Model lipid bilayer with facile diffusion of lipids and integral membrane proteins

A model membrane system is formed by the rupture of giant unilamellar vesicles (GUVs) onto a passivating layer comprising a PEG polymer cushion anchored in a lipid bilayer supported on glass. The novel use of pH-dependent electrostatic interactions between NeutrAvidin in the passivating layer and anionic lipids in the GUV drives vesicle rupture. The resulting "GUV pancakes" are single, planar lipid bilayer patches whose diameters vary from approximately 20 to 50 microm. The pancakes have several potential advantages for the in vitro study of protein-lipid interactions and integral membrane protein function. All components are commercially available. The pancakes resist nonspecific binding of vesicles containing protein. Both lipids and integral membrane proteins exhibit good lateral mobility in the GUV pancakes, as evidenced by single-particle tracking (SPT) of the DiD double-tailed fluorescent probe and of the integral membrane protein syntaxin-1A, labeled with AlexaFluor 633 (AF633-Syx). At least 80% of both probes exhibit free, homogeneous diffusion with a diffusion coefficient of approximately 5.5 microm(2) s(-1), which is more than 10 times faster than diffusion in a GUV pancake supported on bare glass. Atomic force microscopy (AFM) suggests that the polymer cushion has a height of approximately 4 nm. The mobility of a large fraction of the AF633-Syx probe suggests that even integral membrane proteins with large domains on both sides of the lipid bilayer should exhibit free diffusion within a GUV pancake.