title: Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase.
abstract: Nanodiscs (NDs) enable the analysis of membrane proteins (MP) in natural lipid bilayer environments. In combination with cell-free (CF) expression, they could be used for the co-translational insertion of MPs into defined membranes. This new approach allows the characterization of MPs without detergent contact and it could help to identify effects of particular lipids on catalytic activities. Association of MPs with different ND types, quality of the resulting MP/ND complexes as well as optimization parameters are still poorly analyzed. This study describes procedures to systematically improve CF expression protocols for the production of high quality MP/ND complexes. In order to reveal target dependent variations, the co-translational ND complex formation with the bacterial proton pump proteorhodopsin (PR), with the small multidrug resistance transporters SugE and EmrE, as well as with the Escherichia coli MraY translocase was studied. Parameters which modulate the efficiency of MP/ND complex formation have been identified and in particular effects of different lipid compositions of the ND membranes have been analyzed. Recorded force distance pattern as well as characteristic photocycle dynamics indicated the integration of functionally folded PR into NDs. Efficient complex formation of the E. coli MraY translocase was dependent on the ND size and on the lipid composition of the ND membranes. Active MraY protein could only be obtained with ND containing anionic lipids, thus providing new details for the in vitro analysis of this pharmaceutically important protein.
authors.affiliation: Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Roos
authors.forename: Michael
authors.initials: M
authors.lastname: Zocher
authors.forename: Daniel
authors.initials: D
authors.lastname: Müller
authors.forename: Daniela
authors.initials: D
authors.lastname: Münch
authors.forename: Tanja
authors.initials: T
authors.lastname: Schneider
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Sahl
authors.forename: Frank
authors.initials: F
authors.lastname: Scholz
authors.forename: Josef
authors.initials: J
authors.lastname: Wachtveitl
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.forename: Davide
authors.initials: D
authors.lastname: Proverbio
authors.forename: Erik
authors.initials: E
authors.lastname: Henrich
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
mesh_terms: D017920:Antiporters / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D025604:Rhodopsins, Microbial; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups)
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D018832:Molecular Chaperones; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; C080403:sugE protein, E coli; C075436:EmrE protein, E coli; D012243:Rhodopsin; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria