A single-liposome assay that enables temperature-dependent measurement of proton permeability of extremophile-inspired lipid membranes

Anirvan Guha^a, Melissa L. McGuire^a, Geoffray Leriche^b, Jerry Yang^b, Michael Mayer^a

Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland.

Abstract

Thermoacidophilic archaea are able to survive in low pH environments by maintaining large proton gradients across their cell membranes. The unique structure of the unipolar and bipolar lipids that compose these membranes, with branched isoprene chains and ether linkages, may contribute to their reduced permeability to protons at elevated temperature. Here we present a proton permeability assay that interrogates hundreds of surface-tethered single liposomes in parallel using total internal reflection fluorescence (TIRF) microscopy to determine a proton permeability value for each liposome. The large number of liposomes that can be individually interrogated by this microscopy platform allow for the collection and statistical analysis of large quantities of data within a single experiment, as opposed to the single, averaged result produced by a bulk fluorimetry assay. In addition, tethering individual liposomes to a surface prevents liposome aggregation in free solution, which is especially prevalent when performing studies at elevated temperatures. Using a temperature-controlled chamber, we demonstrated a decreased temperature-dependence of proton permeability for bipolar archaea-mimetic membranes in comparison to unipolar eukaryote-mimetic membranes. Eyring-Polanyi analysis revealed a high entropic cost associated with proton flux across the archaea-mimetic membranes. Furthermore, analysis of the distribution of proton permeability values within populations of liposomes revealed a positive correlation between size and permeability in the eukaryote-mimetic liposomes that was absent in the archaea-mimetic liposomes.

Keywords: Proton permeability, Liposome, Total internal reflection fluorescence microscopy, Extremophile archaea.