BPS2026 – Single-molecule fluorescence provides insights into the rate-limiting step in BAM-mediated OMP folding

The β-barrel assembly machinery (BAM) complex is a conserved and essential factor in gram-negative bacteria, making it an attractive target for antibiotic development. While structural and biochemical studies have advanced our understanding of how outer membrane proteins (OMPs) are folded and inserted into the outer membrane (OM), how conformational changes of the OM insertase: the beta barrel assembly machine (BAM), connect to function remains unclear. To address this, we developed a single-molecule FRET (smFRET) construct to monitor the real-time dynamics of the BamA lateral gate, a key functional region of the complex. Dynamics were probed in both detergent micelles and lipid environments, and with mutants that bias its lateral gate equilibrium. Together, smFRET and mechanistic assays reveal how the conformational preference of the BAM lateral gate relates to the rate-limiting step of BAM catalysis and opens the door to revealing new insights into how the BAM complex assembles OMPs in vitro and in vivo.