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Thermolytic fragments of the channel-forming bacterial toxins colicin B and colicin E1 were uniformly labeled with the 15N isotope and reconstituted into uniaxially oriented membranes. These well-aligned samples were investigated by proton-decoupled 15N solid-state 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 15N-labeled amide bonds is nearly identical for colicin B and E1, 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 15N 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 15N resonances of the colicin B channel protein. Approximately 50 15N signals resonate in a region characteristic of transmembrane helical residues, in strong support of the previously suggested umbrella conformation of the closed colicin channel.

An approach is presented to selectively label the methionines of the colicin E1 and B channel domains, each about 200 residues in size, and use them for oriented solid-state NMR investigations. By combining site-directed mutagenesis, bacterial overexpression in a methionine auxotroph E. coli strain and biochemical purification, quantitative amounts of the proteins for NMR structural investigations were obtained. The proteins were selectively labeled with (15)N at only one, or at a few, selected sites. Multidimensional heteronuclear correlation high-resolution NMR spectroscopy and mass spectrometry were used to monitor the quality of isotopic labeling. Thereafter the proteins were reconstituted into oriented phospholipid bilayers and investigated by proton-decoupled (15)N solid-state NMR spectroscopy. The colicin E1 thermolytic fragment that carries a single (15)N methionine within its hydrophobic helix 9 region exhibited (15)N resonances that are characteristic of helices that are oriented predominantly parallel to the membrane surface at low temperature, and a variety of alignments and conformations at room temperature. This suggests that the protein can adopt both umbrella and pen-knife conformations.

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the fulllength toxin is consistent with two endothermic transitions. Whereas peak A (T m ‫؍‬ 55°C) most likely corresponds to the receptor/translocation domain, peak B (T m ‫؍‬ 59°C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18°C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T m ‫؍‬ 65°C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B (Ϸ40%) or its pore-forming fragment (Ϸ80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T m ‫؍‬ 54°C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.

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