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Polymyxin interaction with negatively charged lipid bilayer membranes and the competitive effect of Ca2+
Abstract:
The binding of cationic polymyxin-B to negatively charged phosphatidic acid and phosphatidylglycerol membranes has been investigated by fluorescence polarization study. Competition experiments with Ca2+ were performed. 1. Binding of polymyxin-B to mixed dipalmitoylphosphatidic acid/distearoylphosphatidylcholine membranes leads to a phase separation. Domains of polymyxin-bound phosphatidic acid are formed. 2. Ca2+ is found to be a strong competitor in displacing polymyxin from the complex in the mixed membrane system. Complete displacement is obtained at pH 9.0. With decreasing pH value, Ca2+ becomes a less strong competitor and is ineffective at pH 5.0. 3. Binding of polymyxin to dipalmitoylphosphatidylglycerol membranes is observed. Incorporation of polymyxin lowers the lipid phase transition by 10 degrees C. One polymyxin is found to bind five phosphatidylglycerol molecules. The binding curve is determined and in contrast to phosphatidic acid membranes, a noncooperative binding could be established. 4. Addition of Ca2+ decreases the amount of phosphatidylglycerol bound to polymyxin by about 20%. No complete displacement is achieved even at 10-fold excess of Ca2+ with respect to phosphatidylglycerol.
Insights
Polymyxin-B binding to lipid membranes causes phase separation and alters lipid transitions. Calcium ions compete with polymyxin-B binding, with effectiveness varying by pH and lipid type.
Area of Science:
- Biochemistry
- Membrane Biophysics
- Pharmacology
Background:
- Polymyxin-B is a cationic antibiotic that interacts with bacterial membranes.
- Understanding its binding to anionic phospholipids is crucial for its mechanism of action.
Purpose of the Study:
- To investigate the binding of polymyxin-B to phosphatidic acid and phosphatidylglycerol membranes.
- To explore the competitive binding of calcium ions (Ca2+) with polymyxin-B.
Main Methods:
- Fluorescence polarization studies were employed to analyze binding.
- Competition experiments with Ca2+ were conducted at varying pH levels.
Main Results:
- Polymyxin-B binding induced phase separation in mixed phosphatidic acid/phosphatidylcholine membranes, forming polymyxin-bound phosphatidic acid domains.
- Ca2+ effectively displaced polymyxin-B from mixed membranes at pH 9.0 but was ineffective at pH 5.0.
- Polymyxin-B binding to phosphatidylglycerol membranes lowered the lipid phase transition temperature by 10°C, with a noncooperative binding stoichiometry of 1:5 (polymyxin:phosphatidylglycerol).
- Ca2+ reduced phosphatidylglycerol binding to polymyxin-B by approximately 20% without complete displacement.
Conclusions:
- Polymyxin-B binding to anionic phospholipids is concentration-dependent and can induce membrane domain formation.
- Calcium ions act as competitors for polymyxin-B binding, with pH-dependent efficacy.
- Polymyxin-B incorporation significantly affects the biophysical properties of phosphatidylglycerol-containing membranes.