title: Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation.
abstract: Colicin E1 is an E. coli plasmid-encoded water-soluble protein that spontaneously inserts into lipid membranes to form a voltage-gated ion channel. We have employed a novel approach in which site-directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels. A series of colicin mutants, differing only by the position of a single cysteine residue, were prepared and selectively labeled at that cysteine. A hydrophilic sequence (398-406) within the C-terminal domain of the water-soluble form of the protein was investigated and exhibited an electron paramagnetic resonance (EPR) spectral periodicity strongly suggesting an amphiphilic alpha-helix. After removal of the N-terminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.
authors.affiliation: Jules Stein Eye Institute, University of California, Los Angeles 90024.
authors.forename: A P
authors.initials: AP
authors.lastname: Todd
authors.forename: J
authors.initials: J
authors.lastname: Cong
authors.forename: F
authors.initials: F
authors.lastname: Levinthal
authors.forename: C
authors.initials: C
authors.lastname: Levinthal
authors.forename: W L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D000595:Amino Acid Sequence; D003087:Colicins; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009589:Nitrogen Oxides; D010070:Oxalates; D019815:Oxalic Acid; D011487:Protein Conformation; D013113:Spin Labels; D014357:Trypsin
chemical_list: D003087:Colicins; D009589:Nitrogen Oxides; D010070:Oxalates; D013113:Spin Labels; D019815:Oxalic Acid; D014357:Trypsin; C039900:nitroxyl; D003545:Cysteine