Electrostatics at Peptide-Lipid Interface in Nano-Bio Hybrid Systems by Spin-Labeling EPR

Abstract

The ionization states of individual amino acid residues of membrane proteins are difficult to decipher or assign directly in the lipid membrane environment. The effective pK(a) values of protein groups are determined by a complex interplay between local polarity, Coulomb interactions, and a structural reorganization. The analysis is further complicated by the dearth of information about gradients in polarity, electric potentials, and hydration at the protein-membrane interface. The goal of our work is to develop spin-labeling EPR methods for assessing effects of membrane surface potential, local environment at the protein-membrane interface, and water penetration along this interface on effective pK(a) of membrane-burred ionisable groups. In this work we report on developing pH-sensitive ionizable EPR labels and related methods to 1) profile a heterogeneous dielectric environment along the α-helix of a WALP peptide integrated in a lipid bilayer; 2) asses the effect of solid support, specifically silica nanoparticles, on effective pK(a) of membrane-burred ionisable sidechains; and 3) use DEER to assess the location of spin-labeled sidechains. pH-sensitive EPR labels were attached to cysteine residues positioned at various depth within lipid bilayers at the peptide-lipid interface. We have shown that effective pK(a) of membrane-burred sidechain can be significantly shifted by varying the membrane surface charge density. The silica support exerted pronounced effects on WALP dynamics and the effective pK(a) of the ionizable probe. It was demonstrated that the silica nanoparticles shift the effective pK(a) of the ionizable nitroxide probe in a membrane depth-dependent manner. Upon protonation of the membrane-burred model ionisable sidechain the silica support caused significant changes in the membrane association of WALP peptide that is not observed when WALP is integrated into unilamellar phospholipid vesicles of similar curvature.Supported by NSF 1508607 to TIS.