Effect of Silica Support on Electrostatics at Membrane-Water Interface and Lipid-Protein Interface by EPR-Active Molecular pH Probe

Polarity, electrostatic potentials, and hydration are the major physico-chemical characteristics of lipid membranes that govern membrane-protein and protein-protein interactions as well as transport of small molecules through cellular membranes. At the membrane-water interface, the surface electrostatic potential of a lipid bilayer plays a fundamental role in such key processes of cellular functioning as endo- and exocytosis, membrane fusion and cellular division to name a few. At the lipid-protein interface, local dielectric constant of biomembranes determines stability, folding, and aggregation of membrane receptor proteins that are involved in a myriad of cellular functions. Some of the properties of cellular membranes could be mimicked by supported lipid bilayers (SLB) that serve as very useful model membrane platforms with the lipid bilayer providing a biocompatible interface and the solid support allowing for manipulation of lipid bilayer properties in a controllable manner. Such membrane-mimicking systems are considered to be the promising candidates for a number of biomedical and biotechnological applications. At present, little is understood about the influence of nanostructured support and the nanoconfinement on the properties of the membrane at membrane-water interface and proteinlipid interface. This PhD thesis project reports on employing EPR-active pH sensitive probes to assess the surface electrostatics and to profile a heterogeneous dielectric environment along a transmembrane peptide incorporated into both unsupported unilamellar lipid vesicles and the lipid membranes formed on the surface of silica beads.;EPR titration of spin-labeled pH-sensitive lipids allows us to experimentally determine the terms contributing to the interfacial of the lipid bilayers and calculate the magnitude of the surface electrostatic potential. By using this experimental approach we have investigated the effect of the lipid composition and silica support on the membrane surface potential. EPR titration experiments of spin labeled transmembrane peptide WALP with symmetric positions of the nitroxide-labeled sidechains with respect to the bilayer center revealed two sequential proton dissociations characterized by two pKai values that yielded local dielectric constant as a function of the label position, i.e., a profile of the dielectric constant along the protein-lipid interface using single and double labeled WALP mutants effects of the lipid composition and silica support on the effective pKai of the ionizable sidechain of the transmembrane peptide was examined. Supported lipid bilayer dynamics was also characterized by mobility and order parameters derived from spin label EPR spectra. It was observed that local rotational dynamics of spin-labeled lipids is affected by the silica support resulting in an increased rotational correlation time and a higher ordering of the lipids in the bilayer. Molecular accessibly of the bilayer surface and specific sites of the transmembrane WALP peptide was assessed by an EPR assay based on a reduction of EPR-active nitroxides to an EPR silent hydroxylamine upon reactions with hydrophilic ascorbate/ascorbic acid. In conclusion, the EPR-active pH-sensitive nitroxide spin labels allow for an expansion of the existing arsenal of experimental EPR methods for assessing local electrostatics and dielectric properties of nanoscale heterogeneous systems and are fully suitable for studies of hybrid nanomaterials/nanosystems composed of inorganic nanoscale materials and lipids that are capable of self-assembly.