Nanoporous Microbead Supported Bilayers:  Stability, Physical Characterization, and Incorporation of Functional Transmembrane Proteins

Abstract

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature.Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and 4 fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform. 5

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

“…for lateral diffusion measurements of lipids and shown to have minimal impact on similarly structured dipalmitoylphosphatidylcholine bilayer transitions. [6,20,22,29] Lipid enveloped SBAS materials with 9.1 nm pore diameters were prepared (Supporting Information) to demonstrate that, at even the largest of pore diameters investigated in this study, techniques for traditional lipid bilayer enveloping, in which lipids are not present in the pores ( Figure S4, Supporting Information), are applicable to these particles. [6,20,22,29] Lipid enveloped SBAS materials with 9.1 nm pore diameters were prepared (Supporting Information) to demonstrate that, at even the largest of pore diameters investigated in this study, techniques for traditional lipid bilayer enveloping, in which lipids are not present in the pores ( Figure S4, Supporting Information), are applicable to these particles.…”

“…[3] Mesoporous silica is morphologically temperature and support surface chemistry. [16,[20][21][22] Confocal laser scanning microscopy (CLSM) has been employed for the characterization and visualization of a bilayer formation on nonporous supports, as well as the measurement of membrane fluidity via fluorescence recovery after photobleaching (FRAP) in both large diameter nonporous particles [20,21] and nonporous planar thin films. [12,18,19] Nonporous, micrometer diameter particles and nonporous thin films are common supports for the measurement and characterization of supported lipid bilayer diffusivity due to their respective large particle diameters (micrometer scale), and suitability for microscopic visualization and surface characterization techniques.…”

Double Cushions Preserve Transmembrane Protein Mobility in Supported Bilayer Systems

“…Finally, Brozik and co-workers used nanoporous microbeads rather than a polymer cushion strategy to support lipid membranes with bacteriorhodopsin. 46 High protein mobile factions were obtained (~78%) with D = 3.8 × 10 −10 cm 2 /s for proteins in the pores of the bead. It would be important to determine if the same type of mobility can be extended to systems with planar geometry.…”

Supported Bilayer Electrophoresis under Controlled Buffer Conditions

“…There have been a number of significant successes in these efforts. In particular, the use of cushioned bilayers often results in a substantial fraction of the proteins in the membrane showing mobility by fluorescence recovery after photobleaching (FRAP) measurements 2-7. Other techniques, such as surface acoustic wave generation and QCM-D, have been used to pattern and monitor lipids, lipid-bound proteins and membrane proteins 8-10.…”