Kinetic Analysis of AAA+ Translocases by Combined Fluorescence and Anisotropy Methods
Kinetic Analysis of AAA+ Translocases by Combined Fluorescence and Anisotropy Methods
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结合荧光和各向异性方法对 AAA 转位酶进行动力学分析
DOI：
10.1016/j.bpj.2020.08.018
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发表时间：
2020
影响因子：
3.4
通讯作者：
Lucius, Aaron L.
中科院分区：
文献类型：
--
作者：
Scull, Nathaniel W.;Lucius, Aaron L.
细胞中存在着多种多样的、依赖能量的过程，需要多种多样的大分子机器来维持体内平衡、允许生长和促进繁殖。与各种细胞活性相关的atp酶是一组蛋白质组合，其功能类似于分子马达，将三磷酸核苷结合和水解的能量偶联到沿着聚合物晶格的机械运动中。最近对这些马达结构的深入研究导致了对这些马达如何实现其功能的结构性假设。然而，在许多情况下，我们缺乏对这些电机在观察到的结构状态之间转换时所经历的动态过程的直接动力学测量。因此，有必要改进测试溶液中结构假设的技术。在这里，我们应用瞬态荧光各向异性和全荧光停止流方法来分析这些atp酶马达催化的多肽易位。我们特别关注ClpA的Hsp100-Clp蛋白系统，这是一个研究得很好的模型atp酶，与各种细胞活性系统相关，具有真核生物和古细菌的同源物。使用该系统，我们表明我们可以通过同时分析荧光各向异性和总荧光来重现先前建立的动力学参数，并克服了以前方法的局限性。具体来说，据我们所知，我们首次获得了长度超过100 aa的多肽底物易位的定量解释。
The multitude of varied, energy-dependent processes that exist in the cell necessitate a diverse array of macromolecular machines to maintain homeostasis, allow for growth, and facilitate reproduction. ATPases associated with various cellular activity are a set of protein assemblies that function as molecular motors to couple the energy of nucleoside triphosphate binding and hydrolysis to mechanical movement along a polymer lattice. A recent boom in structural insights into these motors has led to structural hypotheses on how these motors fulfill their function. However, in many cases, we lack direct kinetic measurements of the dynamic processes these motors undergo as they transition between observed structural states. Consequently, there is a need for improved techniques for testing the structural hypotheses in solution. Here, we apply transient-state fluorescence anisotropy and total fluorescence stopped-flow methods to the analysis of polypeptide translocation catalyzed by these ATPase motors. We specifically focus on the Hsp100-Clp protein system of ClpA, which is a well-studied, model ATPases associated with various cellular activity system that has both eukaryotic and archaea homologs. Using this system, we show that we can reproduce previously established kinetic parameters from the simultaneous analysis of fluorescence anisotropy and total fluorescence and overcome previous limitations of our previous approach. Specifically, for the first time, to our knowledge, we obtain quantitative interpretations of the translocation of polypeptide substrates longer than 100 aa.