Scull Nathaniel W, Lucius Aaron L
Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama.
Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama.
Biophys J. 2020 Oct 6;119(7):1335-1350. doi: 10.1016/j.bpj.2020.08.018. Epub 2020 Aug 24.
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.
细胞中存在众多不同的、依赖能量的过程，这就需要各种各样的大分子机器来维持体内平衡、促进生长并推动繁殖。与各种细胞活动相关的ATP酶是一组蛋白质组装体，它们作为分子马达，将核苷三磷酸结合与水解的能量与沿聚合物晶格的机械运动相耦合。最近对这些马达的结构见解激增，引发了关于这些马达如何履行其功能的结构假说。然而，在许多情况下，我们缺乏对这些马达在从观察到的结构状态转变过程中所经历的动态过程的直接动力学测量。因此，需要改进的技术来在溶液中测试这些结构假说。在这里，我们将瞬态荧光各向异性和全荧光停流方法应用于分析这些ATP酶马达催化的多肽转运。我们特别关注ClpA的Hsp100-Clp蛋白系统，它是一个经过充分研究的、与各种细胞活动相关的模型ATP酶系统，有真核生物和古细菌的同源物。利用这个系统，我们表明我们可以通过同时分析荧光各向异性和全荧光来重现先前建立的动力学参数，并克服我们之前方法的局限性。具体而言，据我们所知，我们首次获得了对长度超过100个氨基酸的多肽底物转运的定量解释。
