Use this URL to cite or link to this record in EThOS:
Title: A functional and topological analysis of the E. coli export protein HlyD
Author: Balakrishnan, L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
Availability of Full Text:
Full text unavailable from EThOS.
Please contact the current institution’s library for further details.
Type I bacterial export of E. coli hemolysin (HlyA) is mediated by an inner membrane translocase complex, comprising traffic ATPase HlyB and adaptor protein HlyD, and the outer membrane channel-tunnel TolC. Engagement of HlyA by the translocase triggers recruitment of TolC and the formation of a trans-periplasmic conduit through which HlyA is exported out of the bacterial cell. This thesis presents a functional and topological analysis of HlyD, which is proposed to interact with TolC. HlyD is a trimeric 478-residue protein with cytosolic (amino acid 1-59) and periplasmic (amino acid 81-478) domains, connected by a single transmembrane helix (amino acid 60-80). In vivo chemical cross-linking was used to investigate the function of the small N-terminal cytosolic domain. Deleting most of the c.60 amino acid domain abolished export of HlyA, even though the truncated HlyD was, like wildtype, able to trimerise in the inner membrane and interact with the traffic ATPase. The mutant translocase complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger TolC recruitment. The N-terminal truncated HlyD was specifically unable to bind the substrate, suggesting that substrate engagement by the traffic ATPase is insufficient, and that interaction with the HlyD cytosolic domain is crucial to trigger TolC recruitment. Further analyses indicated that central to this HlyD function is an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues. When the charged amino acids were deleted, substrate engagement by HlyD was unproductive, i.e. TolC recruitment was not triggered. This suggests an uncoupling of the signal transduction mechanism underlying TolC recruitment. The HlyD periplasmic domain is predicted to feature two coiled-coils from amino acid 125-160 (coil I) and 240-300 (coil II). The functional role of coil II was investigated by introducing three specific point mutations, which reduced the probability of coiled-coil formation. This mutated HlyD trimerised in the inner membrane and formed a translocase complex with HlyB which engaged HlyA substrate and recruited TolC. However, the mutant was completely defective in HlyA export, supporting the view that the coiled-coil may be involved in opening the TolC channel-tunnel.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available