Specific isotope labeling of colicin E1 and B channel domains for membrane topological analysis by oriented solid-state NMR spectroscopy

An approach is presented to selectively label the methionines of the colicin El 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 N-15 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 N-15 solid-state NMR spectroscopy. The colicin El thermolytic fragment that carries a single N-15 methionine within its hydrophobic helix 9 region exhibited N-15 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.