Department of Chemical Engineering, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
School of Plant Science and Food Security, Tel Aviv University, Tel Aviv 6997801, Israel.
J Struct Biol. 2020 Jun 1;210(3):107495. doi: 10.1016/j.jsb.2020.107495. Epub 2020 Mar 12.
Polyproteins, comprised from proteins arrayed in tandem, respond to mechanical loads through partial unfolding and extension. This response to tension that enables their physiological function is related to the ability to dynamically regulate their elasticity. The unique arrangement of their individual mechanical components (proteins and polymeric linkers), and the interactions between them eventually determines their performance. The sequential unfolding-times within a polyprotein are inherently assumed to be independent and identically distributed (iid), thus expected to follow an exponential distribution. Nevertheless, a large body of literature using single molecule force spectroscopy (SMFS) provides evidence that forced unfolding-times of N proteins within a polyprotein do not follow the exponential distribution. Here we use SMFS with Atomic Force Microscopy to measure the unfolding kinetics of Poly-(I91) at 180 pN. The unfolding time-intervals were statistically analysed using three common approaches, all exhibiting an N-effect: hierarchical behavior with non-identical unfolding time distributions. Using continuous time random walk approach indicates that the unfolding times display subdiffusive features. Put together with free-energy reconstruction of the whole unfolding polyprotein, we provide physical explanation for this nontrivial behavior, according to which the elongating polypeptide chain with each unfolding event intervenes with the sequential unfolding probabilities and correlates them.
多聚蛋白由串联排列的蛋白质组成，通过部分展开和延伸来响应机械载荷。这种对张力的反应使它们能够发挥生理功能，这与它们动态调节弹性的能力有关。它们各自的机械组件（蛋白质和聚合接头）的独特排列方式以及它们之间的相互作用最终决定了它们的性能。多聚蛋白内的顺序展开时间被假定为独立且同分布（iid），因此预计遵循指数分布。然而，大量使用单分子力谱（SMFS）的文献提供的证据表明，多聚蛋白内的 N 个蛋白质的强制展开时间不遵循指数分布。在这里，我们使用原子力显微镜的 SMFS 来测量 Poly-(I91) 在 180 pN 下的展开动力学。使用三种常见方法对展开时间间隔进行了统计分析，所有方法都表现出 N 效应：具有非相同展开时间分布的层次行为。使用连续时间随机行走方法表明，展开时间显示亚扩散特征。结合整个展开多聚蛋白的自由能重建，我们根据这种非平凡行为提供了物理解释，根据该解释，每个展开事件的伸展多肽链都会干预顺序展开概率并将它们相关联。
