Folding and Lipid Membrane Interactions of BamD, An Essential Component of the β-Barrel Assembly Machine from Escherichia Coli

Abstract

BamD (YfiO) [1] is an essential component of the β-barrel assembly machine of the outer membrane of Escherichia coli. BamD is synthesized in the cytosol and translocated across the cytopasmic membrane in unfolded form for processing and transport to the outer membrane by the LOL system [2].To examine the interactions of BamD with lipid membranes and BamD folding, we have over-expressed BamD into the periplasm and isolated it from membrane fractions in unfolded form in 8 M urea. Circular dichorism spectroscopy indicated secondary structure formation in BamD upon rapid dilution of the denaturant urea in aqeuous buffer. In the absence of lipid bilayers, BamD displayed a lower content of α-helix structure. The CD-data indicated that native-like folding of BamD required the presence of lipid-membranes of phosphatidylglycerol and phosphatidylcholine. An increased content of phosphatidylglycerol facilitated BamD folding. Fluorescence spectroscopy demonstrated BamD binding to both zwitterionic and negatively charged lipid. The lipid/BamD stoichiometry was estimated to 17 phosphatidylglycerol/BamD and to 31 phosphatidylcholine/BamD.[1] Malinverni JC, Werner J, Kim S, Sklar JG, Kahne D, Misra R, Silhavy TJ (2006) YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli. Mol Microbiol 61:151-164.[2] Narita S, Tokuda H (2010) Sorting of bacterial lipoproteins to the outer membrane by the Lol system. Methods Mol Biol 619:117-129.