Insertion of β-Barrel Proteins in Gram-Negative Bacteria

Abstract

Gram-negative bacteria possess two membranes, the inner and outer of which contain primarily α-helix and β-barrel proteins respectively. In recent years, significant progress has been made in understanding insertion and assembly of proteins into the inner membrane, while the same process in the outer membrane has remained elusive. In 2013, the crystal structure of BamA, the central and essential component of the β-barrel assembly machinery (BAM), was released, paving the way for rapid progress in understanding the insertion and assembly process. All-atom molecular dynamics simulations have been performed, revealing many novel features including lateral gate opening between the first and last barrel strands, and a significantly thinner, destabilized membrane region near the putative insertion site. However, many questions remain, including the role of the periplasmic domains, the mode of substrate recognition, and the energetic factors driving function in the absence of both ATP and an electrochemical gradient. We have performed novel equilibrium simulations of the protein in its native lipopolysaccharide environment including its essential periplasmic domain. Here, we present a comparison of free energy associated with lateral-gate opening for native systems, as well as systems with strand modifications and augmentations which yield insight into driving energetics and substrate recognition.