A Flow-Extension Tethered Particle Motion Assay for Single-Molecule Proteolysis.

ABSTRACT: Regulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads not be restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow-extension strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allow assignment of tethered substrates to singly captured and multiply tethered bins, with

SUBMITTER: Drabek AA 

PROVIDER: S-EPMC6607913 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature