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Specific Isotope Labeling of Colicin E1 and B Channel Domains For Membrane Topological Analysis by Oriented Solid‐State NMR Spectroscopy
Author(s) -
Aisenbrey Christopher,
Cusan Monica,
Lambotte Stephan,
Jasperse Pieter,
Georgescu Julia,
Harzer Ulrike,
Bechinger Burkhard
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700507
Subject(s) - colicin , chemistry , nuclear magnetic resonance spectroscopy , solid state nuclear magnetic resonance , heteronuclear single quantum coherence spectroscopy , crystallography , isotopic labeling , helix (gastropod) , spectroscopy , heteronuclear molecule , mass spectrometry , nuclear magnetic resonance spectroscopy of nucleic acids , two dimensional nuclear magnetic resonance spectroscopy , stereochemistry , escherichia coli , transverse relaxation optimized spectroscopy , biochemistry , nuclear magnetic resonance , fluorine 19 nmr , chromatography , biology , organic chemistry , ecology , physics , quantum mechanics , snail , gene
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.