Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membrane-bound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic α-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic α-helix (3.7 ± 0.1 residues per turn and 97 ± 3.0°, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin. Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membrane-bound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic α-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic α-helix (3.7 ± 0.1 residues per turn and 97 ± 3.0°, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin. The colicins are a family of antimicrobial proteins that are secreted by Escherichia coli strains under environmental stress, because of nutrient depletion or overcrowding, and these proteins often target sensitive bacterial strains (1Pugsley A.P. Microbiol. Sci. 1984; 1: 203-205PubMed Google Scholar). The lethal actions of colicins against their target cells are manifested in a number of different modes that include the following: (i) formation of depolarizing ion channels in the cytoplasmic membrane, (ii) inhibition of protein and peptidoglycan synthesis, and (iii) degradation of cellular nucleic acids (1Pugsley A.P. Microbiol. Sci. 1984; 1: 203-205PubMed Google Scholar, 2Cramer W.A. Dankert J.R. Uratani Y. Biochim. Biophys. Acta. 1983; 737: 173-193Crossref PubMed Scopus (77) Google Scholar, 3Stroud R.M. Reiling K. Wiener M. Freymann D. Curr. Opin. Struct. Biol. 1998; 8: 525-533Crossref PubMed Scopus (64) Google Scholar, 4Lakey J.H. Slatin S.L. Curr. Top. Microbiol. Immunol. 2001; 257: 131-161PubMed Google Scholar, 5Gillor O. Kirkup B.C. Riley M.A. Adv. Appl. Microbiol. 2004; 54: 129-146Crossref PubMed Scopus (105) Google Scholar, 6Cramer W.A. Heymann J.B. Schendel S.L. Deriy B.N. Cohen F.S. Elkins P.A. Stauffacher C.V. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 611-641Crossref PubMed Scopus (182) Google Scholar, 7Riley M.A. Wertz J.E. Annu. Rev. Microbiol. 2002; 56: 117-137Crossref PubMed Scopus (824) Google Scholar). In this context, the bacterial machinery responsible for colicin biological activity feature important mechanisms that are fundamental to various biological processes. These mechanisms include protein receptor binding, membrane translocation, membrane binding and protein unfolding, membrane insertion, voltage-gated ion channel formation, catalysis, and inhibition of enzymes. Colicin E1 is a member of the channel-forming subfamily of colicins and is secreted by E. coli that harbors the naturally occurring colE1 plasmid; the whole colicin consists of three functional segments, the translocation, receptor-binding domains, and channel-forming domains. Initially, the receptor-binding domain (8Brunden K.R. Cramer W.A. Cohen F.S. J. Biol. Chem. 1984; 259: 190-196Abstract Full PubMed Google with the receptor of target cells J. Cramer W.A. J. Biol. Chem. 1998; Full Full PubMed Scopus Google Scholar). receptor the domain with the the of colicin E1 the membrane and into the J.R. Uratani Y. Cramer W.A. M. J. Biol. Chem. 257: Full PubMed Google Scholar). In the the channel domain a to an state and into the cytoplasmic membrane of the an ion The channel the of in the of the and of the target of the cytoplasmic In an to for the membrane by the colicin E1 activity is in the in the of Cramer W.A. Stauffacher C.V. Full Full PubMed Scopus Google Scholar). The is A number of to the of the membrane-bound state of colicin of the surface of the colicin E1 channel to and was that the is to to that in W.A. Schendel Stauffacher C.V. Cohen F.S. Microbiol. Immunol. PubMed Google Scholar). The of the membrane-bound colicin E1 channel is or with the C-terminal to The membrane-associated closed state of the channel was also studied by fluorescence J. Biol. Chem. Full PubMed Google Scholar). the and the quencher The this were with the of in the closed channel and were in with by 259: PubMed Scopus Google an of cysteine of the channel the and of the of the colicin E1 channel were studied by Cramer W.A. PubMed Scopus Google and by J.H. Slatin S.L. Curr. Top. Microbiol. Immunol. 2001; 257: 131-161PubMed Google Scholar). the membrane-bound of the colicin E1 channel was analysis and was to that the a membrane-bound Biophys. J. 1995; Full PubMed Scopus Google Scholar). and J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Biochim. Biophys. Acta. 2002; PubMed Scopus Google a of fluorescence to the membrane of the closed channel and that the channel that are in in one state is the M. Y. Cramer W.A. Sci. 1998; PubMed Scopus Google that the colicin E1 closed channel a helical with of the to the bilayer surface. Y. K. Schendel S.L. Cramer W.A. Sci. 1998; PubMed Scopus Google colicin in and that the data were with a single helical into the bilayer each colicin M. Cramer W.A. PubMed Scopus Google the in the binding and for the colicin channel domain and a for the membrane lipid in the of colicin E1 Cramer W.A. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar). Cramer W.A. 2004; PubMed Scopus Google that the channel activity of colicin E1 was sensitive to the membrane of the lipid bilayer that the channel a toroidal pore in the bilayer. A for into was by Elkins Cramer W.A. Stauffacher C.V. Full Full PubMed Scopus Google of the of the channel of colicin A and In this a of cysteine helix 1 of the colicin E1 channel domain, and the residue with the bimane the of site-directed fluorescence labeling to the membrane-bound of the closed state of the channel domain in membrane a number of and fluorescence properties of the bimane fluorophore tethered at different the channel domain, including fluorescence emission maximum, quantum yield, anisotropy, membrane depth, surface accessibility, and apparent polarity. squares harmonic function analysis of the properties of the tethered bimane at various helix 1 that this α-helix is amphipathic and is the membrane bilayer with face bathing the membrane and face with the solvent. and of E1 channel domain with an quantum of by to that by surface channel-forming domain of colicin E1 (1Pugsley A.P. Microbiol. Sci. 1984; 1: 203-205PubMed Google was a of E. coli the that in of the protein. proteins were and J. Biol. Chem. 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In the periodicity and the angular frequency of the fluorescence were a least squares fit of the data the harmonic function in where a is the is the is the and is an were an by The of the colicin E1 channel domain, is in and the protein including helix 1 in the that was is indicated by the the of helix 1 and also the of the residues are that to the of each residue The channel domain is a of that is of helical that include helix 1 with helix a helical that a the and and the C-terminal helix is that the A of the channel domain are in with the membrane, an important in channel formation Cramer W.A. Stauffacher C.V. Full Full PubMed Scopus Google Scholar). The 1 and is and in helix 1 the of the is to the to to to the the cytoplasmic membrane of the bacterial Cramer W.A. Stauffacher C.V. Full Full PubMed Scopus Google Scholar). of and in the channel domain of colicin A and for of S.L. J.H. 2002; PubMed Scopus Google Scholar). and functional of colicin A and channel that the of the the channel properties of these proteins S.L. J.H. 2002; PubMed Scopus Google Scholar). In the fluorescence a of protein in channel activity analysis to the of the the of cysteine and the bimane labeling the and functional of the protein. membrane binding analysis of the revealed that the for membrane of the channel the channel domain of colicin the binding of to under 0.1 lipid for the protein the activity was J. Biol. Chem. Full Full PubMed Scopus Google Scholar, J.B. Cramer W.A. PubMed Scopus Google Scholar). The binding per and are in with for the colicin channel J. Biol. Chem. Full Full PubMed Scopus Google Scholar, J.B. Cramer W.A. PubMed Scopus Google Scholar). the of mutant proteins and that were with of the that the of the was or that of the channel The in channel of and mutant proteins were also with the channel domain J. Biol. Chem. 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Chem. 1998; Scopus Google Scholar). with are residues with are J. Chem. 1998; Scopus Google Scholar). The apparent of the and the of helix 1 residues show is the apparent the apparent and of residues at the and C-terminal of helix 1 and at least partially the was that the of helix 1 was in the and that the the and of the helix Cramer W.A. Stauffacher C.V. Full Full PubMed Scopus Google Scholar). The apparent of the of the helix 1 by the and the bimane labeling at that the residues at the of helix 1, and are a of a turn 1 and that is that the of the at these because of a a of the and Finally, the fluorescence of bimane attached at these A and is with this under and of Helix the apparent of the membrane-bound mutant The apparent of the mutant proteins and are with residues at the of the is in to the apparent of bimane to the mutant proteins and M. Cramer W.A. J. Biol. 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These are binding helix bilayer M. Cramer W.A. PubMed Scopus Google Scholar). The of the protein with model to the of (i) of the and the of these with to the amphipathic (ii) of helix 1 the of the by the the helical by and and helix and (iii) of the M. Cramer W.A. J. Biol. PubMed Scopus Google Scholar). of colicin E1 upon membrane to the membrane of the and the helical the of an the surface of the A number of a of and in the to the of the membrane-bound colicin channel protein. of the for the closed channel state include a that an helical by a single helical and that a and a state J. Biol. Chem. Full PubMed Google Scholar, 259: PubMed Scopus Google Scholar, J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Biochim. Biophys. Acta. 2002; PubMed Scopus Google Scholar, M. Y. Cramer W.A. Sci. 1998; PubMed Scopus Google Scholar, Y. K. Schendel S.L. Cramer W.A. Sci. 1998; PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar, E. J. 1998; Google Scholar, Cramer W.A. Sci. 1: PubMed Scopus Google Scholar). of these upon a of the of the colicin E1 channel domain, a by of the protein in membrane-bound state is the of and Sci. 8: PubMed Scopus Google where a of single cysteine the of the colicin E1 channel was In that the region, and 1, A and was to an and partially single α-helix of residues including Sci. 8: PubMed Scopus Google Scholar). The site-directed fluorescence labeling data for the amphipathic of helix 1 to the membrane bilayer is the harmonic function analysis of the fluorescence data that the periodicity and angular frequency of helix 1 upon binding to the membrane surface. The data also support the of that that helix 1 the surface of the membrane with face bathing the of the bilayer. In single mutant channel that for was in the membrane and the bilayer and ± the J. Biol. Chem. Full PubMed Google Scholar, J. Biol. Chem. Full Full PubMed Scopus Google Scholar). the of the of the measured is to the helix of this the of the a is with the of the to of the membrane-bound closed channel state of the protein Cramer W.A. Sci. 1: PubMed Scopus Google Scholar). a residue by residue of the and bilayer of helix 1, these and the that of this helix is against or of helix in the closed channel The quencher analysis is a of the bilayer of a and this was to the bilayer penetration of helix The this analysis were in with apparent fluorescence anisotropy, and fluorescence quantum that helix 1 is to the membrane surface and is parallel to the bilayer surface. a model of helix 1 in the closed channel The and the of the channels by colicins upon in In is the of the of that the voltage-gated channel a channel with an of of the and the of the membrane and of of the membrane-bound the of the are the helical by and of the protein. to the of the segments, various of indicated the formation of at least Cramer W.A. PubMed Scopus Google Scholar, J. Biol. Chem. Full Full PubMed Scopus Google Scholar, Cramer W.A. Sci. 1: PubMed Scopus Google Scholar, Slatin S.L. J. PubMed Scopus Google Scholar, S.L. D. D. J. Biol. 2004; PubMed Scopus Google Scholar). of these include an helix by the of 1 and of the one of the helix is an of the of colicin E1 membrane and the of the of the protein in the membrane-bound state Sci. 8: PubMed Scopus Google our data the of 1 and into an helix a feature of the closed channel state of colicin E1 and the of the channel These the toroidal model for the colicin E1 channel by Cramer and Cramer W.A. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar, Cramer W.A. 2004; PubMed Scopus Google and the of in an membrane in to a channel with a of The toroidal lipid pore model to the of activity of antimicrobial Cramer W.A. Biochim. Biophys. Acta. 2004; PubMed Scopus Google and this model a for membrane lipid in the formation of membrane by the important for the toroidal model is the of the activity to membrane lipid where with pore formation of the Cramer and Cramer W.A. 2004; PubMed Scopus Google the of colicin E1 activity membrane to a toroidal pore model for this The harmonic function analysis of our fluorescence data of the periodicity of in the and membrane-bound of the protein. In the of colicin the of helix 1 and the of the turn to that helix 1 helix 1, A and is in channel-forming colicins and is in the turn that helix 1 and in of channel-forming our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the our data that the of of helix 1 and into a single amphipathic helix of the channel are by the the of the channels by the colicin and A S.L. D. D. J. Biol. 2004; PubMed Scopus Google Scholar). In this of proteins that to helix 1 of the colicin were to into the membrane with the helix 1 and a the and of the channel state S.L. D. D. J. Biol. 2004; PubMed Scopus Google Scholar). colicin this is to partially to the of the membrane, and the of helix is the to the and of the and of the In to our of the of the colicin our to residue by residue the membrane of colicin E1 in closed channel state and channel is that this to of these and the that the of this in the to residues the colicin E1 channel domain that the is a and for protein and also for membrane protein D. J. Chem. Scopus Google Scholar, E. M. Y. Chem. Scopus Google Scholar). is to protein and is to for into the membrane bilayer at and in a of also the of and PubMed Scopus Google Scholar, 2002; PubMed Scopus Google in the of the fluorescence of bimane to the of membrane-associated In our that the bimane fluorescence and quantum that to a these is our that these in the to the of the of the colicin E1 channel domain in the membrane-bound state by various including fluorescence for in the of this

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