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1 Department of Biology, University of York, York YO10 5YW, United Kingdom
2 Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom
3 School of Chemical Sciences & Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom
(RECEIVED October 7, 2005; FINAL REVISION November 30, 2005; ACCEPTED November 30, 2005)
Nuclease type colicins and related bacteriocins possess the unprecedented ability to translocate an enzymatic polypeptide chain across the Gram-negative cell envelope. Here we use the rRNase domain of the cytotoxic ribonuclease colicin E3 to examine the structural changes on its interaction with the membrane. Using phospholipid vesicles as model membranes we show that anionic membranes destabilize the nuclease domain of the rRNase type colicin E3. Intrinsic tryptophan fluorescence and circular dichroism show that vesicles consisting of pure DOPA act as a powerful protein denaturant toward the rRNase domain, although this interaction can be entirely prevented by the addition of salt. Binding of E3 rRNase to DOPA vesicles is an endothermic process (
H = 24 kcal mol–1), reflecting unfolding of the protein. Consistent with this, binding of a highly destabilized mutant of the E3 rRNase to DOPA vesicles is exothermic. With mixed vesicles containing anionic and neutral phospholipids at a ratio of 1:3, set to mimic the charge of the Escherichia coli inner membrane, destabilization of E3 rRNase is lessened, although the melting temperature of the protein at pH 7.0 is greatly reduced from 50°C to 30°C. The interaction of E3 rRNase with 1:3 DOPA:DOPC vesicles is also highly dependent on both ionic strength and temperature. We discuss these results in terms of the likely interaction of the E3 rRNase and the related E9 DNase domains with the E. coli inner membrane and their subsequent translocation to the cell cytoplasm.
Keywords: conformational changes; enzymes; membrane-associated proteins; circular dichroism; fluorescence; thermodynamics; hydrodynamics; calorimetry
Abbreviations: DOPA, 1,2-dioleoyl-sn-glycero-3-phosphate (monosodium salt) • DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine • E3 rRNase, the isolated 12-kDa rRNase domain of colicin E3 • Im3, the colicin E3 immunity protein • E9 DNase, the isolated 15-kDa endonuclease domain of colicin E9 • KPi, potassium phosphate • RL-P, lipid:protein molar ratio • CD, circular dichroism • ITC, isothermal titration calorimetry
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051890306.
Reprint requests to: Colin Kleanthous, Department of Biology, University of York, York YO10 5YW, UK; e-mail: ck11{at}york.ac.uk; fax: +44 (0) 1904-328825.
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