Challenges in the Development of Functional Assays of Membrane Proteins

Abstract

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes

“…To study protein-mediated transport, functional transmembrane proteins must be incorporated into the membrane of giant liposomes that consist of a single lipid bilayer (giant unilamellar vesicle, GUV). Few methods exist for the production of such giant proteoliposomes with active ion channels or transporter proteins due to the sensitive and fragile nature of many transmembrane proteins and GUVs [6–10]. In addition, existing methods can lead to giant proteoliposomes that enclose many smaller liposomes making it difficult to determine the accessible volume inside these liposomes.…”

Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules

“…They may contain large hydrophobic moieties that anchor them in the membrane, but which consequently limit their solubility in most other media, particularly if their structure has to be preserved. For this reason, biomimetic model lipid membranes have attracted lively interest because of their unique feature to provide an amphiphilic matrix for reconstitution of integral MPs [150,155,156]. Hence, MPs as preferred targets for pharmaceuticals received widespread recognition in drug discovery and protein-ligand screening and are of high interest for the development of biosensor platforms [150,155].…”

“…For this reason, biomimetic model lipid membranes have attracted lively interest because of their unique feature to provide an amphiphilic matrix for reconstitution of integral MPs [150,155,156]. Hence, MPs as preferred targets for pharmaceuticals received widespread recognition in drug discovery and protein-ligand screening and are of high interest for the development of biosensor platforms [150,155]. Very early after developing techniques for planar lipid bilayer generation membrane-active peptides incorporated in lipid membranes were studied [157].…”

“…The most challenging property of model lipid membranes is the feasibility to incorporate membrane-active (antimicrobial) peptides (AMPs) [229,230] and, more important, (complex) integral MPs in a functional state [150].…”

Formation and fluidity measurement of supported lipid bilayer on polyvinyl chloride membrane

“…Platforms and experimental techniques for the researches of membrane proteins using artificial lipid bilayer membranes are demanded, 11,12 because the membrane proteins occupy ~60% of the targets of drug discovery. 13,14 Supported lipid bilayers (SLBs) are artificial lipid bilayer membranes existing at the interfaces between solids with hydrophilic surfaces and aqueous solutions. 11,12,15 The SLBs on solid sensing devices will be valuable as artificial biomembrane platforms for the investigation of lipids and membrane proteins in vitro.…”