Binding of His-tagged molecules to giant vesicles via anchor lipids.

Abstract

Giant unilamellar vesicles (GUVs) are versatile biomimetic membrane compartments made of a lipid bilayer. For the biofunctionalization of the membranes, proteins can be attached peripherally to membranes using anchor lipids. One such anchor lipid is the 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (DGS-NTA) lipid, which has a head group that can bind to metal cations and subsequently to proteins via a six-histidine (6H) chain. The proteins are tagged with fluorescent molecules to visualize them in the confocal microscope. We use two molecules with a 6H tag - fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP). The binding affinity of the protein to the NTA lipid is affected by the size of the molecule and the choice of the metal cation in the head group. In this study, we use a microfluidic device to expose the GUVs with NTA lipids to a constant His-tagged fluorophore concentration in the exterior solution. The equilibrium dissociation constant for the NTA chelated to divalent nickel cation (Ni2+) is measured to be 37.5 nM for 6H-GFP and 18.5 nM for 6H-FITC (Soft matter, 2022, 18, 6372). We also establish a DGS-NTA lipid system that chelates to trivalent cobalt cation (Co3+) and compare it to the Ni2+ system. We achieve such a system without oxidizing reagents, keeping the lipids and the protein unaffected.