Construction of tethered bilayer lipid membrane with oriented membrane proteins on surface modified mica substrate

Abstract Incorporating membrane proteins, which are major targets of drug discovery, into artificial planar lipid bilayer membranes is important. From the observation at the high spatiotemporal resolution in the microscopic region, the intermolecular interactions between lipids and membrane proteins can be revealed. It leads to elucidation of the reaction mechanisms occurring in biological membranes. However, when a lipid membrane supported on a solid substrate (SLB) is used, the following points are interfering with the research. The first point is the structural change due to the contact between the membrane protein and the underlying substrate. The other is disorder of the membrane protein orientation in the SLB. Research on the incorporation of membrane proteins has been done in the field of both physical and biological chemistry, but few reports have achieved detailed measurements with placing emphasis on the plasticity and diversity of membrane proteins. In this study, the tethered bilayer lipid membrane (tBLM) was formed on the atomically flat self-assembled monolayer modified surface which specifically binds to the membrane proteins in the membrane. The fluidity of artificial lipid bilayer and orientation of membrane proteins are observed by the epi-fluorescence microscopy and atomic force microscopy, respectively. The consequent decreased interaction between the substrate and lipid membrane and limited observation for the specific orientation of membrane proteins suggest that it was possible to reproduce the same environment as the biological membrane even in the artificial lipid bilayer membrane. The present tBLM system should be useful for the creation of basic technologies for structural, functional, and dynamical analysis of membrane proteins. It also leads to the understanding the molecular structure of biological membrane which include the various membrane proteins such as ion channels, transporters, pumps and enzymes with addition of more complex structural properties such as the underlying membrane scaffolds, asymmetric lipid composition and domains.