Diffusion and interactions in micropatterned and phase-separated polymer-supported membranes

Polymer-supported membranes (PSM) are valuable models for probing the role of the lipid environment for membrane protein function under highly defined conditions by a multitude of surface-sensitive spectroscopic and microscopic techniques. Here, we present a simple and versatile approach for efficiently reconstituting transmembrane membrane proteins into tethered PSM, which employs a hydrophilic, inert polymer cushion (polyethylene glycol) as a spacer between a glass surface and the membrane. Proteins reconstituted into very small unilamellar vesicle by using detergent extraction with cyclodextrin were captured to these surfaces by means of lipophilic anchors coupled to the PEG polymer brush. Vesicle fusion into continuous PSM as well as the incorporation and the lateral diffusion of single-spanning transmembrane proteins in these PSM was demonstrated by multiparameter solid phase detection and by fluorescence imaging techniques. Moreover, diffusion and interactions of a transmembrane receptor reconstituted into PSM was investigated by single molecule imaging and fluorescence correlations spectroscopy. Upon fusing ternary lipid mixtures into PSM, formation of coexisting liquid-ordered and liquid-disordered lipid phased was observed. The properties of liquid-ordered phases could be manipulated by the lipophilic anchors for membrane tethering. Moreover, photochemical micropattering of PSM was readily achieved based on the bottom-up chemistry. By combining ternary lipid mixtures and functional surface micropatterning with different lipid anchors, not only the properties, but also the geometry of lipid phases could be controlled. Proteins were readily reconstituted into these micropatterned membranes, enabling for probing protein diffusion and partitioning in the lipid phases by single molecule techniques in a highly controlled lipid environment.