Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments.
Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments.
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优化单分子蛋白质展开实验的能量景观参数的计算。
DOI：
10.1103/physreve.91.012710
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发表时间：
2015
影响因子：
2.4
通讯作者：
L. Dougan
中科院分区：
文献类型：
--
作者：
KM. Tych;ML. Hughes;J. Bourke;Y. Taniguchi;M. Kawakami;DJ. Brockwell;L. Dougan
使用原子力显微镜(AFM)的单分子力谱可以用来测量恒速实验中蛋白质的平均去折叠力。结合蒙特卡罗模拟，通过应用Zhurkov-Bell模型，可以获得关于描述蛋白质潜在展开能量图景的参数的信息。使用这种方法，我们已经在一定的拉动速度范围内完成了对多蛋白的蛋白质展开实验。与以前的工作一致，我们发现在每个接近-收回周期中观察到的蛋白质展开事件的数量在1到5个之间变化，这是由于多蛋白、AFM尖端和底物之间相互作用的性质，并且存在不相等的展开概率分布。我们开发了一个蒙特卡罗模拟，该模拟结合了这种不均匀的展开概率分布对中间展开力的影响以及蛋白质展开能量景观参数的计算。这些结果表明，当存在显著的、不相等的展开概率分布时，利用Zhurkov-Bell模型获得的展开能量景观参数受影响不大。这一结果很重要，因为它证明了力扩展实验中通常使用的最小可接受标准是合理的，并且不会歪曲展开能量景观参数的计算。我们通过确定两个极端情况下能量景观参数的误差进一步验证了这种方法，并提供了可以用来提高使用多蛋白质的单分子实验的精度水平的方法的建议。
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 polyproteinover 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.