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Site‐directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation
Author(s) -
Todd A. Paul,
Cong Jianping,
Levinthal Francoise,
Levinthal Cyrus,
Hubell Wayne L.
Publication year - 1989
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340060312
Subject(s) - colicin , chemistry , electron paramagnetic resonance , crystallography , spin label , nitroxide mediated radical polymerization , mutagenesis , cysteine , site directed spin labeling , helix (gastropod) , site directed mutagenesis , biophysics , protein structure , mutant , stereochemistry , biochemistry , plasmid , membrane , biology , dna , nuclear magnetic resonance , copolymer , organic chemistry , ecology , physics , radical polymerization , snail , gene , enzyme , polymer
Colicin E1 is an E. coli plasmid‐lencoded water‐soluble protein that spontaneously inserts into lipid membranes to form a voltage‐gated ion channel. We have employed a novel approach is 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 resonancc (EPR) spectral periodicity strongly suggesting an amphiphilic α‐helix. After removal of the Nterminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.