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Activation Biosensor for G Protein-Coupled Receptors: A FRET-Based m1 Muscarinic Activation Sensor That Regulates Gq Seungwoo Chang, Elliott M. Ross* Department of Pharmacology, Molecular Biophysics Graduate Program, Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America Abstract We describe the design, construction and validation of a fluorescence sensor to measure activation by agonist of the m1 muscarinic cholinergic receptor, a prototypical class I Gq-coupled receptor. The sensor uses an established general design in ¨ which Forster resonance energy transfer (FRET) from a circularly permuted CFP mutant to FlAsH, a selectively reactive fluorescein, is decreased 15–20% upon binding of a full agonist. Notably, the sensor displays essentially wild-type capacity to catalyze activation of Gaq, and the purified and reconstituted sensor displays appropriate regulation of affinity for agonists by Gq. We describe the strategies used to increase the agonist-driven change in FRET while simultaneously maintaining regulatory interactions with Ga , in the context of the known structures of Class I G protein-coupled receptors. q The approach should be generally applicable to other Class I receptors which include numerous important drug targets. Citation: Chang S, Ross EM (2012) Activation Biosensor for G Protein-Coupled Receptors: A FRET-Based m1 Muscarinic Activation Sensor That Regulates G . PLoS q ONE 7(9): e45651. doi:10.1371/journal.pone.0045651 Editor: Karl-Wilhelm Koch, University of Oldenburg, Germany Received June 13, 2012; Accepted August 20, 2012;