Interactions of β-barrel assembly machinery protein D (BamD) with phospholipid bilayers.

Abstract

The β-barrel assembly machinery (BAM) complex from Escherichia coli consists of the transmembrane protein BamA and four peripheral proteins, BamB, BamC, BamD and BamE. We investigated the lipid interactions of BamD (also called YfiO), which is essential for E. coli using site-directed fluorescence spectroscopy. To prepare a range of single-cysteine mutants of BamD for fluorescence labelling at the cysteine, we selected nine amino acid residues that were distributed over the entire surface of BamD. Expression plasmids were prepared and the mutants were then isolated from transformed E. coli strains to analyze interactions of BamD with bilayers composed of DLPC or of DLPG or of mixtures of DLPG and DLPE at a ratio of 4:1. BamD mutants were labeled with the fluorophore IANBD to examine BamD-lipid interactions by fluorescence spectroscopy. Higher fluorescence intensities and blue shifts of the emission spectra indicated that the entire surface of BamD interacted with the lipid bilayer. The fluorescence maxima of IANBD-labeled mutants were shifted towards shorter wavelength by ∼ 6 to 18 nm. Intensities of IANBD fluorescence increased most strongly for negatively charged DLPG bilayers, suggesting stronger binding due to electrostatic interactions. The weakest interactions were observed for lipid bilayers composed of DLPE and DLPG at a ratio of 4:1. For these bilayers, the IANBD-fluorescence spectra were most strongly changed for the IANBD labeled mutants A37C-BamD, G153C-BamD, and V181C-BamD. Fluorescence of all labeled mutants, except G200C-BamD, was quenched when 14-Doxyl-PC was used as a quencher, indicating penetration of BamD into the lipid bilayer. Fluorescence spectra of L75C-BamD and of V181C-BamD also indicated interactions with BamA in the absence of lipid bilayers.