Bischofberger Mirko, Iacovache Ioan, Boss Daniel, Naef Felix, van der Goot F Gisou, Molina Nacho
Global Health Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; The Institute of Bioengineering, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Global Health Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Biophys J. 2016 Apr 12;110(7):1574-1581. doi: 10.1016/j.bpj.2016.02.035.
Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.
许多生物过程依赖于蛋白质复合物的顺序组装。然而，通过直接方法研究此类过程的动力学往往不可行。作为这类蛋白质复合物的一个重要类别，成孔毒素最初是可溶性单体蛋白，然后寡聚形成跨膜复合物，最终在靶细胞膜上形成孔道。在此，我们实时监测了特征明确的细菌成孔毒素气溶素在单细胞中的孔形成动力学，以确定导致每个细胞形成第一个功能性孔道的延迟时间。对这些延迟时间的概率建模表明，一个缓慢反应和七个同等快速的限速反应最能解释整体孔形成动力学。该模型预测单体激活是整个孔形成过程的限速步骤。我们推测这可能是通过前肽的释放实现的，并且确实发现肽的去除消除了这些步骤。这项研究说明了如何利用复杂过程动力学中的随机性来识别多步生物分子组装途径背后的限速机制。
