Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer

Abstract

Investigations of ligand‐binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity‐related limitations impaired by low signal‐to‐noise ratios. Inspired by affinity capturing of water‐soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane‐protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris‐NTA)–biotin conjugate. A polymer‐supported lipid bilayer containing His6‐tagged β‐secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His6–BACE in the Ni2+–NTA‐modified region of the substrate resulted in a stationary three‐fold increase in surface coverage, and an accompanied increase in ligand‐binding response.

Formation of Supported Lipid Bilayers Derived from Vesicles of Various Compositional Complexity on Conducting Polymer/Silica Substrates

“…5 Such vesicles have previously been shown to effectively transfer BACE1 to the resulting bilayer. 5,30 The results of these studies are summarized in Table 4.…”

“…The vesicles were either proteoliposomes, containing POPC lipids with incorporated BACE1 transmembrane proteins (BACE1pl) or native membrane vesicle hybrids (hNMVs) derived from native membranes and POPC vesicles . Such vesicles have previously been shown to effectively transfer BACE1 to the resulting bilayer. , The results of these studies are summarized in Table .…”

Brownian Motion at Lipid Membranes: A Comparison of Hydrodynamic Models Describing and Experiments Quantifying Diffusion within Lipid Bilayers

“…In fact, as these model systems are far less complex than cell membranes, they finally allowed for probing the validity of models describing the diffusion of lipids and proteins within bilayers; the results of these efforts will be summarized in this review. While this, in principle, allows for some of the initial questions to be addressed in well controlled model systems, it was realized, in addition, that the fluidic functionality of bilayers can also be used for other analytical purposes, such as the (electrophoretically or hydrodynamically driven) isolation of membrane-associated compounds or transmembrane proteins [ 18 , 19 , 20 , 21 ], the characterization of bilayer-linked objects [ 20 , 22 , 23 , 24 ], or the quantification of multivalent interactions [ 10 ]. The validity of these new analytical approaches also requires a profound knowledge about the principle of diffusion processes that take place within bilayers and hence, this field benefited strongly from the theoretical considerations mostly done in the 1980s and 1990s.…”

Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations

“…As an emerging solution to this challenge, in-membrane separation has been demonstrated for separation of membrane components based on general physicochemical properties such as charge, , size, and partitioning kinetics in different membrane phases. , In other approaches, differentiation was achieved based on a combination of such properties and the strength of the frictional coupling to the lipid membrane, − where the latter may be enhanced by the use of surface-immobilized, high-affinity ligands to specifically capture His-tagged membrane proteins . Yet, these examples were limited to proof-of-principle demonstrations of lipid or protein accumulation in already pure, synthetic systems.…”

“…18,19 In other approaches, differentiation was achieved based on a combination of such properties and the strength of the frictional coupling to the lipid membrane, 20−22 where the latter may be enhanced by the use of surfaceimmobilized, high-affinity ligands to specifically capture Histagged membrane proteins. 23 Yet, these examples were limited to proof-of-principle demonstrations of lipid or protein accumulation in already pure, synthetic systems. Consequently, the corresponding methods cannot be directly translated to provide the biomolecular specificity required to purify rare, predefined integral membrane proteins from a native cellular membrane preparation.…”