Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation
| Autor: | Wayne L. Hubbell, Jianping Cong, Cyrus Levinthal, Françoise Levinthal, A. P. Todd |
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| Rok vydání: | 1989 |
| Předmět: |
Nitroxide mediated radical polymerization
Protein Conformation Stereochemistry Molecular Sequence Data Colicins Sequence (biology) Biology Biochemistry law.invention Structural Biology law Escherichia coli Trypsin Amino Acid Sequence Cysteine Binding site Electron paramagnetic resonance Site-directed mutagenesis Molecular Biology Oxalates Oxalic Acid Mutagenesis Electron Spin Resonance Spectroscopy Colicin Mutation Nitrogen Oxides Spin Labels |
| Zdroj: | Proteins: Structure, Function, and Genetics. 6:294-305 |
| ISSN: | 1097-0134 0887-3585 |
| DOI: | 10.1002/prot.340060312 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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