Membrane proteins (MPs) are the gatekeepers between different biological compartments separated by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing number of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extraction and purification of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the experimental system. In this review, we focus on proteoliposomes that have become an indispensable experimental system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resolution. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymatic cascades, a very promising experimental tool considering our increasing knowledge of the interplay of different cellular components.

Giant unilamellar vesicles (GUVs) are micrometer-sized model membrane systems that can be viewed directly under the microscope. They serve as scaffolds for the bottom-up creation of synthetic cells, targeted drug...

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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Giant unilamellar vesicles (GUVs) are micrometer-sized model membrane systems that can be viewed directly under the microscope. They serve as scaffolds for the bottom-up creation of synthetic cells, targeted drug delivery and have been used in many in vitro studies of membrane related phenomena. GUVs are also of interest for the functional investigation of membrane proteins that carry out many key cellular functions. A major hurdle to a wider application of GUVs in this field is the diversity of existing protocols that are optimized for individual proteins. Here, we compare PVA assisted and electroformation techniques for GUV formation under physiologically relevant conditions, and analyze the effect of immobilization on vesicle structure and membrane tightness towards small substrates and protons. There, differences in terms of yield, size, and leakage of GUVs produced by PVA assisted swelling and electroformation were found, dependent on salt and buffer composition. Using fusion of oppositely charged membranes to reconstitute a model membrane protein, we find that empty vesicles and proteoliposomes show similar fusion behavior, which allows for a rapid estimation of protein incorporation using fluorescent lipids.