Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 106 V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.
展开▼
机译:单分子方法为生物分子在平衡和非平衡条件下的行为提供了全新的视角。最值得注意的是通过原子力显微镜和激光镊子进行的拉伸实验。在这里,我们提出了一种替代的单分子方法,该方法可以展开蛋白质结构域,在大于10 6 sup> V / m的电场中观察到,并且可以通过在整个孔膜上施加增加的电压来完全控制。此外,这种展开机制的特征在于测量蛋白质在纳米孔中的停留时间和当前的阻滞。展开数据支持逐步展开机制,而不是经典尿素变性实验观察到的协同转变。最后,通过比较蛋白质的两个突变不稳定的变体,表明电压介导的展开是蛋白质稳定性的函数。
展开▼