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Probing the mechanism of membrane translocation by bacterial toxin nuclease domains through ensemble and single-molecule approaches

Reference	BB/C516236/1
Principal Investigator / Supervisor	Professor Colin Kleanthous
Co-Investigators / Co-Supervisors	Dr Christoph Baumann
Institution	University of York
Department	Biology
Funding type	Research
Value (£)	215,210
Status	Completed
Type	Research Grant
Start date	01/04/2005
End date	31/07/2008
Duration	40 months

Abstract

The proposed research will investigate whether colicin nucleases (the DNase domain of colicin E9 and the rRNase domain of colicin E3) have a natural propensity to translocate across membranes, determine if the essential inner membrane protein FtsH is involved in the translocation and establish the structural constraints on membrane translocation and import in the bacterial cytoplasm. The proposal will address these objectives through a multidisciplinary programme using ensemble and single molecule methods. In the first part of the programme we will establish methods that will be able to identify the unaided translocation of colicin nucleases across membranes in vitro. This will be based on the entrapment of either substrate DNA (for the Dnase) or specific immunity proteins (that bind with very high affinity to the nucleases) within anionic small unilamellar vesicles (SUVs). Both molecules will be fluorescently tagged. In the cast of DNA the fluorescence will be quenched until cleavage occurs. Nuclease-Im protein complexes will be detected by the use of FRET following association. Detection of translocated DNA will be based on fluorescence observed through a variety of methods, including static fluorescence, confocal microscopy and Total Internal Reflection Fluorescence Microscopy (TIRFM). TIRFM allows individual fluorophores to be visualised in real time. We plan to follow membrane translocation of colicin nucleases using biotinylated SUVs, tethered to a surface, containing fluorescently tagged DNA substrates or immunity proteins. The programme will also investigate, using traditional ensemble methods such as fluorescence quenching and CD, the structural changes to the colicin rRNase E3 since preliminary data presented in the proposal indicate very similar changes to those already reported for the colicin E9 DNase. At the same time, SUV experiments will also be established, using trapped Im3 as the reporter of translocation. The second part of the programme will look at the possible involvement of essential inner membrane protease FtsH mutants in which are known to make bacteria resistant to colicins. FtsH degrades membrane proteins by translocating them into the cytoplasm and then proteolysing them, and the hypothesis being tested is that colicin nucleases may resemble membrane proteins destined for destruction but which escape proteolysis following translocation. We will test this comparing the cytotoxic effects of colicin nuclease domains on spheroplasts of bacteria with and without endogenous FtsH. We will also attempt to construct an assay based on the reconstitution of purified FtsH into proteoliposomes and their use in import assays, developed in the first part of the programme. The third part of the programme will test the capacity of colicin nucleases to translocate cargo molecules either into vesicles or bacteria, as a test of the structural constraints on the import process.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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