title: Global structural rearrangement of the cell penetrating ribonuclease colicin E3 on interaction with phospholipid membranes.
abstract: 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 (DeltaH=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 degrees C to 30 degrees 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.
authors.affiliation: Department of Biology, University of York, York YO10 5YW, UK.
authors.forename: Khédidja
authors.initials: K
authors.lastname: Mosbahi
authors.forename: Daniel
authors.initials: D
authors.lastname: Walker
authors.forename: Richard
authors.initials: R
authors.lastname: James
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Moore
authors.forename: Colin
authors.initials: C
authors.lastname: Kleanthous
mesh_terms: D000838:Anions / Q000737:chemistry; D002151:Calorimetry; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism; D009994:Osmolar Concentration; D010743:Phospholipids / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D000838:Anions; D003087:Colicins; D010743:Phospholipids; D012260:Ribonucleases