Surface Electrostatics and Lipid-Substrate Interactions of Nanopore-Confined Lipid Bilayers

Abstract

Substrate-supported lipid bilayers serve many purposes: from acting as versatile models of cellular membranes to biotechnological applications including substrate functionalization and stabilizing membrane proteins in functional conformations. While adsorption and subsequent reorganization of phospholipid vesicles on solid substrates were studied in the past, the exact nature of physicochemical interactions between the lipids and substrate surfaces remain largely unknown. Here we employed recently synthesized pH-sensitive spin-labeled phospholipids - derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PTE) with pH-reporting nitroxides that are covalently attached to the lipid's headgroup - to investigate surface electrostatics of nanotubular lipid bilayers confined in cylindrical nanopores. The lipid nanotubes were formed by self-assembling phospholipids inside ordered nanochannels of anodic aluminum oxide with pore diameters from 60 to 170 nm and diameter-to-pore length ratio of up to 1:1000. 31P NMR confirmed formation of macroscopically aligned lipid nanotubes with just 1-2° mosaic spread from zwitterionic DMPC, anionic DMPG, and their mixtures. Interfacial potentials were measured by carrying out titration experiments and observing the protonation state of the nitroxide tag by EPR. For nanopore-confined DMPC:DMPG (1:1) bilayers the protonation equilibrium was shifted to more acidic values: when the single lipid bilayer was deposited per nanopore the pKa of the nitroxide probe was shifted by (−0.91±0.05) pH units but only by (−0.34±0.05) when three bilayers per nanopore were present. Notably, the nitrogen hyperfine coupling constant for non-protonated nitroxides remained the same in all the samples indicating essentially the same interfacial dielectric environment. Thus, these shifts in pKa must come from changes in the lipid bialyer surface potential that was estimated to increase by 52±3 mV. EPR data on the lipid-substrate interface were combined with differential scanning calorimetry to elucidate effects of pore curvature, surface modification, and binding of antibacterial peptides on lipid-substrate interactions. Supported by DE-FG02-02ER15354.