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 conformational stabilities of full-length colicin B and its isolated C-terminal domain were studied by guanidine hydrochloride induced unfolding. The unfolding/refolding was monitored by far-UV CD and intrinsic tryptophan fluorescence spectroscopies. At pH 7.4, the disruption of the secondary structure of full-length colicin B is monophasic, while changes in tertiary structure occur in two separate transitions. The intermediate species, which is well-populated around 2.2 M guanidine hydrochloride, exhibits secondary and tertiary structures distinct from both native and unfolded states. Whereas the domain structure of native full-length colicin B is reflected in its DSC profile, the folding intermediate of the same protein exhibits a single unresolved peak. These observations have led us to propose an unfolding model for full-length colicin B where the first transition between 0 and 2.5 M GuHCl with an associated free energy of 3 kcal/mol correlates with the partial unfolding of the R/T domain. The stability of full-length colicin B is weakened due to the presence of the R/T domain in both the native [Ortega, A., Lambotte, S., and Bechinger, B. (2001) J. Biol. Chem. 276 (17), 13563-13572] and the intermediate states. The second transition between 2.5 and 5 M GuHCl involves unfolding of the C-terminal domain (Delta = 7 kcal/mol). The isolated colicin B C-terminal domain consists of two subdomains, and the two parts of this protein fragment unfold sequentially through the formation of at least one intermediate. The significance of these results for membrane insertion of colicin B is discussed.