Scholl Zackary N, Marszalek Piotr E
Department of Physics, University of Alberta, Edmonton, AB, Canada.
Department of MEMS, Duke University, Durham, NC, USA.
Methods Mol Biol. 2018;1814:35-47. doi: 10.1007/978-1-4939-8591-3_3.
Single-molecule force spectroscopy by AFM (AFM-SMFS) is an experimental methodology that allows unequivocal sensitivity and control for investigating and manipulating the mechanical properties of single molecules. The past 20 years of AFM-SMFS has provided numerous breakthroughs in the understanding of the mechanical properties and force-induced structural rearrangements of sugars, DNA, and proteins. Here, we focus on the application of AFM-SMFS to study proteins, since AFM-SMFS has succeeded in providing abundant information about protein folding pathways, kinetics, interactions, and misfolding. In this chapter we describe the experimental procedures for conducting a SMFS-AFM experiment-including purification of protein samples, setup and calibration of the AFM instrumentation, and the thorough and unbiased analysis of resulting AFM data.
原子力显微镜单分子力谱（AFM-SMFS）是一种实验方法，能够以明确的灵敏度和控制能力来研究和操纵单分子的力学性质。过去20年的AFM-SMFS在理解糖类、DNA和蛋白质的力学性质以及力诱导的结构重排方面取得了众多突破。在此，我们聚焦于AFM-SMFS在研究蛋白质方面的应用，因为AFM-SMFS已成功提供了有关蛋白质折叠途径、动力学、相互作用和错误折叠的丰富信息。在本章中，我们描述了进行SMFS-AFM实验的实验步骤，包括蛋白质样品的纯化、AFM仪器的设置和校准，以及对所得AFM数据进行全面且无偏差的分析。
