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| - Orientational Distribution of α-Helices in the Colicin B and E1 Channel Domains: A One and Two Dimensional 15N Solid-State NMR Investigation in UniaxiallyAligned Phospholipid Bilayers
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| - Thermolytic fragments of the channel-forming bacterial toxinscolicin B and colicin E1 wereuniformly labeled with the 15N isotope and reconstitutedinto uniaxially oriented membranes. These well-aligned samples were investigated by proton-decoupled 15Nsolid-state NMR spectroscopy at 40.5 and71.0 MHz. The one dimensional spectra indicate a predominantorientation of the colicin α-helices parallelto the bilayer surface but also the presence of a considerableproportion of peptide bonds that align in atransmembrane direction. The orientational distribution of15N-labeled amide bonds is nearly identicalfor colicin B and E1, each a representative of a different group ofmembrane-active colicins. Thiscomparison indicates common structural features of the water-soluble aswell as the bilayer-associatedproteins. When the pH is lowered, the orientational distributionof amide vectors exhibits only a smallshift from in-plane to transmembrane orientations, in agreement withincreased affinity and activity ofcolicins at acidic conditions. The 15N spectral lineshape was independent of the bilayer phospholipidcomposition (100−75 mol % phosphatidylcholine/0−25 mol %phosphatidylglycerol) or the protein-to-lipid ratio in the range 1.7−12 wt %. Two dimensional separatedlocal field spectroscopy (PISEMA)resolves almost 200 15N resonances of the colicin B channelprotein. Approximately 50 15N signalsresonatein a region characteristic of transmembrane helical residues, in strongsupport of the previously suggestedumbrella conformation of the closed colicin channel.
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