Application of Fluorescence Resonance Energy Transfer Techniques to Establish Ligand–Receptor Orientation

Abstract

Fluorescence resonance energy transfer (FRET) has been utilized to determine distances between a fluorescence donor and a fluorescence acceptor having appropriately overlapping spectra. In this chapter, we utilize this approach to establish distances between a fluorescence donor situated in a distinct position within a docked ligand and a fluorescence acceptor situated in a distinct position within its receptor. This technique is applicable to receptor expressed in the environment of an intact cell containing the full complement of signaling and regulatory proteins. A number of controls are necessary, including those establishing the normal function of the modified ligand and receptor, the absence of energy transfer to non-receptor proteins, and the specificity of transfer between the donor of interest and the acceptor of interest. We have utilized the example of FRET between a secretin peptide incorporating Alexa(488) and a secretin receptor construct derivatized with Alexa(568). The latter was prepared by the derivatization of a mono-cysteine-reactive receptor construct with a fluorescent methanethiosulfonate reagent. This approach can provide important spatial information that can be useful in the meaningful docking of a ligand at its receptor.

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…A similar observation was made for the secretin receptor, for which it was also possible to obtain structural constraints that allowed modeling of ligand/receptor interactions within the extracellular region of the receptor (Harikumar et al, 2007). A detailed protocol about how to perform such studies was published and should enable other groups to use this approach (Harikumar and Miller, 2009). Although the studies mentioned so far were performed as steady-state measurements, our own group used a tetramethylrhodamine-labeled PTH and a PTH receptor, which was N-terminally tagged with GFP, to study dynamic receptor-ligand interaction with FRET in real time ( Fig.…”

Tau liquid–liquid phase separation is modulated by the Ca2+‐switched chaperone activity of the S100B protein

“…4 μM of Alexa Fluor 488 5‐SDP Ester labeled tau (Alexa488‐tau) was phase‐separated in the presence of 14% w/v PEG‐10000 in 10 mM HEPES pH 7.4, with 1 mM DTT, 10 mM NaCl in the presence or absence of 240 μM CaCl 2 , at 37°C. Experiments were also performed in the presence or absence of Alexa Fluor 568 NHS Ester labeled S100B (Alexa568‐S100B); this fluorophore has spectral overlay with Alexa488‐tau and is therefore amenable to FRET studies (Harikumar & Miller, 2009) ( n = 3–5 fields per sample). 20 μl samples were deposited in a μ‐Slide 18 Well—Flat (Cat.…”