomic force microscopy (AFM) is an ideal technique to address questions that are unanswered by other approaches. For example, AFM is perfectly suited to study allosteric regulation of proteasome, the role of protein dynamics in enzymatic catalysis, and the spatial organization of modules and subunits in assemblies. Here, we present a method that probes the conformational diversity and dynamics of yeast core particle using the oscillating mode AFM in liquid. We are taking advantage of the observation that the tube-shaped core particle is equipped with a swinging gate leading to the catalytic chamber. We demonstrate how to identify distinct gate conformations in AFM images and how to characterize the gate dynamics controlled with ligands and disturbed by mutations.