Single-Molecule Assay for Proteolytic Susceptibility: Force-Induced Collagen Destabilization

蛋白水解敏感性的单分子测定:力诱导胶原不稳定

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作者:Michael W H Kirkness, Nancy R Forde

Abstract

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.

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