Sumbul Fidan, Marchesi Arin, Takahashi Hirohide, Scheuring Simon, Rico Felix
LAI, Aix-Marseille Université, INSERM UMR_S 1067, CNRS UMR 7333, 13009, Marseille, France.
Department of Anesthesiology, Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA.
Methods Mol Biol. 2018;1814:243-264. doi: 10.1007/978-1-4939-8591-3_15.
Single-molecule force spectroscopy (SMFS) measurements allow for quantification of the molecular forces required to unfold individual protein domains. Atomic force microscopy (AFM) is one of the long-established techniques for force spectroscopy (FS). Although FS at conventional AFM pulling rates provides valuable information on protein unfolding, in order to get a more complete picture of the mechanism, explore new regimes, and combine and compare experiments with simulations, we need higher pulling rates and μs-time resolution, now accessible via high-speed force spectroscopy (HS-FS). In this chapter, we provide a step-by-step protocol of HS-FS including sample preparation, measurements and analysis of the acquired data using HS-AFM with an illustrative example on unfolding of a well-studied concatamer made of eight repeats of the titin I91 domain.
单分子力谱（SMFS）测量能够对展开单个蛋白质结构域所需的分子力进行量化。原子力显微镜（AFM）是力谱（FS）领域长期使用的技术之一。尽管传统AFM拉伸速率下的FS能提供有关蛋白质展开的有价值信息，但为了更全面地了解其机制、探索新领域，并将实验与模拟相结合并进行比较，我们需要更高的拉伸速率和微秒级时间分辨率，如今可通过高速力谱（HS-FS）实现。在本章中，我们提供了HS-FS的详细操作流程，包括样品制备、测量以及使用HS-AFM对采集数据的分析，并以一个由肌联蛋白I91结构域的八个重复序列组成的串联体展开为例进行说明。
