Insertion and folding of beta-barrel membrane proteins into lipid bilayers - Functions of BamA (YaeT) and of membrane anchored Bam lipoproteins in membrane protein folding.

• Applicant: Professor Dr. Jörg H. Kleinschmidt
• Subject Area: Biochemistry ; Biophysics
• Term: from 2008 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 88778146

Insertion and folding of transmembrane proteins are not well understood, although these processes are important for membrane biogenesis and cell growth. For insertion and folding of outer membrane proteins (OMPs) in bacteria and mitochondria of eukaryotic cells, the barrel assembly machinery (BAM) complex is vital. Based on our previous work on the mechanism of folding and insertion of OMPs like OmpA and FomA, we are investigating the biophysical and biochemical functions of the BAM proteins of Escherichia coli. This BAM complex is composed of the essential transmembrane protein BamA (also called YaeT or Omp85) and of four peripheral lipoproteins, BamB, C, D, and E. Only BamD (YfiO) is essential, but the simultaneous deletion of BamB and BamE is also lethal. In the previous funding period, we found that BamA, BamD, and BamB independently improve the folding rates of OmpA into lipid bilayers from an unfolded form prepared in 8 M urea. BamA also facilitated folding of OmpA from a complex with its periplasmic chaperone Skp. The analysis indicated further that the periplasmic domain (PD) of BamA has a principal contribution to the observed folding rates. Lipids, the lipid anchors of the lipoproteins, or bound client OMPs are not resolved in any of the known structures of the BAM complex, which is one of the reasons why the structure-function relationships of the complex are not understood. Two aims of the present project are to examine how proteins of the BAM interact with OMPs like OmpA and OmpG in a lipid environment and how these interactions facilitate folding and membrane insertion. The rates of folding and insertion of OMPs can be modulated by temperature and by the lipid composition of the membrane and we recently identified a folding intermediate of OmpA bound to bilayers of dilauroyl phospholipids containing BamA. A slower folding process and trapped folding intermediates of client OMPs in the presence of BAM proteins will now allow us to investigate how BAM proteins function. We will use site-directed spectroscopy to determine interactions of the BAM proteins in the folding of OmpA and investigate the relevant protein-protein and protein-lipid interactions. The soluble PD of BamA and BamD form a large ring-like structure in the periplasm and we could demonstrate that both bind to lipid membranes. Site-directed fluorescence methods and fluorescence quenching with lipid-bound quenchers will be used to investigate which regions of the PD of BamA and of BamD interact with lipids. We will also examine the lipid-selectivity of both proteins and a possible formation of a lipid microdomain in their vicinity. The results of this work will greatly advance our understanding of the principles how protein insertion machinery works in membrane protein folding. Since there are differences in the protein insertion machineries of bacteria und eukaryotic cells, this basic knowledge could also be useful for future developments of antibiotics.

• DFG Programme: Research Grants