Decrease of anion selectivity caused by mutation of Thr501 and Gly502 to Glu in the hydrophobic domain of the colicin E1 channel.
Structure-function relations of the colicin E1 ion channel were studied through the effects of mutations in the 35-residue hydrophobic region of the channel polypeptide and neighboring residues in the channel domain. Mutation of neutral residues threonine 501 and glycine 502 to a more polar or charged glutamic acid generated a protein whose channel conductance properties in each case had a decreased selectivity for anions. There was no significant effect on ion selectivity caused by mutations that changed residue charge outside the hydrophobic domain at the neighboring aspartic acid 509 or at glycine 439. The Thr501----Glu and Gly502----Glu mutants possessed lower cytotoxic and in vitro activity. An altered thermolysin cleavage pattern and a greater binding to membrane vesicles at pH greater than 4.5 of the Gly502----Glu mutant indicated greater exposure of its COOH-terminal hydrophobic domain in solution. It is concluded that the hydrophobic nature of threonine 501 and glycine 502 is important in the structure of the channel lumen and the soluble colicin. Altering proline 462, a residue conserved in five sequenced channel-forming colicins, had no significant effect on channel properties. These conclusions are discussed in the context of sequence-structure-function concepts for channel proteins.
Shirabe
K
K
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Cohen
F S
FS
Xu
S
S
Peterson
A A
AA
Shiver
J W
JW
Nakazawa
A
A
Cramer
W A
WA
D000595:Amino Acid Sequence; D002712:Chlorides; D003087:Colicins / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine; D007700:Kinetics; D008051:Lipid Bilayers; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009154:Mutation; D010713:Phosphatidylcholines; D010743:Phospholipids; D013912:Threonine
D002712:Chlorides; D003087:Colicins; D005971:Glutamates; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010743:Phospholipids; D013912:Threonine; D018698:Glutamic Acid; C024990:asolectin; D005998:Glycine