Maintenance of the cell envelope is essential for the viability of Gram-negative bacteria and requires translocation of outer membrane proteins (OMPs) from the cytoplasm to the outer membrane (OM). Posttranslational secretion of unfolded OMPs through the conserved SecYEG channel permits trans-periplasmic trafficking by chaperones, which escort and deposit those OMPs into the β-Barrel Assembly Machinery (BAM) for folding and OM insertion. The SecYEG channel, in complex with ancillary membrane proteins SecDF, YidC and YajC, forms the holotranslocon (HTL), a 250 kDa complex associated with OMP trafficking. Recent data suggest that periplasmic domains of the HTL contact BAM, forming a trans-periplasmic super-complex posited to enhance the efficiency of OMP trafficking. To investigate the diversity of HTL:chaperone interactions in OMP trafficking, a HTL:chaperone network was generated with STRING. Coevolutionary analysis software GREMLIN was used to assess the probability of HTL:BAM interactions, and screen for novel interactors. One such protein identified was YfgM, which was homology-modelled to assess the spatial plausibility of HTL/BAM interactions. SurA, a model OMP chaperone, interacts with both HTL and BAM. Though low-resolution structures of HTL:SurA and HTL:SurA:OmpA are available, single particle reconstruction using cryo-EM data will determine precise interactions with the HTL, and whether SurA binding is spatially compatible or competitive with HTL:BAM complex formation. Here, a strategy for HTL:SurA and HTL:SurA:OmpA purification by in vitro crosslinking and gel filtration is outlined, with a preliminary SurA:OmpA structure generated from a negative stain dataset. The collation of structural models dissecting protein translocation will heighten our understanding of this process and may delineate novel interaction targets for antimicrobial compounds.
|Date of Award||11 May 2021|
- The University of Bristol
|Supervisor||Ian R Collinson (Supervisor) & Mark S Dillingham (Supervisor)|