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

Area of Science:

• Biochemistry and Molecular Biology
• Cellular Mechanics
• Biophysics

Background:

• Regulated proteolysis of signaling proteins under mechanical tension is crucial for cellular communication.
• Previous single-molecule studies using magnetic tweezers faced challenges with multiple tethers, limiting accuracy.
• Understanding force-induced proteolytic sensitivity is vital in various physiological contexts.

Purpose of the Study:

• To develop a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem.
• To enable robust assessment of protease activity as a function of applied magnetic force.
• To provide a generally applicable method for studying force-dependent proteolysis.

Main Methods:

• A novel flow-extension strategy combined with magnetic tweezers was employed.
• Particle tracking and computational sorting were used to differentiate singly and multiply tethered substrates.
• Computational exclusion of multiple-tether beads ensured accurate analysis of proteolysis.

Main Results:

• The assay successfully overcomes the multiple-tether limitation in single-molecule force assays.
• The fraction of single-tethered substrates was found to be inversely dependent on substrate loading concentration.
• Robust assessment of proteolysis by tobacco etch virus protease and ADAM17 was achieved.

Conclusions:

• The developed multiplexed assay provides a reliable method for studying single-molecule proteolysis under mechanical tension.
• This technique allows for accurate evaluation of protease sensitivity as a function of applied force.
• The method is broadly applicable to various proteases and future investigations into mechanobiology.