title: Fluorescence energy transfer distance measurements. The hydrophobic helical hairpin of colicin A in the membrane bound state.
abstract: The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this study fluorescence resonance energy transfer spectroscopy was used to determine the position of this helical hairpin in the membrane bound state. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulpho-1-naphthyl)ethylenediamine (I-AEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Five mutants I26C (helix 1), F105C (between helices 4 and 5), G166CJ (helix 8), A169C (helix 8-9), G176C (helix 9) were used. All mutants show wild-type binding activity to phosphatidylglycerol vesicles as judged by fluorescence polarization anisotropy, emission wavelength changes and brominated lipid quenching. The three tryptophan residues were used as a compound donor to AEDANS in resonance energy transfer distance determinations. The distances obtained for the soluble form were equal to those found in the crystal structure. On adding vesicles under conditions where intermolecular transfer was avoided the indicated distances increased; I26(10.9 A) F105(3.4 A), G166(3.3 A), A169(1.9 A) and G176(2.9 A). This confirms that, in the absence of a membrane potential, helices 1 and 2 open out onto the membrane surface whilst the helical hairpin remains closely packed against the rest of the structure. The insertion of this hairpin is thus not the driving force behind colicin membrane binding.
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, F.R.G.
authors.forename: J H
authors.initials: JH
authors.lastname: Lakey
authors.forename: D
authors.initials: D
authors.lastname: Duché
authors.forename: J M
authors.initials: JM
authors.lastname: González-Mañas
authors.forename: D
authors.initials: D
authors.lastname: Baty
authors.forename: F
authors.initials: F
authors.lastname: Pattus
mesh_terms: D003087:Colicins / Q000737:chemistry* / Q000235:genetics; D005454:Fluorescence Polarization; D007473:Ion Channels / Q000737:chemistry; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates; D010715:Phosphatidylglycerols; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012995:Solubility; D014867:Water / Q000737:chemistry
chemical_list: D003087:Colicins; D007473:Ion Channels; D008565:Membrane Proteins; D009282:Naphthalenesulfonates; D010715:Phosphatidylglycerols; D014867:Water; C006421:1,5-I-AEDANS; C002773:dimyristoylphosphatidylglycerol