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Liposomes, Exosomes, and Virosomes: From Modeling Complex
Membrane Processes to Medical Diagnostics and Drug Delivery
Poster Abstracts
61
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.