Investigating Interactions Of The SecYEG Holotranslocon With Periplasmic Chaperones And Outer Membrane Proteins

  • Danny P Williams-Jones

Student thesis: Master's ThesisMaster of Science (MSc)


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 Award11 May 2021
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorIan R Collinson (Supervisor) & Mark S Dillingham (Supervisor)

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