TY - JOUR
T1 - Application of fluorescence resonance energy transfer techniques to establish ligand-receptor orientation.
AU - Harikumar, Kaleeckal G.
AU - Miller, Laurence J.
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=67650394863&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=67650394863&partnerID=8YFLogxK
U2 - 10.1007/978-1-60327-317-6_21
DO - 10.1007/978-1-60327-317-6_21
M3 - Article
C2 - 19513658
AN - SCOPUS:67650394863
SN - 1064-3745
VL - 552
SP - 293
EP - 304
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -