A 136-amino-acid-residue COOH-terminal fragment of colicin A is endowed with ionophoric activity.
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 NH2-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 alpha-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.
Baty
D
D
Centre de Biochimie et de Biologie Moléculaire, Centre National de la Recherche Scientifique, Marseille, France.
Lakey
J
J
Pattus
F
F
Lazdunski
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003087:Colicins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D007473:Ion Channels; D007476:Ionophores; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D015183:Restriction Mapping
D003087:Colicins; D007473:Ion Channels; D007476:Ionophores; D008051:Lipid Bilayers; D015345:Oligonucleotide Probes; D010446:Peptide Fragments