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Proteins. 1989;6(3):294-305.

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

Author information

1
Jules Stein Eye Institute, University of California, Los Angeles 90024.

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.

PMID:
2560193
DOI:
10.1002/prot.340060312
[Indexed for MEDLINE]

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