Limitations of Constant-Force-Feedback Experiments
Limitations of Constant-Force-Feedback Experiments
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DOI：
10.1016/j.bpj.2012.06.051
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发表时间：
2012-10-03
影响因子：
3.4
通讯作者：
Marqusee, Susan
中科院分区：
文献类型：
--
作者：
Elms, Phillip J.;Chodera, John D.;Marqusee, Susan
单分子力谱为生物分子和系统的性质和机制提供了重要的见解。一个常见的实验是测量平衡时构象变化的力依赖性。在这里，我们证明了常用的力反馈技术在用于评估快速大分子构象转变时具有严重的局限性。通过使用恒定力反馈和恒定陷阱位置技术比较三大类大分子（DNA、RNA 和蛋白质）的力依赖性动力学，我们证明了力反馈实验中的问题。仪器力反馈的有限响应时间可以改变分子的行为，导致所报告的参数错误，例如构象转变的速率常数和到过渡态的距离。我们阐明了这个问题的原因，并提供了一个简单的测试来识别和评估影响的程度。我们建议避免使用恒力反馈作为研究大分子快速构象变化的方法。
Single-molecule force spectroscopy has provided important insights into the properties and mechanisms of biological molecules and systems. A common experiment is to measure the force dependence of conformational changes at equilibrium. Here, we demonstrate that the commonly used technique of force feedback has severe limitations when used to evaluate rapid macromolecular conformational transitions. By comparing the force-dependent dynamics of three major classes of macromolecules (DNA, RNA, and protein) using both a constant-force-feedback and a constant-trap-position technique, we demonstrate a problem in force-feedback experiments. The finite response time of the instrument's force feedback can modify the behavior of the molecule, leading to errors in the reported parameters, such as the rate constants and the distance to the transition state, for the conformational transitions. We elucidate the causes of this problem and provide a simple test to identify and evaluate the magnitude of the effect. We recommend avoiding the use of constant force feedback as a method to study rapid conformational changes in macromolecules.