Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes |
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Authors: | Nicolas Dolder Prof?Dr Christoph von Ballmoos |
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Affiliation: | Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland |
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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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Keywords: | carboxyfluorescein DNA conjugates liposomes membrane proteins pH sensors |
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