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 (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.
Aisenbrey
Christopher
C
Institut de Chimie, Université Louis Pasteur Strasbourg - CNRS, UMR 7177, 4, Rue Blaise Pascal, 67000 Strasbourg, France.
Cusan
Monica
M
Lambotte
Stephan
S
Jasperse
Pieter
P
Georgescu
Julia
J
Harzer
Ulrike
U
Bechinger
Burkhard
B
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry*; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010743:Phospholipids / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011522:Protons; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*
D003087:Colicins; D009587:Nitrogen Isotopes; D010743:Phospholipids; D011522:Protons; D008715:Methionine