G protein-coupled receptors (GPCRs) are a large superfamily of membrane proteins that are activated by extracellular small molecules or photons. Neurotensin receptor 1 (NTSR1) is a GPCR that is activated by neurotensin, i.e. a 13 amino acid peptide. Binding of neurotensin induces conformational changes in the receptor that trigger the intracellular signaling processes. While recent single-molecule studies have reported a dynamic monomerdimer equilibrium of NTSR1 in vitro, a biophysical characterization of the oligomerization status of NTSR1 in living mammalian cells is complicated.Here we report on the oligomerization state of the human NTSR1 tagged with mRuby3 by dissolving the plasma membranes of living HEK293T cells into 10 nm-sized soluble lipid nanoparticles by addition of styrene-maleic acid copolymers (SMALPs). Single SMALPs were analyzed one after another in solution by multi-parameter single molecule spectroscopy including brightness, fluorescence lifetime and anisotropy for homoFRET. Brightness analysis was improved using single SMALP detection in a confocal ABELtrap for extended observation times in solution. A bimodal brightness distribution indicated a significant fraction of dimeric NTSR1 in SMALPs or in the plasma membrane, respectively, before addition of neurotensin.

FoF1-ATP synthases in mitochondria, in chloroplasts, and in most bacteria are proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes’ reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthases in solution were extended up to several seconds using a confocal anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes, we revealed that ADP inhibition did not decrease the catalytic turnover of all FoF1-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios.

The human neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor. The receptor is activated by a small peptide ligand neurotensin. NTSR1 can be expressed in HEK cells by stable transfection. Previously we used the fluorescent protein markers mRuby3 or mNeonGreen fused to NTSR1 for EMCCD-based structured illumination microscopy (SIM) in living HEK cells. Ligand binding induced conformational changes in NTSR1 which triggered the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer/dimer equilibrium of this receptor in artificial lipid bilayers. Here we report on the oligomerization state of human NTSR1 from living cells by trapping them into lipid nanodiscs. Briefly, SMALPs (styrene-maleic acid copolymer lipid nanoparticles) were produced directly from the plasma membranes of living HEK293T FlpIn cells. SMALPs with a diameter of 15 nm were soluble and stable. NTSR1 in SMALPs were analyzed by single-molecule intensity measurements one membrane patch at a time using a custom-built confocal anti-Brownian electrokinetic trap (ABEL trap) microscope. We found oligomerization changes before and after stimulation of the receptor with its ligand neurotensin.

The human neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor. The receptor is activated by a small peptide ligand neurotensin. NTSR1 can be expressed in HEK cells by stable transfection. Previously we used the fluorescent protein markers mRuby3 or mNeonGreen fused to NTSR1 for EMCCD-based structured illumination microscopy (SIM) in living HEK cells. Ligand binding induced conformational changes in NTSR1 which triggered the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer/dimer equilibrium of this receptor in artificial lipid bilayers. Here we report on the oligomerization state of human NTSR1 from living cells by trapping them into lipid nanodiscs. Briefly, SMALPs (styrene-maleic acid copolymer lipid nanoparticles) were produced directly from the plasma membranes of living HEK293T FlpIn cells. SMALPs with a diameter of 15 nm were soluble and stable. NTSR1 in SMALPs were analyzed by single-molecule intensity measurements one membrane patch at a time using a custom-built confocal anti-Brownian electrokinetic trap (ABEL trap) microscope. We found oligomerization changes before and after stimulation of the receptor with its ligand neurotensin.

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