Substrate binding to BamD triggers a conformational change in BamA to control membrane insertion.

The ß-barrel assembly machine (Bam) complex folds and inserts integral membrane proteins into the outer membrane of Gram-negative bacteria. The two essential components of the complex, BamA and BamD, both interact with substrates, but how the two coordinate with each other during assembly is not clear. To elucidate aspects of this process we slowed the assembly of an essential ß-barrel substrate of the Bam complex, LptD, by changing a conserved residue near the C terminus. This defective substrate is recruited to the Bam complex via BamD but is unable to integrate into the membrane efficiently. Changes in the extracellular loops of BamA partially restore assembly kinetics, implying that BamA fails to engage this defective substrate. We conclude that substrate binding to BamD activates BamA by regulating extracellular loop interactions for folding and membrane integration.

Solvent-Free Synthesis and Fluorescence of a Thiol-Reactive Sensor for Undergraduate Organic Laboratories.

A green organic laboratory experiment was developed in which students synthesize a sensor for thiols using a microscale, solventless Diels-Alder reaction at room temperature or 37 °C. The molecular probe is easily purified by column chromatography in a Pasteur pipet and characterized by thin-layer chromatography and NMR spectroscopy. The thiol-reactive sensor becomes intensely fluorescent upon exposure to thiols from N-acetylcysteine, bovine serum albumin, or human hair (pretreated with a reducing agent to reveal cysteine thiols in α-keratin). This fluorescence is observable even with micrograms of probe.

Formation of a ß-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA.

The ß-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane ß-barrel proteins. BamA, an essential component of the complex, is itself a ß-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control ß-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA ß-barrel. Importantly, we identify contacts between the assembling ß-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze ß-barrel assembly.