A study of the structure and dynamics of complexes between polymyxin B and phosphatidylglycerol in monolayers by fluorescence

Abstract

Interactions between the antibiotic polymyxin B and monolayers of dipalmitoylglycerophosphoglycerol have been reinvestigated through a study of the structure and dynamics of the complexes by means of an interface fluorimeter of our fabrication. A fluorescence technique has been developed where the use of linearly polarized incident beams gives the simultaneous determination of the orientation and the lateral diffusion rate of a fluorescent probe inserted in the film. The present investigation was carried out with 12-(9-anthroyloxy)-stearic acid, a fluorescent compound which forms non-fluorescent photodimers upon illumination. Orientation of the probe was studied by computing the ratio of the two dimerization constants K,, and the ratio of the fluorescence intensities obtained with crossed linearly polarized incident lights. The lateral diffusion rate of the probe was obtained by measuring fluorescence recovery after photobleaching (photodimerization) of the probe. Control experiments, carried out with dimyristoylglycerophosphocholine, a lipid which does not interact with polymyxin B, show that the antibiotic does not significantly modify the behaviour of the probe. Both in terms of orientation and dynamics, with respect to dipalmitoylglycerophosphoglycerol, when the antibiotic is present in the subphase (1 pM, saturating conditions), data indicate that the lipid remains in a liquid-expanded state. This is true even at a high surface pressure ( T C Z 37 mN 'm-'), above the apparent 'transition' which can be observed at 30-35 mN .m-' on its compression isotherm. Computation of the contribution of polymyxin B to the film expansion leads to the conclusion that this 'transition' would be a structural transition between two models of interaction: one, below the 'transition', where the polypeptide ring penetrates between the film-forming lipid molecules and another one, above the 'transition', were the antibiotic is adsorbed at the lipid-water interface with only its hydrocarbon chain penetrating the film.Polymyxins are antibiotics isolated from various strains of Bacillus polymyxa, the activity of which is directed mainly against gram-negative bacteria [I]. Polymyxins consist of one fatty acid residue attached through an amide bond to a linear tripeptide linked to a heptapeptide ring. The presence of five or six 2,4-diaminobutyric acid residues confers a net positive charge to these molecules. It is now well recognized that the primary site of action of these antibiotics is the bacterial membrane [I]. As shown by studies carried out with lipids in membrane models, polymyxin B displays a marked preference for acidicphospholipids [I -71. Its interactions are directed by both electrostatic and hydrophobic forces [I].In the case of phosphatidylglycerol, monolayer studies have shown that, at saturation, these interactions achieve a phosphatidylglycerol-polymyxin B complex in the molar ratio 5:l [4]. No interaction was detected with the zwitterionic phosphatidylcholine [2 -41. In bilayer systems and in nonsaturating conditi...

Lateral proton conduction at a lipid/water interface. Effect of lipid nature and ionic content of the aqueous phase

“…This is found at the end of the liquid-expandedlliquid-condensed plateau where we observed a decrease in lateral diffusion and a reorientation of the fatty acid chains in Pam2GroPGro monolayers [20]. As far as the polar moiety of Pam2GroPCho is concerned, there are few reports on monolayer models.…”

“…Phase transitions of lipids (Pam2GroPCho, Pam2Gro-PGro) induce the abolishment of the facilitated lateral proton diffusion when the matrix is in the gel state ( Figs 5 and 6). This is found at the end of the liquid-expandedlliquid-condensed plateau where we observed a decrease in lateral diffusion and a reorientation of the fatty acid chains in Pam2GroPGro monolayers [20]. As far as the polar moiety of Pam2GroPCho is concerned, there are few reports on monolayer models.…”

Endotoxins: Relationships between Structure, Function, and Activity

“…A rapid increase of membrane permeability with respect to charged or polar molecules of low molecular weight [74] and visible morphological alterations have been observed [75]. The action of PMB has been in the focus of numerous studies [76][77][78][79][80][81][82][83][84][85][86][87][88][89]. Strong interactions have been observed with LPS [75,90] as should be expected from the polycationic character of the antibiotic.…”

Evidence for a homogeneous lateral distribution of lipids in a bacterial membrane

“…Structure and organization of biomembranes are certainly very subtle and complex and new tools are required for describing the membrane assembly at a microscopic scale, both in terms of organization and dynamics of the various components. Remembering that anthracene is fluorescent and that its photo-dimers are not [lo], it is worth noting that this group can be used not only for topological studies of lipids in membranes but also for investigating their lateral diffusion rate [22,23].…”