Interaction of the colicin-A pore-forming domain with negatively charged phospholipids.
The interaction of colicin-A thermolytic fragment with negatively charged liposomes was studied by fluorescence spectroscopy. 1,2-Dioleoyl-sn-glycero-3-phospho-1-sn-glycerol (Ole2GroPGro) containing liposomes do not significantly alter the fluorescence properties of the protein, and thus cannot give much information about this interaction. 1,2-Bis(9,10-dibromooleoyl-sn-glycero-3-phospho-1-sn-glycerol (Br4Ole2GroPGro) is easily synthesized by addition of bromine atoms to the double bond located at the mid-point of the fatty-acid acyl chain of Ole2GroPGro. The brominated phospholipid forms vesicles that strongly quench the protein fluorescence emission. The results presented here show that conversion of Ole2GroPGro to Br4Ole2GroPGro does not change either the affinity for the protein or the extent of lipid binding. This observation allows for the estimation of the distribution of the quenching phospholipid molecules around the fluorophores [Yeager, M. D. & Feigenson G. W. (1990) Biochemistry 29, 4380-4392]. Binding of the protein to the vesicles is an irreversible process, since inserted molecules do not dissociate from the vesicle. From steady-state measurements, it can be concluded that in the membrane-bound form, the tryptophans are located within quenching distance of the bromine atoms, i.e. close to the lipid head-group/hydrocarbon boundary, completely accessible to the quencher, protected from the polar phase and that the maximum number of phospholipid molecules in contact with the fluorescent domain of the protein is nine.
González-Mañas
J M
JM
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Lakey
J H
JH
Pattus
F
F
D001966:Bromine; D003087:Colicins / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004563:Electrochemistry; D005454:Fluorescence Polarization; D008081:Liposomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism*; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000235:genetics
D003087:Colicins; D008081:Liposomes; D010446:Peptide Fragments; D010715:Phosphatidylglycerols; D010743:Phospholipids; D013912:Threonine; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D003545:Cysteine; D001966:Bromine