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Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Dolder Nicolas, Ballmoos Christoph,
Project Functional investigations of bacterial and eukaryotic membrane proteins
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Original article (peer-reviewed)

Journal ChemBioChem
Publisher Wiley
Volume (Issue) 21(15)
Page(s) 2219 - 2224
Title of proceedings ChemBioChem
DOI 10.1002/cbic.202000146

Abstract

Enzyme‐mediated proton transport across biological membranes is critical for many vital cellular processes. pH‐sensitive fluorescent dyes are an indispensable tool for investigating the molecular mechanism of proton‐translocating enzymes. Here, we present a novel strategy to entrap pH‐sensitive probes in the lumen of liposomes that has several advantages over the use of soluble or lipid‐coupled probes. In our approach, the pH sensor is linked to a DNA oligomer with a sequence complementary to a second oligomer modified with a lipophilic moiety that anchors the DNA conjugate to the inner and outer leaflets of the lipid bilayer. The use of DNA as a scaffold allows subsequent selective enzymatic removal of the probe in the outer bilayer leaflet. The method shows a high yield of insertion and is compatible with reconstitution of membrane proteins by different methods. The usefulness of the conjugate for time‐resolved proton pumping measurements was demonstrated by using two large membrane protein complexes.
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