Enhanced Fluorescence Resonance Energy Transfer in G-Protein-Coupled Receptor Probes on Nanocoated Microscopy Coverslips
Schreiber B, Kauk M, Heil HS, Emmerling M, Tessmer I, Kamp M, Hoefling S, Holzgrabe U, Hoffmann C, Heinze KG (2018)
Publication Type: Journal article
Publication year: 2018
Journal
Book Volume: 5
Pages Range: 2225-2233
Journal Issue: 6
DOI: 10.1021/acsphotonics.8b00072
Abstract
The G-protein-coupled receptor (GPCR) superfamily mediates cellular responses and communication across cellular membranes and is the largest known class of molecular targets with proven therapeutic value. For probing conformational changes of GPCRs and others in a live cell setting, fluorescence resonance energy transfer (FRET) is usually the method of choice. FRET probes often require careful labeling procedures, elaborate characterization, and assay optimization to provide both physiologically relevant probes with unaltered pharmacology and a sufficient dynamic range of the FRET changes. Here, we present an approach to optimize the energy transfer without changing the design of the FRET probe. We show that gold-coated glass coverslips reinforce the otherwise forbidden donor-acceptor energy transfer by virtual optimization of the dipole orientation. First, we confirm the resulting enhanced FRET efficiency on our nanocoatings for the inactive M1 muscarinic acetylcholine receptor (mAChR) labeled with a FRET pair of cyan fluorescent protein and fluorescein arsenical hairpin binder in classical bleaching experiments. Second, we show the advantage of this enhanced FRET technique for ligand binding studies in live cells, by the increased dynamic FRET response between the inactive and active states of the muscarinic acetylcholine receptor M1 subtype. Our method is not limited to GPCRs and thus has general potential for surface-bound FRET approaches. We believe our technique is particularly suited for pharmaceutical drug screening to boost FRET probes, in which it is highly desired to amplify signal responses without interfering with the well-characterized assay.
Involved external institutions
Germany (DE)How to cite
APA:
Schreiber, B., Kauk, M., Heil, H.S., Emmerling, M., Tessmer, I., Kamp, M.,... Heinze, K.G. (2018). Enhanced Fluorescence Resonance Energy Transfer in G-Protein-Coupled Receptor Probes on Nanocoated Microscopy Coverslips. ACS Photonics, 5(6), 2225-2233. https://doi.org/10.1021/acsphotonics.8b00072
MLA:
Schreiber, Benjamin, et al. "Enhanced Fluorescence Resonance Energy Transfer in G-Protein-Coupled Receptor Probes on Nanocoated Microscopy Coverslips." ACS Photonics 5.6 (2018): 2225-2233.
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