Membrane-inserted colicin E1 channel domain: a topological survey by fluorescence quenching suggests that model membrane thickness affects membrane penetration.
The topography of the closed-state membrane-associated, colicin E1 channel domain was examined using depth-dependent fluorescence quenching to determine the membrane location of various single Trp residues introduced into the sequence by site-directed mutagenesis. We have extended previous studies (Palmer, L. R., and Merrill, A. R. (1994) J. Biol. Chem. 269, 4187-4193) with additional single Trp residues in the helix 8/9 region, and with an additional quencher located in the polar region of the membrane to detect shallowly located Trp residues. Quenching data for seven single Trp mutants examined in the previous study, but without the shallow quencher, confirmed the previously reported depths. Mutants containing single Trp at residues 355, 460, or 507 were found to be more shallowly located than those at 404, 443, 484, or 495. In addition, analysis of fluorescence in the presence of the shallow quencher eliminated the possibility that there is a predominant population of these residues residing near the membrane-aqueous interface. The fluorescence quenching of three new single Trp at residues 478, 492, or 499 introduced into the channel domain was also evaluated. These residues were found at either medium or deep locations in the bilayer. Of special interest was the position of the Trp at residue 492 (W492), which is within the loop region connecting hydrophobic helices 8 and 9. If helices 8 and 9 were fully transmembraneous, then the predicted W492 location would have been shallow. Instead the quenching pattern demonstrated W492 to be deeply embedded in the lipid bilayer. We also studied the effect of altering bilayer width on protein conformation. Membrane width had little effect on most residues, but Trp at residues 478 and 507 were located more shallowly in thin bilayers. We also examined the effect of bilayer width on the position of Cys 505 labeled with bimane, an environmentally sensitive fluorophore. As the membrane width was decreased, C505-bimane shifted into a more nonpolar environment, as judged by fluorescence emission lambda max and quenching. Models for the conformation of helices 8/9 and the effect of membrane width on these helices are considered. We conclude that helices 8 and 9 probably do not adopt a fully transmembraneous state under the conditions examined in this report.
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215, USA.
S E
SE
Malenbaum
A R
AR
Merrill
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E
London
D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry; D003087:Colicins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010539:Permeability; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D014357:Trypsin / Q000737:chemistry
D019086:Bridged Bicyclo Compounds, Heterocyclic; D003087:Colicins; D008051:Lipid Bilayers; D013113:Spin Labels; C021149:bimanes; D014357:Trypsin; D003545:Cysteine