Hydrogel-supported protein-tethered bilayer lipid membranes: a new approach toward polymer-supported lipid membranes

Abstract

Polymer-supported bilayer lipid membranes offer great opportunities for the investigation of functional membrane proteins. Here we present a new approach in this direction by introducing a thin hydrogel layer as a soft 'cushion' on indium–tin oxide (ITO), providing a smooth, functional surface to form the protein-tethered BLM (ptBLM). ITO was used as a transparent electrode, enabling simultaneous implementation of electrochemical and optical waveguide techniques. The hydrogel poly(N-(2-hydroxyethyl)acrylamide-co-5-acrylamido-1-carboxypentyl-iminodiacetate-co-4-benzoylphenyl methacrylate) (P(HEAAm-co-NTAAAm-co-MABP)) was functionalized with the nickel chelating nitrilotriacetic acid (NTA) groups, to which cytochrome c oxidase (CcO) from Paracoccus denitrificans was bound in a well defined orientation via a his-tag attached to its subunit I. Given that the mesh size of P(HEAAm-co-NTAAAm-co-MABP) was smaller than the protein size, binding to the hydrogel occurred only on the top of the layer. The lipid bilayer was formed around the protein by in situdialysis. Electrochemical impedance spectroscopy showed good electrical sealing properties with a resistance of ∼1 MΩ cm2. Furthermore, surface plasmon resonance optical waveguide spectroscopy (SPR/OWS) indicated an increased anisotropy of the system after formation of the lipid bilayer. Cyclic voltammetry in the presence of reduced cytochrome c demonstrated that CcO was incorporated into the gel-supported ptBLM in a functionally active form.