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.
ASJC Scopus subject areas
- Molecular Biology