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Enhanced Fluorescence Resonance Energy Transfer in G-Protein-Coupled Receptor Probes on Nanocoated Microscopy Coverslips

Schreiber, Benjamin ; Kauk, Michael ; Heil, Hannah S. LU orcid ; Emmerling, Monika ; Tessmer, Ingrid ; Kamp, Martin ; Höfling, Sven ; Holzgrabe, Ulrike ; Hoffmann, Carsten and Heinze, Katrin G. (2018) In ACS Photonics 5(6). p.2225-2233
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... (More)

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.

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author
; ; ; ; ; ; ; ; and
publishing date
type
Contribution to journal
publication status
published
keywords
fluorescence enhancement, fluorescence spectroscopy, FRET, membrane biophysics, receptor dynamics
in
ACS Photonics
volume
5
issue
6
pages
2225 - 2233
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:85048813469
ISSN
2330-4022
DOI
10.1021/acsphotonics.8b00072
language
English
LU publication?
no
additional info
Publisher Copyright: © 2018 American Chemical Society.
id
784050d5-a939-4d76-86dd-1fce64d375ab
date added to LUP
2025-04-26 12:24:06
date last changed
2025-04-28 08:15:08
@article{784050d5-a939-4d76-86dd-1fce64d375ab,
  abstract     = {{<p>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.</p>}},
  author       = {{Schreiber, Benjamin and Kauk, Michael and Heil, Hannah S. and Emmerling, Monika and Tessmer, Ingrid and Kamp, Martin and Höfling, Sven and Holzgrabe, Ulrike and Hoffmann, Carsten and Heinze, Katrin G.}},
  issn         = {{2330-4022}},
  keywords     = {{fluorescence enhancement; fluorescence spectroscopy; FRET; membrane biophysics; receptor dynamics}},
  language     = {{eng}},
  month        = {{06}},
  number       = {{6}},
  pages        = {{2225--2233}},
  publisher    = {{The American Chemical Society (ACS)}},
  series       = {{ACS Photonics}},
  title        = {{Enhanced Fluorescence Resonance Energy Transfer in G-Protein-Coupled Receptor Probes on Nanocoated Microscopy Coverslips}},
  url          = {{http://dx.doi.org/10.1021/acsphotonics.8b00072}},
  doi          = {{10.1021/acsphotonics.8b00072}},
  volume       = {{5}},
  year         = {{2018}},
}