Immobilized nanodisks for study of ligand binding interactions

The SpyCatcher/SpyTag system represents a unique technology that allows facile conjugation of proteins via formation of a covalent isopeptide bond between the 113 residue SpyCatcher protein and a 16 residue SpyTag peptide. Herein this technology was adapted to incorporate miniature bilayer membranes, termed nanodisks (ND). Fusion proteins comprised of apolipoprotein (apo) A-I/SpyTag peptide and SpyCatcher/maltose binding protein (MBP), respectively, were expressed and purified. Upon incubation of apoA-I:SpyTag fusion protein with SpyCatcher:MBP fusion protein, a covalent adduct was formed. ApoA-I:SpyTag formulated into ND particles with cardiolipin (CL) or phosphatidylcholine retained the ability to form an adduct with SpyCatcher:MBP. This adduct was then immobilized on amylose agarose resin beads through a binding interaction with the MBP component. Upon incubation of cytochrome c with immobilized CL ND, but not with phosphatidylcholine ND, cytochrome c binding occurred. When immobilized cytochrome c CL ND were incubated with buffer containing CaCl₂, cytochrome c dissociated and was recovered in the supernatant fraction obtained after pelleting the amylose agarose beads. Subsequent incubation of the amylose agarose beads with 10 mM maltose revealed that nearly all of the cytochrome c had been released from the beads. The data are consistent with the known ability of calcium to form an ionic interaction with the two negatively charged phosphates in the polar head group of CL. Given the number of ligand-membrane interactions that occur in nature, immobilized ND provide a novel means to probe them.