Biophysical investigations of the mechanism of colicin translocation

Hands, Sarah Louise (2005) Biophysical investigations of the mechanism of colicin translocation. PhD thesis, University of Nottingham.

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Abstract

Colicins are a family of bacterial toxins, which kill Escherichia coli cells and other closely related species. Their mode of action requires binding to an outer membrane receptor, translocation across the outer membrane and periplasm and cytotoxic action on a specific target. Colicins usually kill cells either by attacking the bacterial RNA or DNA or by forming pores in the inner membrane of the cell. Their cytotoxic activity can be inhibited by the high affinity binding of an immunity protein.

For Group A colicins, translocation requires interaction between the N-terminal domain of the colicin and a series of membrane bound and periplasmic proteins called the Tol system (TolB, TolR, TolA, TolQ and Pal).

Three residues of colicin E9 have previously been shown to be essential for an interaction with TolB. This study suggests that these residues play differing roles in the interaction with TolB. Other residues surrounding these previously identified residues are also shown to be involved in the interaction with TolB.

In order to allow cytotoxicity, the immunity protein of colicins E3 and E9 must be lost on entry of the colicin to a target cell. This work has demonstrated by Surface Plasmon Resonance and Atomic Force Microscopy that the affinity of colicins E3 and E9 for TolB is increased when the immunity protein is removed. This observation has implications for the mechanism by which the immunity protein dissociates from the colicin.

Finally this study has used Surface Plasmon Resonance to explore differences between pore-forming and enzymatic colicins in their interactions with Tol proteins. Although the pore-former colicin A interacts with TolR, TolA and TolB, the endonuclease colicins E3 and E9 were shown only to interact with TolB. This suggests that pore-forming and endonuclease colicins use the Tol system in different ways in order to translocate across the periplasm.

Item Type:Thesis (PhD)
Supervisors:James, R.
Roberts, C.J.
Davies, M.C.
Allen, S.
Uncontrolled Keywords:colicin, Atomic Force Microscopy, AFM, Surface Plasmon Resonance, SPR, Tol proteins, protein translocation
Faculties/Schools:UK Campuses > Faculty of Science > School of Pharmacy
ID Code:102
Deposited By:Sarah Hands
Deposited On:09 May 2005
Last Modified:06 Feb 2009 14:43

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