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Title: The mechanism of the ATP-independent periplasmic chaperone SurA in outer membrane protein biogenesis
Author: Humes, Julia Rose
ISNI:       0000 0004 7654 6616
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
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Outer membrane proteins (OMPs) of Gram-negative bacteria travel from their site of synthesis in the cytoplasm, across the inner membrane and through the periplasm to the outer membrane (OM) prior to their folding to a functional form. To protect OMPs from misfolding or aggregation while traversing the periplasm a network of chaperones are employed. The OMPs must then reach the essential β-barrel assembly machinery (BAM) complex, which is involved in inserting OMPs into the OM. This process occurs in the absence of chemical energy as the periplasm is devoid of ATP and in a highly dynamic environment as the 'leaky' OM allows small molecules (< 600 Da) to enter from the extracellular milieu. The major periplasmic chaperone for OMPs, SurA, is known to interact with a number of substrates and has been crosslinked to the BAM complex in vivo. SurA is composed of four domains, an N-terminal domain, two peptidyl-prolyl isomerase (PPIase) domains and a short C-terminal helical domain. In this work wild-type E. coli SurA and SurA truncation variants lacking one (P2) or both (N-Ct) of the PPIase domains have been studied. Using microscale thermophoresis, light scattering, native mass spectrometry and other biophysical techniques how each domain is involved in OMP binding, chaperoning and delivery to BAM is investigated. The results demonstrate that SurA binds unfolded OMPs, tOmpA and OmpT with μM affinity, agreeing with previous findings. The core domain (SurA N-Ct) is sufficient for this interaction, but the addition of the PPIase domains leads to a tighter binding. Light scattering experiments shows that SurA WT can prevent aggregation of the two model OMPs, but the removal of the PPIase domains reduces the chaperoning ability for the larger, more aggregation-prone OMP, OmpT. These observations demonstrate that the acquisition of the PPIase domains is advantageous for both OMP binding and chaperoning. An interaction between SurA and the BAM complex is also observed for the first time in vitro. Overall, the results reveal new insights into how SurA binds and chaperones OMPs before delivering them to the BAM complex for folding in the OM.
Supervisor: Radford, Sheena ; Brockwell, David Sponsor: US National Institutes of Health
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available