Conformation of the closed channel state of colicin A in proteoliposomes: an umbrella model |
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Authors: | Padmavathi Pulagam V L Steinhoff Heinz-Jürgen |
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Institution: | Department of Physics, University of Osnabrück, Barbarastrasse 7, D-49069 Osnabrück, Germany |
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Abstract: | Colicin A (ColA) is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of Escherichia coli. Until now, two models were proposed for the closed channel state: the umbrella model and the penknife model. Mutants of ColA, each containing a single cysteine, were labeled with a nitroxide spin label, reconstituted into liposomes, and studied by electron paramagnetic resonance (EPR) spectroscopy to study the membrane-bound closed channel state. The spin-labeled ColA variants in solution and in liposomes of native E. coli lipid composition were analyzed in terms of the mobility of the nitroxide, its accessibility to paramagnetic reagents, and the polarity of its microenvironment. The EPR data determined for the soluble ColA pore-forming domain are in agreement with its crystal structure. Moreover, the EPR results show that ColA has a conformation in liposomes different from its water-soluble conformation. Residues that belong to helices H8 and H9 are significantly accessible for O2 but not for nickel-ethylene diamine diacetic acid, indicating their location inside the membrane. In addition, the polarity values determined from the hyperfine tensor component Azz of residues 176, 181, and 183 (H9) indicate the location of these residues close to the center of the lipid bilayer, supporting a transmembrane orientation of the hydrophobic hairpin. Furthermore, the accessibility and polarity data suggest that the spin-labeled side chains of the amphipathic helices (H1-H7 and H10) are located at the membrane-water interface. Evidence that the conformation of the closed channel state in artificial liposomes depends on lipid composition is given. The EPR results for ColA reconstituted into liposomes of E. coli lipids support the umbrella model for the closed channel state. |
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Keywords: | ColA colicin A EPR electron paramagnetic resonance FRET fluorescence resonance energy transfer MTS methanethiosulfonate sol-ColA water-soluble ColA mem-ColA ColA reconstituted into membranes Ni-EDDA nickel-ethylene diamine diacetic acid LUV large unilamellar vesicle |
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