LRET Methods for Investigating Conformational Changes in Functional Ionotropic Glutamate Receptors

Abstract

Luminescence resonance energy transfer (LRET) is an invaluable tool to study the conformational changes of proteins induced by ligands or protein–protein interactions at an Ångstrom-level resolution. LRET experiments require a pair of fluorophores with overlapping spectra, where the emission of the “donor” overlaps with the excitation of the “acceptor” fluorophore. The efficiency of non-radiative energy transfer can be determined by measuring the sensitized emission of the acceptor fluorophore, upon exciting the donor fluorophore. The efficiency of transfer, in turn, can be correlated to the distance between the two fluorophores. A modification of the LRET technique, whereby the LRET lifetime is measured before and after protease cleavage of the receptor, allows for the quantification of background fluorescence and for measurements to be made in functional receptors expressed in mammalian cells (Rambhadran et al., J Biol Chem 286:16953–16957, 2011; Sirrieh et al., J Biol Chem 288:22555–22564, 2013; Pal et al., Biochemistry 44:13638–13649, 2005; Gonzalez et al., Biochemistry 47:10027–10032, 2008). The specific LRET lifetime that can be measured in this system can be used to describe conformational changes associated with gating, allosteric modulation, partial agonism, protein–protein interactions, and inter-subunit interactions associated with motions of the receptor. By subsequently coupling this technique with functional characterization of the receptor, a direct connection can be drawn between structural changes and the functional consequences.