Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1818, Issue 3, Pages 349-358Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2011.10.020
Keywords
Membrane protein; Membrane mimetics; Nanodisc; Cell-free expression; NMR; Protein folding
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Funding
- Russian Academy of Sciences
- Russian Foundation for Basic Research
- Russian Federation [MK 8404.2010.4]
- Federal Target Program Scientific and Science-Educational Personnel of Innovative Russia
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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 similar to 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 N-15-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. (C) 2011 Elsevier B.V. All rights reserved.
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