A simple method for probing the mechanical unfolding pathway of proteins in detail
A simple method for probing the mechanical unfolding pathway of proteins in detail
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DOI：
10.1073/pnas.192351899
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发表时间：
2002-09-17
影响因子：
11.1
通讯作者：
Clarke, J
中科院分区：
文献类型：
--
作者：
Best, RB;Fowler, SB;Clarke, J
原子力显微镜是一种令人兴奋的新单分子技术，可添加到蛋白质（解）折叠方法工具箱中。然而，到目前为止，对蛋白质在外力作用下的展开的详细分析依赖于蛋白质分子动力学模拟或对突变数据的定性解释。在这里，我们描述了如何蛋白质工程（D值分析可以适用于表征蛋白质的机械解折叠的过渡态。单分子研究也比批量实验具有优势，因为过渡态部分结构产生的部分Phi值可以与替代途径的平均值明显区分开来。我们表明，展开速率常数在标准的方式通过使用Monte Carlo模拟是不可靠的，因为所涉及的错误。然而，它是可能的，以规避这些问题，提供的展开机制是不改变突变，无论是通过修改的Monte Carlo程序或通过比较突变体和野生型数据直接。该方法的适用性进行了测试的模拟数据集和实验数据的突变体的肌联蛋白127。
Atomic force microscopy is an exciting new single-molecule technique to add to the toolbox of protein (un)folding methods. However, detailed analysis of the unfolding of proteins on application of force has, to date, relied on protein molecular dynamics simulations or a qualitative interpretation of mutant data. Here we describe how protein engineering (D value analysis can be adapted to characterize the transition states for mechanical unfolding of proteins. Single-molecule studies also have an advantage over bulk experiments, in that partial Phi values arising from partial structure in the transition state can be clearly distinguished from those averaged over alternate pathways. We show that unfolding rate constants derived in the standard way by using Monte Carlo simulations are not reliable because of the errors involved. However, it is possible to circumvent these problems, providing the unfolding mechanism is not changed by mutation, either by a modification of the Monte Carlo procedure or by comparing mutant and wild-type data directly. The applicability of the method is tested on simulated data sets and experimental data for mutants of titin 127.