Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes.

Dolder, Nicolas; von Ballmoos, Christoph (2020). Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes. ChemBioChem, 21(15), pp. 2219-2224. Wiley-VCH 10.1002/cbic.202000146

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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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