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

Biochemistry. 2019; 
DrabekAndrew A,LoparoJoseph J,BlacklowSteph
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摘要

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 as... More

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