Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes

Abstract

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering. However, observing the precise elemental reactions and structures of membrane proteins is difficult, despite their functioning through interactions with various biomolecules in living cells. To investigate these properties, methodologies have been developed to study the functions of membrane proteins that have been purified from biological cells. In this paper, we introduce various methods for creating liposomes or lipid vesicles, from conventional to recent approaches, as well as techniques for reconstituting membrane proteins into artificial membranes. We also cover the different types of artificial membranes that can be used to observe the functions of reconstituted membrane proteins, including their structure, number of transmembrane domains, and functional type. Finally, we discuss the reconstitution of membrane proteins using a cell-free synthesis system and the reconstitution and function of multiple membrane proteins.

Keywords: artificial cell model; giant lipid vesicles; ion channels; liposome; membrane proteins; nanopores; planar bilayer lipid membrane; proteoliposome.

Figure 2

Illustration of giant liposome formation (a) Gentle hydration method. (b) Electroformation method. (c) Droplet emulsion transfer method. Reproduced with permission from * [45]. Copyright (2003) National Academy of Sciences (d) Microfluidic devices of cell-sized lipid vesicle formations based on the droplet transfer method * [48]. (e) Pulsed-jet flow method * [64]. * Reproduced with permission from.

Figure 1

Schematic illustration of functional studies and applications of membrane proteins on artificial lipid membranes.

Figure 3

Illustration of proteo-GUVs formation. (a) Rehydration method. (b,c) Detergent-mediated reconstitution and membrane fusion method * [74]. (d) Direct reconstitution method * [88]. (e) Cell-free protein synthesis with liposome * [91]. * Reproduced with permission from.

Figure 4

(a) Reconstituted protein GL1-Alexa488 is cleaved from the GUV by RavZ, an antiautophagy factor [85] *. (b) light-induced proton pumping of BR induced an internal acidification of GUVs [74] *. (c) OmpLA transported ions and small molecules. The phospholipase activity caused the budding of small vesicles [77] *. (d) N-terminal hydrophobic domain of PR is necessary and sufficient for recruitment of ribosomes to the membrane and membrane insertion of PR [78] *. * Reproduced with permission from.

Figure 5

Illustration of BLM formation and the reconstituted ion channel. (a) Painting method. (b) Langmuir-Blodgett method. (c) Droplet contact method. (d) Vesicle Fusion method. (e) Nystatin/Ergosterol method.

Figure 6

(a) Building a new constricted region AeL for heterogeneously charged peptide sensing * [145]. (b) Peptides are pre-hydrolyzed by a specific protease and the resulting peptides are measured as they translocate the FraC nanopore [148] *. (c) Detection of various structure of DNA depending on the nanopore size using OmpG WT or mutated OmpG with different number of β−hairpins [142] *. (d) Detection of the protein analyze outside the FhuA nanopore by means of tethered protein bait [151] *. * Reproduced with permission from.

Figure 7

(a) Modular assembly of a functional electron transport chain by delivering ATP-synthase and bo3-oxidase into target membrane [21] *. (b) Schematic illustration of the artificial photosynthetic cell encapsulating artificial organelle containing BR and ATP synthase. Green fluorescent protein was synthesized inside GUV driven by light [22] *. * Reproduced with permission from.