Extreme Dynamics in the BamA ß-Barrel Seam.

BamA is an essential component of the ß-barrel assembly machine (BAM) that is responsible for insertion and folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. BamA is an OMP itself, and its ß-barrel transmembrane domain is thought to catalyze OMP insertion and folding, although the molecular mechanism remains poorly understood. Crystal structures of BamA and complementary molecular dynamics simulations have shown that its ß-barrel seam (the interface between the first and last barrel strands) is destabilized. This has led to mechanistic models in which the BamA barrel seam functions as a lateral gate that opens and successively accepts ß-hairpins from a nascent OMP such that a nascent barrel can bud from BamA. Consistent with this model, disulfide locking of the BamA barrel seam is lethal in Escherichia coli. Here we show that disulfide locking of the BamA barrel has no effect on its ability to catalyze folding of a model OMP into liposomes. However, disulfide trapping experiments indicate that the BamA barrel is highly dynamic in the liposome membranes, with the ß-strands at the barrel seam undergoing "register sliding" by more than 14 Å both up and down the membrane. Remarkably, these extreme dynamics were also observed in the BamA barrel in the context of the native E. coli outer membrane. These results are consistent with a model in which the BamA barrel dynamics induce defects in the outer membrane that facilitate insertion of nascent OMPs.

Small Angle X-ray Scattering (SAXS) Characterization of the POTRA Domains of BamA.

BamA is the central component of the BAM complex and contains a C-terminal ß-barrel domain embedded in the outer membrane, and a soluble, periplasmic domain, made out of five polypeptide transport associated (POTRA) motifs. Structural characterization of the POTRA domains was carried out by a combination of crystallographic, NMR and solution Small Angle X-ray Scattering (SAXS) approaches. Despite its limited resolution, SAXS is an excellent complement to NMR and crystallography. It is well suited to validate high-resolution models in solution and is particularly useful to characterize flexible systems such as the POTRA domains of BamA. Here we present a protocol for sample preparation and discuss the considerations of SAXS data collection and quality control, which is applicable to most soluble proteins.