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Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes
Author(s) -
Dolder Nicolas,
Ballmoos Christoph
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000146
Subject(s) - oligomer , chemistry , liposome , moiety , biophysics , lipid bilayer , bilayer , dna , conjugate , membrane , bifunctional , biological membrane , biochemistry , combinatorial chemistry , stereochemistry , organic chemistry , biology , mathematical analysis , mathematics , catalysis
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.