Orientational distribution of α-helices in the colicin B and E1 channel domains:: A one and two dimensional 15N solid-state NMR investigation in uniaxially aligned phospholipid bilayers

Thermolytic fragments of the channel-forming bacterial toxins colicin B and colicin El were uniformly labeled with the N-15 isotope and reconstituted into uniaxially oriented membranes, These well-aligned samples were investigated by proton-decoupled N-15 solid-stale NMR spectroscopy at 40.5 and 71.0 MHz. The one dimensional spectra indicate a predominant orientation of the colicin alpha-helices parallel to the bilayer surface but also the presence of a considerable proportion of peptide bonds that align in a transmembrane direction, The orientational distribution of N-15-labeled amide bonds is nearly identical for colicin B and El, each a representative of a different group of membrane-active colicins. This comparison indicates common structural features of the water-soluble as well as the bilayer-associated proteins, When the pH is lowered, the orientational distribution of amide vectors exhibits only a small shift from in-plane to transmembrane orientations, in agreement with increased affinity and activity of colicins at acidic conditions, The N-15 spectral line shape was independent of the bilayer phospholipid composition (100-75 mol % phosphatidylcholine/0-25 mol % phosphatidylglycerol) or the protein-to-lipid ratio in the range 1.7-12 wt %. Two dimensional separated local field spectroscopy (PISEMA) resolves almost 200 N-15 resonances of the colicin B channel protein. Approximately 50 N-15 signals resonate in a region characteristic of transmembrane helical residues, in strong support of the previously suggested umbrella conformation of the closed colicin channel.