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

Area of Science:

• Biophysics
• Biochemistry
• Structural Biology

Background:

• Mechanical forces are critical for biomolecular dynamics and interactions.
• Existing techniques lack high-throughput methods for measuring force-dependent biomolecular responses.

Purpose of the Study:

• To develop and validate a novel enzymatic assay for assessing force-dependent structural accessibility.
• To enable real-time, high-throughput readout of biomolecular force responses using a specialized centrifuge microscope.

Main Methods:

• Utilized a wireless mini-radio centrifuge force microscope for high-throughput, video-rate analysis.
• Employed an enzymatic assay measuring proteolytic cleavage events on tethered collagen molecules.
• Applied varying mechanical loads to thousands of individual collagen molecules.

Main Results:

• Demonstrated load-enhanced trypsin sensitivity in collagen.
• Observed destabilization of the collagen triple helix under applied force.
• Achieved real-time kinetic readout of individual molecular events.

Conclusions:

• The developed centrifuge force microscope provides a powerful tool for studying force-dependent biomolecular dynamics.
• Mechanical force significantly impacts the stability of collagen triple helices.
• This technique offers a new avenue for high-throughput investigation of molecular mechanics.