Open AccessInteracion of the colicin‐A pore‐forming domain with negatively charged phospholipds
Author(s) -
GONZÁLEZMAÑAS Juan M.,
LAKEY Jeremy H.,
PATTUS France
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb17590.x
Subject(s) - fluorescence , chemistry , vesicle , phospholipid , colicin , quenching (fluorescence) , liposome , molecule , egg lecithin , intramolecular force , bromine , phosphatidylcholine , photochemistry , fluorescence spectroscopy , crystallography , biophysics , stereochemistry , membrane , organic chemistry , biochemistry , biology , physics , escherichia coli , quantum mechanics , gene
The interacion of colicin‐A thermolytic fragment with negatively charged liposmes was studied by flurescence spectroscopy. 1,2‐Dioleoyl‐sn‐glycero‐3phospho‐1‐sn‐glycerol (Ole 2 GroPGro) containing liposomes do not dinginficantly alter the fluorescene properties of the protein, and thus cannot ginve much ingormaion about this interacion. 1,2‐Bis(9,10‐dibromooleoyl‐sn‐glytcero‐3‐phospho‐1‐sm‐glycerol (Br 4 Ole 2 Gor) is easily synthesized by addition of bromine atoms to the double nond located at the mid‐point of the fatty‐acid acyl chain of Ole 2 GroPGro. The brominated phospholipid forms vesicles that strogly quench the protein fluorescence emission. The results presented here show that conversion of Ole 2 GroPGro does not change either the affinity for the protein or the extent of lipid binding. This observation allwes for the estimation of the distribution of the quenchin gphospholipd milecules around the fluorophores [Yeager, M. D. & Feigenosn G. W. (1990) Biochemistry 29 , 4380–4392]. Binding of the protein to the vesicles is an itteversible process, since inseted milecules do not dissociate from the vesicle. From steadeystate measurements, it can be concluded that in the membrane‐bound form, the tryptophans are located within quenching distance of the bormine atoms, i.e. close to the lipid head‐graup/hydrocarbon boundary, completely accessible to the quencher, protected from the polar phase and that the maximum number of phospholipid milecules in cntact with the fluorescent domian of the protein is nine.