Tych Katarzyna M, Hughes Megan L, Bourke James, Taniguchi Yukinori, Kawakami Masaru, Brockwell David J, Dougan Lorna
Astbury Centre for Structural Molecular Biology and School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, United Kingdom.
School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, United Kingdom.
Phys Rev E Stat Nonlin Soft Matter Phys. 2015 Jan;91(1):012710. doi: 10.1103/PhysRevE.91.012710. Epub 2015 Jan 26.
Single-molecule force spectroscopy using an atomic force microscope (AFM) can be used to measure the average unfolding force of proteins in a constant velocity experiment. In combination with Monte Carlo simulations and through the application of the Zhurkov-Bell model, information about the parameters describing the underlying unfolding energy landscape of the protein can be obtained. Using this approach, we have completed protein unfolding experiments on the polyprotein (I27)(5) over a range of pulling velocities. In agreement with previous work, we find that the observed number of protein unfolding events observed in each approach-retract cycle varies between one and five, due to the nature of the interactions between the polyprotein, the AFM tip, and the substrate, and there is an unequal unfolding probability distribution. We have developed a Monte Carlo simulation that incorporates the impact of this unequal unfolding probability distribution on the median unfolding force and the calculation of the protein unfolding energy landscape parameters. These results show that while there is a significant, unequal unfolding probability distribution, the unfolding energy landscape parameters obtained from use of the Zhurkov-Bell model are not greatly affected. This result is important because it demonstrates that the minimum acceptance criteria typically used in force extension experiments are justified and do not skew the calculation of the unfolding energy landscape parameters. We further validate this approach by determining the error in the energy landscape parameters for two extreme cases, and we provide suggestions for methods that can be employed to increase the level of accuracy in single-molecule experiments using polyproteins.
使用原子力显微镜（AFM）的单分子力谱可用于在恒速实验中测量蛋白质的平均解折叠力。结合蒙特卡罗模拟并通过应用朱尔科夫-贝尔模型，可以获得有关描述蛋白质潜在解折叠能量景观的参数的信息。使用这种方法，我们在一系列拉伸速度下完成了对多聚蛋白（I27）(5)的蛋白质解折叠实验。与先前的工作一致，我们发现由于多聚蛋白、AFM 探针和底物之间相互作用的性质，在每个接近-回缩循环中观察到的蛋白质解折叠事件数量在一到五个之间变化，并且存在不平等的解折叠概率分布。我们开发了一种蒙特卡罗模拟，该模拟纳入了这种不平等解折叠概率分布对中值解折叠力和蛋白质解折叠能量景观参数计算的影响。这些结果表明，虽然存在显著的、不平等的解折叠概率分布，但使用朱尔科夫-贝尔模型获得的解折叠能量景观参数并未受到很大影响。这一结果很重要，因为它表明在力延伸实验中通常使用的最小接受标准是合理的，并且不会扭曲解折叠能量景观参数的计算。我们通过确定两种极端情况下能量景观参数的误差进一步验证了这种方法，并为可用于提高使用多聚蛋白的单分子实验准确性的方法提供了建议。
