Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

28-POS Board 14 Customized Lipid Bilayers in Cell-Free Synthetic Biology: From Mechanisms to Applications Erik Henrich 1 , Oliver Peetz 2 , Yi Ma 3 , Ina Engels 4,5 , Tanja Schneider 4,5 , Nina Morgner 2 , Frank Löhr 1 , Volker Dötsch 1 , Frank Bernhard 1 . 1 Goethe University Frankfurt am Main, Frankfurt am Main, Hessen, Germany, 2 Goethe University Frankfurt am Main, Frankfurt am Main, Hessen, Germany, 3 South China University of Technology, Guangzhou, China, 4 University of Bonn, Bonn, Germany, 5 German Centre for Infection Research (DZIF), partner site Cologne-Bonn, Bonn, Germany. In contrast to soluble proteins, membrane protein research is often complicated by the hydrophobic character of the target protein. Cell-free synthetic biology provides new platforms for the efficient production of membrane proteins by adjusting and refining artificial hydrophobic expression environments for the functional folding of synthesized membrane proteins. We demonstrate the synergistic combination of nanodiscs and cell-free production, which allows for systematic lipid screening of detergent sensitive membrane proteins to generate high quality samples. Using MraY translocase homologues from various pathogenic bacteria, we demonstrate that MraY has very strict lipid requirements for its activity. The co-translational insertion of MraY into preformed nanodiscs enables the complete in vitro reconstitution of designed biosynthetic pathways for essential cell-wall precursors by comprising MraY homologues as well as whole sets of soluble enzymes in detergent free environments. Selective inhibition at different pathway steps demonstrates the potential of these synthetic in vitro pathways as a new drug screening platform. Furthermore, nanodisc based cell-free expression was combined with non-covalent LILBID mass spectrometry to characterize insertion mechanisms as well as oligomeric state formation of membrane proteins in nanodiscs. We have analyzed the complex formation of a variety of membrane proteins including proteorhodopsin, the multidrug transporter EmrE and the enzymes MraY and LspA. By implementing isotope labelling, we show monomer and up to hexamer formation of membrane proteins in nanodiscs, and we identify parameters suitable to trigger complex assembly. We furthermore give evidence that MraY dimer formation depends on its lipid environment and is essential for enzymatic activity. Moreover, we show the first details of the molecular mechanisms of membrane protein insertion into the size restricted nanodisc bilayers.

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