Two-photon excitation fluorescence cross-correlation assay for ligand-receptor binding: cell membrane nanopatches containing the human micro-opioid receptor.
Current ligand-receptor binding assays for G-protein coupled receptors cannot directly measure the system's dissociation constant, Kd, without purification of the receptor protein. Accurately measured Kd's are essential in the development of a molecular level understanding of ligand-receptor interactions critical in rational drug design. Here we report the introduction of two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to the direct analysis of ligand-receptor interactions of the human micro opioid receptor (hMOR) for both agonists and antagonists. We have developed the use of fluorescently distinct, dye-labeled hMOR-containing cell membrane nanopatches ( approximately 100-nm radius) and ligands, respectively, for this assay. We show that the output from TPE-FCCS data sets can be converted to the conventional Hill format, which provides Kd and the number of active receptors per nanopatch. When ligands are labeled with quantum dots, this assay can detect binding with ligand concentrations in the subnanomolar regime. Interestingly, conjugation to a bulky quantum dot did not adversely affect the binding propensity of the hMOR pentapeptide ligand, Leu-enkephalin.
Department of Chemistry, University of Calgary, Calgary, Alberta, Canada.
Jody L
JL
Swift
Melanie C
MC
Burger
Dominique
D
Massotte
Tanya E S
TE
Dahms
David T
DT
Cramb
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004743:Enkephalin, Leucine / Q000737:chemistry / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D009270:Naloxone / Q000737:chemistry; D009271:Naltrexone / Q000737:chemistry; D049329:Nanostructures / Q000737:chemistry*; D009292:Narcotic Antagonists; D017785:Photons; D045663:Quantum Dots; D011957:Receptors, Opioid / Q000819:agonists / Q000032:analysis* / Q000378:metabolism*; D012996:Solutions; D018411:Spodoptera; D017186:Titrimetry
D003432:Cross-Linking Reagents; D008024:Ligands; D009292:Narcotic Antagonists; D011957:Receptors, Opioid; D012996:Solutions; D009270:Naloxone; D004743:Enkephalin, Leucine; D009271:Naltrexone; D019793:Fluorescein