Single-turnover kinetics of BAM activity with bOmpA-A488

The B-barrel assembly machine is an essential, multiprotein complex that folds and inserts outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. The BAM complex is composed of the essential BamA OMP, which contains a transmembrane B-barrel domain and five periplasmic polypeptide transport-associated (POTRA) domains in E. coli. The BamA POTRA domains scaffold the essential lipoprotein BamD and the nonessential lipoproteins BamBCE. While an abundance of cell-based phenotypic assays and structural data have elucidated important insight into BAM-catalyzed OMP folding, methods for quantitative analysis of BAM function have been challenging. We use a quantitative fluorescent reporter of OMP folding, bOmpA-A488, to obtain single-turnover kinetic parameters for wild-type BAM complex in vitro, which agree well with estimates necessary for BAM function in vivo. Our fluorescence-based single-turnover activity assay was further used to investigate the roles of the lipoproteins and the POTRA domains 1-3 on BAM activity. The data file provided includes relevant raw data for SDS-PAGE densitometry, fluorescence measurements, and circular dichroism measurements.