Black-lipid-membrane platform with diffusion suppression for confocal single-molecule FLIM-FRET analysis of gated membrane receptors and transporters.
Signal transduction and material exchange across the boundaries of lipid membranes are coupled to conformational changes of embedded membrane proteins, i.e. receptors, transporters and gated channels. These processes are activated by ligands and depend on compositions of the aqueous phases, lipids, and an electric potential across the lipid bilayer. To foster mechanistic understanding of these essential processes that are crucial for health or diseases, distinct spatiotemporal scales have to be addressed within this CRC/TR: for the primary steps associated with ligand binding, for dynamics of protein distributions in the membrane, and for transmembrane signaling cascades occurring inside specialized cells and across tissue.
Our aim is to develop a high-resolution microscopy platform for quantitative analyses of conformational dynamics of individual ligand-dependent receptors and channels under controlled conditions in vitro. First we focus on the neurotensin receptor type 1 (NTSR-1) as a prototypical G protein-coupled receptor (GPCR). NTSR-1 is triggered by a small peptide neurotensin which modulates dopaminergic systems, analgesia and inhibition of food intake in the brain and digestive processes in the gut. NTSR-1 is chosen because it is one of few GPCRs which can be expressed in bacterial membranes, purified and reconstituted functionally in liposomes. The structure of NTSR-1 with bound ligand has been solved by X-ray crystallography in 2012 by Dr. Reinhard Grisshammer [White et al. (2012) Nature 490, 508-513] indicating a tilt of transmembrane helix VI upon activation, and enabling site-directed mutations to study the dynamics of ligand-induced conformational changes as the initial step of signaling. In addition we will study hyperpolarization activated cyclic nucleotide-gated channels (HCN2) for ligand- and voltage-dependent ion translocation.
We propose to set up an optical black-lipid-membrane (BLM) platform for time-resolved confocal single-molecule Förster resonance energy transfer (smFRET/FLIM) analysis [Ide et al. (2002) Jap J of Physiology 52, 429-434; Tabata et al. (2009) EMBO J 28, 3279-3289]. A novel microfluidic chip system with integrated patch clamp electronics enables exchange of buffers on both sides of the planar membrane plus adjustment of electric membrane potentials. Planar BLMs with 400 µm in diameter comprising arbitrary lipid compositions are positioned 100 µm away from the cover glass of the chip, i.e. ideal for single-molecule imaging [Weiss et al. (2013) Biophys J 105, 455-462]. Native membrane vesicles with HCN2 or proteoliposomes with labeled NTSR-1 are integrated into the BLM with defined orientation by facilitated fusion. Finally, building an additional two-dimensional protein network with annexin V on top of the BLM will suppress diffusion of receptors or channels and allows for long-term smFRET/FLIM recordings but maintains their local rotational mobility, conformational dynamics and function.
Michael Börsch will coordinate and supervise the project together with other members of the team.
Dr. Heitkamp, Thomas
Thomas Heitkamp prepares all membrane proteins (NTSR1,FoF1-ATP synthase, KdpFABC, ABC transporter Pgp). This includes mutations, dye labeling, reconstitution methods, receptor assays, enzyme activities.
Sonja Rabe is responsible for bacterial cell growth including operation of the fermenter, protein analytics including SDS-PAGE, liposome preparations and general lab work.
Dr. Starke, Ilka
Ilka Starke is building the widefield single-molecule FRET microscope for combination with the BLM system. She also works on the confocal single-molecule FRET FLIM microscope.
|Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models||2014||Zarrabi, N., Ernst, S., Verhalen, B., Wilkens, S., and Börsch, M.||Methods||More|
|Twisting and subunit rotation in single FoF1-ATP synthase||2013||Sielaff, H., and Börsch, M.||Phil Trans Royal Soc B||More|
|Real time observation of single membrane protein insertion events by the Escherichia coli insertase YidC||2013||Winterhalter, S., Ernst, S., Börsch, M., Gerken, U., and Kuhn, A.||PLos ONE||More|
|Three-color Förster resonance energy transfer within single FoF1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time||2012||Ernst, S., Düser, M. G., Zarrabi, N., and Börsch, M.||J Biomed Opt||More|
|Elastic deformations of the rotary double motor of single FoF1-ATP synthases detected in real time by Förster resonance energy transfer||2012||Ernst, S., Düser, M. G., Zarrabi, N., Dunn, S. D., and Börsch, M.||Biochim Biophys Act - Bioenergetics||More|
|Dynamic Ligand Induced Conformational Rearrangements in P-glycoprotein as probed by Fluorescence Resonance Energy Transfer Spectroscopy||2012||Verhalen, B., Ernst, S., Börsch, M., and Wilkens, S.||J Biol Chem||More|
|36° step size of proton-driven c-ring rotation in FoF1-ATP synthase||2009||Düser, M. G., Zarrabi, N., Cipriano, D. J., Ernst, S., Glick, G. D., Dunn, S. D., and Börsch, M.||EMBO J||More|
|Movements of the ε-subunit during catalysis and activation in single membrane-bound H+-ATP synthase||2005||Zimmermann, B., Diez, M., Zarrabi, N., Gräber, P., and Börsch, M.||EMBO J||More|
|Proton-powered subunit rotation in single FoF1-ATP synthase||2004||Diez, M., Zimmermann, B., Börsch, M., König, M., Schweinberger, E., Steigmiller, S., Reuter, R. Felekyan, S., Kudryavtsev, V., Seidel, C.A.M., and Gräber, P.||Nat Struct Mol Biol||More|
|Stepwise rotation of the γ-subunit of EFoF1 ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer||2002||Börsch, M., Diez, M., Zimmermann, B., Reuter, R., and Gräber, P.||FEBS lett||More|