Using Force Spectroscopy to Probe Coiled-Coil Assembly and Membrane Fusion

Area of Science:

• Biophysics
• Molecular Biology
• Materials Science

Background:

• Force spectroscopy is crucial for studying single molecule interactions in biological sciences.
• Atomic force microscopy (AFM) offers high force resolution (piconewton to micronewton) for molecular manipulation.
• Existing techniques are limited for studying processes at and between lipid bilayers.

Purpose of the Study:

• To introduce membrane probe spectroscopy as an advanced force spectroscopy technique.
• To investigate lipid bilayer interactions, SNARE protein complex formation, and membrane fusion.
• To detail the preparation and experimental procedures for membrane probe spectroscopy.

Main Methods:

• Utilizing AFM with a lipid-coated glass sphere (colloidal probe) instead of a sharp tip.
• Preparing solid-supported membranes on a flat surface and a glass sphere attached to an AFM cantilever.
• Bringing membranes into contact to observe fusion and measuring separation forces to analyze SNARE complex formation.

Main Results:

• Successfully applied membrane probe spectroscopy to study coiled-coil formation in SNARE proteins.
• Observed and identified lipid membrane fusion processes.
• Gained insights into fusion kinetics and the energy landscape of membrane fusion by varying contact pressure.

Conclusions:

• Membrane probe spectroscopy expands the capabilities of force spectroscopy to lipid bilayer interactions.
• The technique provides a method to study the molecular mechanisms of membrane fusion.
• This approach allows for the characterization of fusogenic peptides and proteins in a biologically relevant context.