Substrate-supported lipid bilayers attract an interest as models of cell membranes as well as self-assembled hybrid nanostructures for various biotechnological applications. Despite extensive ongoing efforts, incorporation of membrane proteins into such hybrid lipid-based systems remains largely problematic. We hypothesize that nano-confinement and solid support of lipid membranes can alter lipid or membrane protein dynamics as well as physico-chemical characteristics of the membrane-lipid interface. In this work we report on employing pH-sensitive ionizable EPR labels to profile a heterogeneous dielectric environment along the α-helix of a transmembrane WALP peptide integrated into lipid bilayers formed on the surface of silica microbeads. Nitroxide EPR spin labels were attached to two cysteine residues positioned equidistant from the center of the peptide, thus, ensuring symmetric location of the labels with respect to the bilayer center. Spin-labeled WALP was incorporated into large unilamellar vesicles and substrate-supported lipid bilayers were prepared via a spontaneous fusion upon mixing of liposomes containing spin labeled WALP and an aqueous suspension of silica beads. The change in the protonation state of the nitroxide was directly observed by EPR. Q-band (35 GHz) double electron-electron resonance (DEER) experiments were carried out to determine the distance between spin labels when embedded in lipid bilayers to provide information about the label location. Two pH sensitive spin labels, S-4-(4-(dimethylamino)-2-ethyl-5,5-dimethyl-1-oxyl-2,5-dihydro-1H-imidazol-2-yl) benzylmethanethiosulfonate (IKMTSL) and methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethyl-imidazolidin-4-ylmethyl) ester (IMTSL), were used to expand the pH range of the titration experiments. We have observed a systematic shift in the depth profile of the effective pKa of the EPR probe at the WALP-lipid interface for silica-supported system as compared to data obtained for WALP incorporated in large unilamellar lipid vesicles. We have shown that i) negative charges on the silica surface stabilize the protonated form of the EPR probe and ii) altering pH conditions is required for protonation of the ionisable protein side chains at the membrane-lipid interface. In addition, water penetration at the peptide-membrane interface was assessed by a hyperfine sublevel correlation spectroscopy (HYSCORE) experiment. Supported by NSF- 1508607 to TIS.

Profiling the dielectric constant at the membrane-peptide interface of silica- nanoparticle-supported lipid bilayer using ionizable EPR probes

251st National Meeting (2016)

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