Bartsch Tobias F, Villasante Camila M, Hengel Felicitas E, Touré Ahmed, Firester Daniel M, Oswald Aaron, Hudspeth A J
Howard Hughes Medical Institute and Laboratory of Sensory Neuroscience, The Rockefeller University, New York, NY, 10065, USA.
Faculty of Medicine, Ruprecht-Karls-Universität, 69120, Heidelberg, Germany.
Sci Rep. 2021 Jan 26;11(1):2196. doi: 10.1038/s41598-021-81593-x.
In a high-speed single-molecule experiment with a force probe, a protein is tethered between two substrates that are manipulated to exert force on the system. To avoid nonspecific interactions between the protein and nearby substrates, the protein is usually attached to the substrates through long, flexible linkers. This approach precludes measurements of mechanical properties with high spatial and temporal resolution, for rapidly exerted forces are dissipated into the linkers. Because mammalian hearing operates at frequencies reaching tens to hundreds of kilohertz, the mechanical processes that occur during transduction are of very short duration. Single-molecule experiments on the relevant proteins therefore cannot involve long tethers. We previously characterized the mechanical properties of protocadherin 15 (PCDH15), a protein essential for human hearing, by tethering an individual monomer through very short linkers between a probe bead held in an optical trap and a pedestal bead immobilized on a glass coverslip. Because the two confining surfaces were separated by only the length of the tethered protein, hydrodynamic coupling between those surfaces complicated the interpretation of the data. To facilitate our experiments, we characterize here the anisotropic and position-dependent diffusion coefficient of a probe in the presence of an effectively infinite wall, the coverslip, and of the immobile pedestal.
在一项使用力探针的高速单分子实验中，一种蛋白质被拴在两个被操控以对系统施加力的底物之间。为避免蛋白质与附近底物之间的非特异性相互作用，蛋白质通常通过长的柔性连接子附着到底物上。这种方法排除了以高空间和时间分辨率测量机械性能的可能性，因为快速施加的力会耗散到连接子中。由于哺乳动物的听力在高达数十至数百千赫兹的频率下运作，转导过程中发生的机械过程持续时间非常短。因此，对相关蛋白质进行的单分子实验不能涉及长连接子。我们之前通过将单个原钙黏蛋白15（PCDH15，一种对人类听力至关重要的蛋白质）单体通过非常短的连接子拴在光学阱中的探针珠和固定在玻璃盖玻片上的基座珠之间，来表征其机械性能。由于两个限制表面仅被拴系蛋白质的长度隔开，这些表面之间的流体动力耦合使数据解释变得复杂。为便于我们的实验，我们在此表征了在存在有效无限大壁（盖玻片）和固定基座的情况下探针的各向异性和位置依赖性扩散系数。
