Single-molecule force spectroscopy of protein-membrane interactions

Area of Science:

• Biophysics
• Cell Biology
• Molecular Interactions

Background:

• Protein-membrane interactions are crucial for many biological processes, often involving mechanical forces.
• Existing high-resolution methods to quantify these interactions are limited.

Purpose of the Study:

• To develop and apply a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains from Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2).
• To measure binding and unbinding transitions of these proteins at the single-molecule level with high spatiotemporal resolution.

Main Methods:

• Utilized single-molecule force spectroscopy with optical tweezers.
• Attached single proteins to membranes supported on silica beads and applied pulling forces.
• Measured binding and unbinding events at the single-molecule level.

Main Results:

• Quantified membrane binding of Syt1 and E-Syt2 C2 domains.
• Measured unbinding forces in the range of 2-7 pN and binding energies of 4-14 k_BT per C2 domain.
• Demonstrated that bilayer composition and Ca²⁺ regulate binding, consistent with known protein properties.

Conclusions:

• The developed method provides unprecedented spatiotemporal resolution for studying protein-membrane interactions.
• This technique is broadly applicable for investigating the mechanics of various protein-membrane systems.
• Offers new insights into the role of mechanical forces in regulating protein-membrane interactions vital for cellular functions.