Department of Biological Sciences, Columbia University, New York, USA.
Nat Protoc. 2013;8(7):1261-76. doi: 10.1038/nprot.2013.056. Epub 2013 Jun 6.
Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. A calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. The use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. These experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. These data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.
在这里，我们描述了一种使用力钳光谱法精确量化力对生化反应影响的方案。通过使用校准力来控制由重复结构域组成的单个多蛋白底物中反应位点的暴露。多蛋白的使用允许从明确的机械展开指纹中识别成功的单分子记录。然后通过检测在恒定力下保持的底物的长度变化直接测量生化反应。我们展示了力钳光谱仪的布局以及设计和进行实验的方案。这些实验测量了施加力的函数下的反应动力学。我们展示了两种不同反应（蛋白质展开和二硫键还原）的力依赖性的示例数据。这些数据仅需几天即可获得，揭示了目前任何其他技术都无法解决的反应的机械细节。
