title: Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes.
abstract: Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.
authors: Russian Academy of Sciences, Moscow, Russian Federation. ekaterina-lyukmanova@yandex.ru
authors: E N
authors: EN
authors: Lyukmanova
authors: Z O
authors: ZO
authors: Shenkarev
authors: N F
authors: NF
authors: Khabibullina
authors: G S
authors: GS
authors: Kopeina
authors: M A
authors: MA
authors: Shulepko
authors: A S
authors: AS
authors: Paramonov
authors: K S
authors: KS
authors: Mineev
authors: R V
authors: RV
authors: Tikhonov
authors: L N
authors: LN
authors: Shingarova
authors: L E
authors: LE
authors: Petrovskaya
authors: D A
authors: DA
authors: Dolgikh
authors: A S
authors: AS
authors: Arseniev
authors: M P
authors: MP
authors: Kirpichnikov
mesh_terms: D001406:Bacillaceae / Q000737:chemistry*; D001426:Bacterial Proteins; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D017510:Protein Folding
chemical_list: D001426:Bacterial Proteins; D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D008823:Micelles