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 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 15N 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 15N solid-state NMR spectroscopy. The colicin E1 thermolytic fragment that carries a single 15N methionine within its hydrophobic helix 9 region exhibited 15N 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

NATURVETENSKAP  -- Kemi -- Fysikalisk kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences -- Physical Chemistry (hsv//eng)

Nyckelord

Bcl-2 proteins

isotopic labeling

membrane proteins

NMR spectroscopy

transmembrane helical loop

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