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Protein Folding01:22

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Atomic force microscopy (AFM) is a type of scanning probe microscopy that can analyze topographic details of various specimens like ceramics, glass, polymers, and biological samples. AFM offers over 1000 times more resolution than the optical imaging system. Images generated from AFM are three-dimensional surface profiles, offering an advantage over the flat, two-dimensional images from other imaging techniques.
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The cytoskeletal architecture can be studied using different microscopic and biochemical techniques. Electron microscopy was instrumental in discovering the cytoskeletal architecture around the 1960s, which allowed obtaining structural information at a high-resolution level. However, the sample preparation procedure often limits this ability in biological samples. Several protocols have been developed over the years to optimize sample preparation. In one of the protocols known as rotary...
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Related Experiment Video

Updated: Jul 13, 2025

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope
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Unfolding and Refolding Proteins Using Single-Molecule AFM.

Marc Mora1, Rafael Tapia-Rojo1, Sergi Garcia-Manyes2

  • 1Department of Physics, Randall Centre for Cell and Molecular Biophysics and London Centre for Nanotechnology, King's College London, London, UK.

Methods in Molecular Biology (Clifton, N.J.)
|October 12, 2023
PubMed
Summary
This summary is machine-generated.

This study details using atomic force microscopy (AFM) to observe individual protein unfolding and refolding dynamics under mechanical force. These experiments reveal molecular mechanisms and kinetics governing protein mechanical behavior.

Keywords:
Atomic force spectroscopy (AFM)Protein foldingProtein nanomechanicsSingle-molecule force spectroscopy

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Area of Science:

  • Biophysics
  • Molecular Mechanobiology

Background:

  • Single-molecule atomic force microscopy (AFM) enables direct observation of molecular conformational changes.
  • Understanding protein mechanical properties is crucial for molecular biology and disease research.

Purpose of the Study:

  • To provide a protocol for protein nanomechanical experiments using AFM.
  • To detail the application of force-extension and force-clamp modes for studying protein dynamics.

Main Methods:

  • Utilizing atomic force microscopy (AFM) in force-extension mode to measure protein unfolding forces.
  • Employing AFM in force-clamp mode to analyze the kinetics of protein unfolding and refolding.
  • Applying mechanical load to individual protein molecules.

Main Results:

  • Demonstrated the capability of AFM to capture real-time conformational dynamics of single proteins.
  • Provided insights into the molecular mechanisms driving protein mechanical unfolding and refolding.
  • Characterized the kinetics associated with these mechanical transitions.

Conclusions:

  • AFM offers a powerful approach to investigate protein nanomechanics at the single-molecule level.
  • Combined force-extension and force-clamp modes yield a comprehensive understanding of protein mechanical responses.
  • This protocol facilitates detailed studies of protein folding landscapes under force.