Dwell-time distribution analysis of polyprotein unfolding using force-clamp spectroscopy
Dwell-time distribution analysis of polyprotein unfolding using force-clamp spectroscopy
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DOI：
10.1529/biophysj.106.099481
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发表时间：
2007-04-01
影响因子：
3.4
通讯作者：
Fernandez, Julio M.
中科院分区：
文献类型：
--
作者：
Brujic, Jasna;Hermans, Rodolfo I. Z.;Fernandez, Julio M.
利用近年来发展起来的单分子力钳技术，定量测量了多蛋白分子在恒力作用下的构象变化动力学。在110 pN的作用力下，我们测量了每个蛋白链中单个泛素模块的1647个展开事件的停留时间。然后，我们建立了一个严格的方法来分析力钳数据使用顺序统计。这使我们能够测试一个与历史无关的两态模型在描述展开过程的动力学方面是否成功。我们发现，平均展开轨迹与每条轨迹中的蛋白质模块数N无关，N在3到12（工程蛋白长度）之间变化，这表明每条链中的展开事件是不相关的。然后，我们推导出停留时间的二项分布来描述蛋白质展开的随机动力学。该分布成功地描述了81%的数据，所有n的单一速率常数为α = 0.6 s（-1）。其余无法解释的数据表明，蛋白质的能量格局中存在其他展开障碍。该方法研究了超出单个速率常数平均测量的展开的统计特征，从而为力钳光谱测量动力学提供了一种有吸引力的替代方法。
Using the recently developed single molecule force-clamp technique we quantitatively measure the kinetics of conformational changes of polyprotein molecules at a constant force. In response to an applied force of 110 pN, we measure the dwell times of 1647 unfolding events of individual ubiquitin modules within each protein chain. We then establish a rigorous method for analyzing force-clamp data using order statistics. This allows us to test the success of a history-independent, two-state model in describing the kinetics of the unfolding process. We find that the average unfolding trajectory is independent of the number of protein modules N in each trajectory, which varies between 3 and 12 (the engineered protein length), suggesting that the unfolding events in each chain are uncorrelated. We then derive a binomial distribution of dwell times to describe the stochastic dynamics of protein unfolding. This distribution successfully describes 81% of the data with a single rate constant of alpha = 0.6 s(-1) for all N. The remainder of the data that cannot be accounted for suggests alternative unfolding barriers in the energy landscape of the protein. This method investigates the statistical features of unfolding beyond the average measurement of a single rate constant, thus providing an attractive alternative for measuring kinetics by force-clamp spectroscopy.