Measurement of hindered diffusion in complex geometries for high-speed studies of single-molecule forces.

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.