Abstract
Accurate real-time measurements of the dynamics of proton concentration gradients are crucial for detailed molecular studies of proton translocation by membrane-bound enzymes. To reduce complexity, these measurements are often carried out with purified, reconstituted enzyme systems. Yet the most paramount problem to detect pH changes in reconstituted systems is that soluble pH reporters leak out of the vesicle system during the reconstitution procedure. This requires loading of substantial amounts of pH-sensors into the lumen of unilamellar liposomes during reconstitution. Here, we report the synthesis and detailed characterisation of two lipid-linked pH sensors employing amine-reactive forms of seminaphthorhodafluors (SNARF®-1 dye) and rhodamine probes (pHrodo™ Red dye). Lipid-conjugation of both dyes allowed for efficient detergent-based reconstitution of these pH indicators into liposomes. Vesicle-embedded pHrodo™ displayed excellent photostability and an optimal pH-response between 4 and 7. The suitability of the lipid-linked pHrodo™ probe as a pH reporter was demonstrated by assaying the activity of a plant plasma membrane H(+)-ATPase (proton pump) reconstituted in proteoliposomes.
Highlights
Cells and organelles depend on electrochemical gradients across their membranes
Under the reaction conditions used, the SNARF-NHS ester reacts readily with the amino group of DOPE yielding SNARF-DOPE; the aryl acetyl group was removed due to the known susceptibility of aryl acetates toward bases (Fig. 1C; Electronic supplementary information (ESI), Fig. S1†)
In this study we describe the synthesis of two lipid-linked pH sensors, and demonstrate their application to monitor pH changes in reconstituted liposomal systems
Summary
Cells and organelles depend on electrochemical gradients across their membranes. Membrane-embedded biological pumps are key players in maintaining these gradients through primary active transport of ions, but the precise mechanisms underlying electrogenic transport are not yet completely understood. Lipid-conjugated fluorescent pH sensors for monitoring pH changes in reconstituted membrane systems†
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