Functional Characterization of the Urea Transporter UreI from Helicobactor Pylori
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More than 50% of the world's population is infected with Helicobactor pylori, a pathogenic bacterium responsible for numerous gastroduodenal disorders such as chronic gastritis, peptic ulcer disease, and gastric cancer. A small proton-gated inner membrane channel HpUreI represents H.pylori's life insurance. It ensures survival in the acidic gastric juice, by means of urea transport from the periplasm to the cytoplasm, where urea is hydrolyzed by urease. In turn, the hydrolysis products ammonia and carbon dioxide buffer the periplasm to pH 6.1. MD simulations suggested that water is transported along with urea through the channel. We tested that hypothesis by estimating the unitary transport rates for both molecules. To this end we overexpressed HpUreI in E.coli, purified the transporter and reconstituted the protein into large unilamellar vesicles (LUVs). We observed water flux by osmotically deflating the vesicles or inducing urea transport under isosmotic conditions at neutral and acidic conditions. We extracted the unitary transport rates from the intensity of scattered light by (i) extending our recent treatment of the Rayleigh-Gans-Debye equation for solvent transport (1) across vesicular membranes to solute transport and (ii) determining the number of reconstituted transporters (2) and their oligomeric state (1) with fluorescence correlation spectroscopy before and after detergent-mediated vesicle solubilization. In addition we confirmed the hexameric arrangement by acquiring high-speed AFM images of the proteoliposomes spread on mica.
(1) Horner A, et al. (2015) The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues. Science Advances 1(2):e1400083. (2) Hoomann T, Jahnke N, Horner A, Keller S, & Pohl P (2013) Filter gate closure inhibits ion but not water transport through potassium channels. Proc. Natl. Acad. Sci. U. S. A. 110(26):10842-10847.