Immobilized nanodisks for study of ligand binding interactions

Area of Science:

• Biochemistry
• Membrane Biophysics
• Protein Engineering

Background:

• The SpyCatcher/SpyTag system enables covalent protein conjugation.
• Nanodisks (NDs) are miniature bilayer membranes used to study membrane proteins.
• Investigating ligand-membrane interactions is crucial in biological research.

Purpose of the Study:

• To adapt the SpyCatcher/SpyTag system for immobilizing nanodisks.
• To develop a method for studying ligand-membrane interactions using immobilized NDs.
• To demonstrate the utility of this system for probing calcium-cardiolipin interactions.

Main Methods:

• Fusion proteins of apolipoprotein A-I (apoA-I)/SpyTag and SpyCatcher/maltose binding protein (MBP) were engineered.
• ApoA-I:SpyTag was formulated into nanodisks (NDs) with cardiolipin (CL) or phosphatidylcholine.
• Immobilization of NDs was achieved via MBP binding to amylose resin, followed by ligand binding assays.

Main Results:

• SpyCatcher/SpyTag mediated covalent adduct formation between fusion proteins.
• Immobilized CL NDs, but not phosphatidylcholine NDs, bound cytochrome c.
• Calcium ions (CaCl2) induced cytochrome c dissociation from CL NDs, and maltose released the NDs from the resin.

Conclusions:

• The SpyCatcher/SpyTag system can be used to immobilize nanodisks for studying ligand-membrane interactions.
• This method allows for the controlled binding and release of ligands, such as cytochrome c.
• Immobilized NDs offer a novel platform for investigating various ligand-membrane binding events.