Baytshtok Vladimir, Baker Tania A, Sauer Robert T
Department of Biology and.
Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139.
Proc Natl Acad Sci U S A. 2015 Apr 28;112(17):5377-82. doi: 10.1073/pnas.1505881112. Epub 2015 Apr 13.
ATP-dependent molecular machines of the AAA+ superfamily unfold or remodel proteins in all cells. For example, AAA+ ClpX and ClpA hexamers collaborate with the self-compartmentalized ClpP peptidase to unfold and degrade specific proteins in bacteria and some eukaryotic organelles. Although degradation assays are straightforward, robust methods to assay the kinetics of enzyme-catalyzed protein unfolding in the absence of proteolysis have been lacking. Here, we describe a FRET-based assay in which enzymatic unfolding converts a mixture of donor-labeled and acceptor-labeled homodimers into heterodimers. In this assay, ClpX is a more efficient protein-unfolding machine than ClpA both kinetically and in terms of ATP consumed. However, ClpP enhances the mechanical activities of ClpA substantially, and ClpAP degrades the dimeric substrate faster than ClpXP. When ClpXP or ClpAP engage the dimeric subunit, one subunit is actively unfolded and degraded, whereas the other subunit is passively unfolded by loss of its partner and released. This assay should be broadly applicable for studying the mechanisms of AAA+ proteases and remodeling chaperones.
AAA+超家族的ATP依赖性分子机器在所有细胞中负责蛋白质的解折叠或重塑。例如，AAA+ ClpX和ClpA六聚体与自我分隔的ClpP肽酶协作，在细菌和一些真核细胞器中使特定蛋白质解折叠并降解。尽管降解分析很直接，但一直缺乏在无蛋白水解情况下测定酶催化蛋白质解折叠动力学的可靠方法。在此，我们描述了一种基于荧光共振能量转移（FRET）的分析方法，其中酶促解折叠将供体标记和受体标记的同型二聚体混合物转化为异型二聚体。在该分析中，无论是在动力学方面还是在所消耗的ATP方面，ClpX都是比ClpA更高效的蛋白质解折叠机器。然而，ClpP显著增强了ClpA的机械活性，并且ClpAP比ClpXP更快地降解二聚体底物。当ClpXP或ClpAP作用于二聚体亚基时，一个亚基被主动解折叠并降解，而另一个亚基则因失去其伙伴而被动解折叠并释放。该分析方法应广泛适用于研究AAA+蛋白酶和重塑伴侣的作用机制。
