Open AccessA 136‐amino‐acid‐residue COOH‐terminal fragment of colicin A is endowed with ionophoric activity
Author(s) -
BATY Daniel,
LAKEY Jeremy,
PATTUS Franc,
LAZDUNSKI Claude
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb15503.x
Subject(s) - colicin , residue (chemistry) , amino acid , chemistry , biophysics , stereochemistry , peptide sequence , methionine , biochemistry , gene , biology , escherichia coli
DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a ‘STOP‐Shine‐Dalgarno‐sequence‐ATG’ cassette encoding a termination codon, a Shine‐Dalgano sequence and an initiation codon within the structural gene. This technique has been used to obtain a 137‐amino‐acid‐residue pore‐forming protein designated DA70C comprising the final 136‐amino‐acid‐residue COOH‐terminal of colicin A preceded by an NH 2 ‐terminal methionine. Da70C was correctly expressed but poorly released to the extracellular medium. Its purification involved, as a final step, a partition in Triton X‐114 thus demonstrating that hydrophobic regions are exposed in this protein. The ability of DA70C to form ion channels in planar lipid bilayers was investigated and pore properties were analyzed. The results indicate that helices 1–3 of the 204‐amino‐acid‐residue colicin pore‐forming domain (containing 10 α‐helices) are not involved in ion conduction through the channel. However, they are important in maintaining the stability of the soluble state of the COOH‐terminal domain.