Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin.
The paucity of crystallographic data on the structure of intrinsic membrane proteins necessitates the development of additional techniques to probe their structures. The colicin E1 ion channel domain contains one prominent hydrophobic region near its COOH terminus that has been proposed to be an anchor for the assembly of the channel. Saturation site-directed mutagenesis of the hydrophobic anchor region of the colicin E1 ion channel was used to probe whether it spanned the bilayer once or twice. A nonpolar amino acid was replaced by a charged residue in 29 mutations made at 26 positions in the channel domain. Substitution of the charged amino acid at all positions except those in the center of the hydrophobic region and the periphery of the hydrophobic region caused a large decrease in the cytotoxicity of the purified mutant colicin E1 protein. This result implies that the hydrophobic domain spans the membrane bilayer twice in a helical hairpin loop, with the center of this domain residing in an aqueous or polar phase. The lengths of the trans-membrane helices appear to be approximately 18 and 16 residues. The absence of significant changes in ion selectivity in five of nine mutants indicated that these mutations did not cause a large change in the channel structure. The ion selectivity changes in four mutants and those previously documented for the flanking Lys residues imply that the hydrophobic hairpin is part of the channel lumen. Water may "abhor" the hydrophobic side of the channel, explaining the small effects of residue charge changes on ion selectivity.
Song
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Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Cohen
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Cramer
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D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000648:ultrastructure*; D003087:Colicins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D007473:Ion Channels / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D003087:Colicins; D007473:Ion Channels