Identifying the Oligomerization State of DegP in the Absence and Presence of Substrate

Abstract

Bacterial outer membrane proteins (OMPs) are synthesized in the cytoplasm, cross the inner membrane, and are then transported across the periplasm before folding into the outer membrane. Similar pathways are present in both mitochondria and chloroplasts. While unfolded outer membrane proteins (uOMPs) are prone to aggregation, they are able to avoid this off pathway reaction with the help of periplasmic chaperones and proteases. The primary protease that interacts with uOMPs is DegP. DegP is a member of the high temperature requirement A (HTRA) protease family and has been implicated in both the Cpx and σE stress response pathways. DegP sequesters and degrades uOMPS when they accumulate in the periplasm. It has been suggested that DegP functions by transitioning from an inactive hexameric state to an active cage-like oligomeric state of either 12 or 24 subunits when substrate is present. In order to further investigate the relationship between the oligomeric state of DegP and the presence of substrate, we performed sedimentation velocity experiments with and without various uOMPs. This allows us to identify the oligomeric populations of DegP at biological concentrations. Results suggest that DegP has significant populations of multiple oligomeric states even in the absence of substrate. This work contributes to our understanding of OMP biogenesis by identifying the nature of uOMP interactions with an important pathway component.