Single-molecule force spectroscopy on polyproteins and receptor-ligand complexes: The current toolbox

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Single-molecule force spectroscopy (SMFS) probes free energy landscapes of protein folding and molecular recognition.
• It identifies rare molecular conformations and customizes unbinding pathways by precise force application.

Purpose of the Study:

• To review recent innovations in AFM-based SMFS experiments.
• To highlight advancements in construct design, bioconjugation, measurement techniques, instrumentation, and data analysis.

Main Methods:

• Focus on innovations in construct design, including customized fingerprint domains and polyproteins.
• Discuss site-specific bioconjugation using peptide tags for covalent surface attachment.
• Present recent improvements in measurement stability, signal-to-noise ratio, and force precision.

Main Results:

• Highlight trends like customized fingerprint domains and polyproteins for mechanostable interactions.
• Detail advancements in measurement techniques and instrumental improvements.
• Discuss theoretical considerations and algorithms for analyzing force-extension curves.

Conclusions:

• Recent innovations significantly improve workflow, throughput, and data yield in AFM-SMFS.
• These advancements provide a foundation for researchers to further advance SMFS capabilities.
• The review serves as a resource for researchers aiming to push the boundaries of single-molecule force spectroscopy.