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Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13 C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13 C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp281 6.48 of the highly conserved SWLP motif and Trp327 7.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp116 23.50 was identified., (© 2020 The Authors. Published by Wiley-VCH GmbH.)