Mapping the membrane topology of the closed state of the colicin E1 channel.
The membrane-associated closed channel state of the colicin E1 thermolytic peptide was studied by the Parallax Method of depth-dependent fluorescence quenching. A number of single Trp-containing peptides of colicin E1 were prepared to facilitate the use of Trp as a probe for the topography of the channel peptide in the membrane-bound state. The bound form of the channel peptide was studied by binding channel peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-pal-mitoyl -2-oleoyl-sn- glycero-3-phosphatidylglycerol large unilamellar vesicles (60:40, mol/mol, approximately 0.1 microns diameter vesicles prepared by an extrusion technique) at low pH (pH 3.5). Depth-dependent fluorescence quenching studies using two nitroxide-labeled phospho-lipids (1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphatidylcho++ +-line and 1-palmitoyl-2-(12-doxylstearoyl)-sn-glycero-3-phosphatidylcholine) were conducted to determine the membrane location of each Trp residue for the vesicle-bound peptide. The three naturally occurring Trp residues in the colicin channel peptide, Trp-424, Trp-460, and Trp-495, were found to reside at membrane depths (from the C-2 carbon of the fatty acyl chain) of 7.4, 3.1, and 8.4 A, respectively. Three Trp residues (Trp-355, Trp-460, and Trp-507) in the channel peptide were classified as shallow (0-5.0 A from C-2 carbon). The remaining 9 Trp residues were classified as moderately buried (5.1-10.0 A). None of the dozen tryptophyls were classified as deeply buried in the membrane bilayer (10.1-15.0 A). A model for the colicin E1 channel based on these measurements along with previous data obtained from proteolysis, chemical labeling, ESR quenching, and mutagenesis experiments is proposed. This model for the closed state of the channel has as its central feature of the presence of only two trans-membrane segments. The membrane-associated portion of the channel includes the hydrophobic membrane anchor domain, Ala-474 to Ile-508. Furthermore, the fluorescence quenching data are consistent with the NH2-terminal helices (helices 1-7) lying on the surface of the membrane with the helical axis being oriented parallel to the membrane plane.
Palmer
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LR
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
Merrill
A R
AR
D000595:Amino Acid Sequence; D003087:Colicins / Q000648:ultrastructure*; D066298:In Vitro Techniques; D007473:Ion Channels / Q000648:ultrastructure*; D008051:Lipid Bilayers; D008081:Liposomes; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000648:ultrastructure; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D003087:Colicins; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D008567:Membranes, Artificial; D014364:Tryptophan