Site‐directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation

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Abstract
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 Nterminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.