Observing monomer: dimer transitions of neurotensin receptors 1 in single SMALPs by homoFRET and in an ABELtrap

Abstract

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.

Fast ATP-Dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution

“…The completed knight’s tour was repeated at a 5 or a 7 kHz rate and was controlled by a field-programmable gate array (FPGA) card. The published FPGA-based ABEL trap software from the Cohen group was slightly modified for utilizing up to four detectors in our setup . Here we used the combined photon counts from two single photon counting avalanche photodiodes (APD), i.e., from the FRET donor and the FRET acceptor channel.…”

“…The published FPGA-based ABEL trap software from the Cohen group 45 was slightly modified for utilizing up to four detectors in our setup. 53 Here we used the combined photon counts from two single photon counting avalanche photodiodes (APD), i.e., from the FRET donor and the FRET acceptor channel. The FPGA software directed the laser focus stepwise and allocated the recorded photons to each focus position.…”

Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution

“…In preliminary smFRET measurements of F o F 1 -ATP synthase in the ABEL trap we noticed that changing the excitation from 491 nm to longer wavelengths resulted in reduced background count rates using the PDMS/glass ABEL trap chips 42, 45 . To choose the optimal FRET donor for 532 nm excitation we compared the photophysical behavior of previously used tetramethylrhodamine (TMR) with Cy3B or Atto R6G bound to F o F 1 -ATP synthase.…”

“…The completed knight tour was repeated at 5 or 7 kHz rate and was controlled by a field- programmable gate array (FPGA) card. The published FPGA-based ABEL trap software from the A. E. Cohen group 45 was slightly modified for utilizing up to four detectors in our setup 53 . Here we used the combined photon counts from two single photon counting avalanche photodiodes (APD), i.e., from FRET donor and FRET acceptor channel.…”

“…We have built different ABEL traps with APD detectors using a variety of lasers and associated optical filter sets 15–17 . Here, the beam diameter of a continuous-wave 532 nm laser (Compass 315M, Coherent) was collimated and adjusted to less than 1 mm by a two-lens telescope and linearly polarized by a polarizing beam splitter cube 18 .…”