Sung Jongmin, Nag Suman, Mortensen Kim I, Vestergaard Christian L, Sutton Shirley, Ruppel Kathleen, Flyvbjerg Henrik, Spudich James A
1] Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA [2] Department of Applied Physics, Stanford University, Stanford, California 94305, USA.
Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.
Nat Commun. 2015 Aug 4;6:7931. doi: 10.1038/ncomms8931.
Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using 'harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.
分子马达负责众多细胞过程，从货物运输到心脏收缩。它们与其他细胞成分的相互作用通常是短暂的，并且表现出依赖于负载的动力学。在这里，我们使用“谐波力谱”来测量这种相互作用。在这种方法中，激光阱样品台的谐波振荡会立即、自动且随机地向与单个轨道相互作用并沿其移动的单个马达分子施加正弦变化的负载。实验方案和数据分析简单、快速且高效。该方案积累统计数据的速度足够快，能够从单分子实验中得出单分子结果。我们通过测量在生理ATP浓度下与肌动蛋白丝相互作用的单个人类β - 心脏肌球蛋白分子的力依赖性动力学来证明该方法的性能。我们表明，分子的ADP释放速率与所施加的负载呈指数关系，这与心肌收缩速度与外部负载成反比的定性结果一致。
