Reconstitution of Membrane Proteins into Polymer-Supported Membranes for Probing Diffusion and Interactions by Single Molecule Techniques

Abstract

We have established a robust and versatile analytical platform for probing membrane protein function in a defined lipid environment on solid supports. This approach is based on vesicle capturing onto an ultrathin poly(ethylene glycol) (PEG) polymer brush functionalized with fatty acid moieties and subsequent vesicle fusion into a contiguous membrane. In order to ensure efficient formation of these tethered polymer-supported membranes (PSM), very small unilamellar vesicles (VSUV) containing fluorescent lipids or model transmembrane proteins were generated by detergent depletion with cyclodextrin. Thus, very rapid reconstitution of membrane proteins into PSM was possible in a format compatible with microfluidics. Moreover, surfaces could be regenerated with detergent solution and reused multiple times. Lipid and protein diffusion in these membranes was investigated by fluorescence recovery after photobleaching, single molecule tracking, and fluorescence correlation spectroscopy. Full mobility of lipids and a high degree of protein mobility as well as homogeneous diffusion of both were observed. Quantitative ligand binding studies by solid phase detection techniques confirmed functional integrity of a transmembrane receptor reconstituted into these PSM. Colocomotion of individual ligand-receptor complexes was detected, demonstrating the applicability for single molecule fluorescence techniques.

Diffusion and Interaction Dynamics of Individual Membrane Protein Complexes Confined in Micropatterned Polymer‐Supported Membranes

“…Similar experiments were carried out with DY‐649‐labeled maltose binding protein fused to the transmembrane helix of the type I interferon (IFN) receptor subunit IFNAR1 ( DY649 MBP‐TM1), which was reconstituted into proteoliposomes. After vesicle fusion, free diffusion was observed for about 60% of the reconstituted protein with a diffusion constant of 0.51 μm 2 /s (Figure S1), which is in good agreement with previous studies 7. The reduced mobile fraction of reconstituted proteins can probably be ascribed to the fraction of MBP oriented towards the polymer cushion 7…”

“…While no significant vesicle binding was detected without illumination, increasing binding amplitudes were observed with increasing illumination times confirming efficient coupling of HDVE under these conditions ( Figure ). A maximum amplitude of ∼6 ng/mm 2 was obtained, similar to what has been previously observed for a PEG polymer brush functionalized with palmitic acid by amide bond formation 7…”

Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

“…PSMs were assembled on glass coverslips, which were coated with diamino-poly(ethylene glycol) and functionalized with palmitic acid73 and then mounted into an incubation chamber for imaging experiments. Proteoliposomes complemented with DOPC/DOPS liposomes without proteins to a final lipid concentration of 250 μM in HBS were incubated on the coverslip surface for 30 min.…”

Effects of Pore Size and Tethering on the Diffusivity of Lipids Confined in Mesoporous Silica

“…[1] Mimicking these individual membrane functions in synthetic supported lipid bilayers (SLBs) is being explored for drug discovery, small molecule separations, targeted drug delivery, and catalysis. [16,17] The tethering of lipid bilayers effects bilayer mobility, reducing bilayer diffusivity as a function of tether density. [3] Mesoporous silica is morphologically temperature and support surface chemistry.…”