Department of Physics, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA.
Int J Biol Macromol. 2010 Mar 1;46(2):159-66. doi: 10.1016/j.ijbiomac.2009.12.001. Epub 2009 Dec 23.
Single molecule methods are becoming routine biophysical techniques for studying biological macromolecules. In mechanical unfolding of proteins, an externally applied force is used to induce the unfolding of individual protein molecules. Such experiments have revealed novel information that has significantly enhanced our understanding of the function and folding mechanisms of several types of proteins. To obtain information on the unfolding kinetics and the free energy landscape of the protein molecule from mechanical unfolding data, a Monte Carlo simulation based on a simple two-state kinetic model is often used. In this paper, we provide a detailed description of the procedure to perform such simulations and discuss the approximations and assumptions involved. We show that the appearance of the force versus extension curves from mechanical unfolding of proteins is affected by a variety of experimental parameters, such as the length of the protein polymer and the force constant of the cantilever. We also analyze the errors associated with different methods of data pooling and present a quantitative measure of how well the simulation results fit experimental data. These findings will be helpful in experimental design, artifact identification, and data analysis for single molecule studies of various proteins using the mechanical unfolding method.
单分子方法正成为研究生物大分子的常规生物物理技术。在蛋白质的机械解折叠中,外部施加的力用于诱导单个蛋白质分子的解折叠。此类实验揭示了新颖的信息,极大地增强了我们对几种类型蛋白质的功能和折叠机制的理解。为了从机械解折叠数据中获得有关蛋白质分子解折叠动力学和自由能景观的信息,通常使用基于简单两态动力学模型的蒙特卡罗模拟。在本文中,我们提供了执行此类模拟的详细过程描述,并讨论了所涉及的近似和假设。我们表明,蛋白质机械解折叠产生的力与延伸曲线的出现受到各种实验参数的影响,例如蛋白质聚合物的长度和悬臂的力常数。我们还分析了不同数据汇总方法相关的误差,并提出了一种定量方法来衡量模拟结果与实验数据的拟合程度。这些发现将有助于使用机械解折叠方法对各种蛋白质进行单分子研究的实验设计、伪影识别和数据分析。