Selective evaluation of surface potentials of the inner and the outer lipid bilayer leaflets by epr of a ph-sensitive nitroxide-modified phospholipid selective evaluation of surface potentials of the inner and the outer

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  • Publicado : 28 de noviembre de 2011
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Selective Evaluation of Surface Potentials of the Inner and the Outer Lipid Bilayer Leaflets by EPR of a pH-Sensitive Nitroxide-Modified Phospholipid

Electrostatic potential of lipid bilayers determines local concentrations of ions in the vicinity of the membrane surface and governs electrostatic interactions between oppositely charged protein residues and the membrane - the process involvedin regulating essential cell functions. Local electric fields in phospholipid assemblies can be assessed by placing a molecular probe, such as a stable nitroxide containing an ionizable functionality, at a specific location with respect to the bilayer interface, and measuring the pKa value of the nitroxide by EPR spectroscopy. Here we describe the use of a synthetic phospholipid with a pH-sensitivenitroxide attached to the polar head group to distinguish surface electrostatics of the inner and outer leaflets of the phospholipid bilayer. This spin labeled-lipid (SSL) allows for positioning of the reporter nitroxide directly at the lipid bilayer-water interface. Such a pH-sensitive nitroxide tag reports on the local electrostatics in two different ways. Firstly, local tumbling of the labelis affected by the charge developed on the protonatable group. Secondly, magnetic parameters of the nitroxide also change upon reversible protonation. Here we report on i) a method to selectively introduce a SLL into the outer leaflet of the phospholipid vesicle, ii) a method to distinguish between the SLLs incorporated into the inner and outer leaflets, and iii) EPR titration studies of SLL invesicles prepared from anionic POPG lipids. EPR titration experiments were performed at 0 oC to prevent the lipid flip-flop between the leaflets.


Introduction
The membranes are primarily composed of lipids and proteins that are responsible for many aspects of cellular function, including cell adhesion, signaling, and transport of ions and other compounds in and out of the cells. Many ofthese functions are thought to be modulated

by electrical charges of the lipid head groups and the associated diffuse double-layer potential that is formed in the aqueous phase adjacent to the membrane. Thus, electrostatic properties of lipid bilayers are considered to be directly involved in such fundamental phenomena as insertion of proteins and viruses into the membranes, interaction withDNA, and fusion of the vesicles and membranes. Experimental methods capable to accurately measuring the membrane surface potential at well-defined sites would allow one to gain a detailed knowledge on electrostatic phenomena at the lipid bilayer interface.
Several analytical methods, including fluorescent spectroscopy,1 NMR,2 and spin probe EPR3,4 can be used to evaluate electrostaticproperties of the lipid bilayer surfaces. Spin probe EPR methods are based on the use of molecular probes – persistent organic molecules bearing unpaired electron – and provide a unique opportunity to gain structural and dynamic information precisely from the location of the molecular probe.
Surface electrostatics and local pH in lipid membranes and protein-lipid complexes can be observed from changesin EPR (Electron Paramagnetic Resonance) spectra of pH-sensitive nitroxide spin probes positioned at the lipid bilayer interface. Nitroxides are stable free radicals that possess an unpaired electron on the π orbital of the N-O bond. Some nitroxides, containing ionizable groups (pH-sensitive spin probes) were shown to be a valuable tool to study the proton transfer-related phenomena and localelectric fields in macromolecules and molecular assemblies, due to strict dependence of EPR spectral parameters on reversible protonation.5 The pKa of such a nitroxide positioned at the lipid bilayer-water interface is determined by properties of the local environment, such as surface electrostatic potential and the effective interfacial dielectric constant.
Spin-labeled phospholipids, bearing...
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