Abstract
Proton gradients are utilized by cells to power the transport activity of many membrane proteins. Synthetic cells, such as proteo-giant unilamellar vesicles, offer an advanced approach for studying the functionality of membrane proteins in isolation. However, understanding of protein-based transport in vitro requires accurate measurements of proton flux and its accompanying electrochemical gradient across the lipid bilayer. We present an approach to directly quantify the flux of protons across single cell-sized lipid vesicles under modulated electrochemical gradients. Our measurements reveal the corresponding association between proton permeation and transmembrane potential development and its relation to the chemical nature of the conjugated anion (base). In the case of formic acid, we showed that, out of the total amount of permeated protons, a fraction of ≈0.2 traverse the lipid bilayer as H+, with the rest (≈0.8) in the form of a neutral acid. For strong acids (HCl or HNO3), proton permeation was governed by translocation of H+. Accordingly, a larger proton motive force (pmf) was generated for strong acids (pmf=14.2 mV) relative to formic acid (pmf=1.3 mV). We anticipate that our approach will guide the development of protein-based transport driven by proton gradient in artificial cell models and enable a deeper understanding of how vital acids, such as fatty acids, amino acids, bile acids, and carboxylic acid-containing drugs, traverse the lipid bilayer.