Abstract
The surface of Gram-negative bacteria is perforated by β-barrel proteins called outer membrane proteins (OMPs). These β-barrels are integral to the outer membrane (OM) and are essential for the viability of the bacterial cell. OMPs often have large loops that protrude into the extracellular environment. These loops show promise for biotechnological applications and as therapeutic targets. The loops can be utilized to attach heterologous proteins at the surface of bacteria. Understanding how modifications to these loops affect the stability and folding of outer membrane proteins is essential for their efficient utilization for biotechnological purposes. In this work, the small outer membrane protein OmpX was used a model system to examine the effects of loop insertions on folding and stability. The insertions were varied according to hydrophobicity and size. The effects of the loop inserts were determined by assaying folding into detergent micelles in vitro by SDS-PAGE. The folding capacity of the constructs were also examined in vivo, by isolating the OM of cells expressing the constructs. The results indicate that folding of OMPs is effected by the hydrophobic character of the extracellular loops. Small insertions of five residues were found to improve the folding efficiency of OmpX, while large hydrophilic inserts reduced folding efficiency. All the constructs that were found to fold in vitro, could also do so in their native environment. One construct that could not fold in vitro, was transported to OM in vivo, but remained unfolded. The results have important biotechnological implications as they could improve the design and efficiency of recombinant OMPs used for surface display.