G protein-coupled receptors (GPCRs) are sensory integral membrane proteins that recognize an enormous range of extracellular stimuli and interact with numerous intracellular partner proteins to initiate cellular signaling events. It is widely appreciated that the functions of GPCRs are enabled by their inherent structural plasticity, i.e., conformational dynamics, and a complete view of GPCR function must also include knowledge of their dynamic behavior [1,2]. While crystallography and cryo-EM have made tremendous progress determining GPCR structures, concurrently, great advances investigating conformational dynamics of GPCRs have been made by spectroscopic methods, especially nuclear magnetic resonance (NMR) spectroscopy [3]. Indeed, current understanding of GPCR molecular recognition mechanisms are highly informed from NMR studies [3].

NMR spectroscopy provides several significant advantages for studying GPCR conformational dynamics, including that experiments can be carried out at physiological temperatures, do not require bulky tags, and frequently utilize proteins with native or nearly-native amino acid sequences. Importantly, NMR data provide information on GPCR structures and dynamics at the level of individual nuclei. This unique capability is enabled by stable-isotopes, which act as “probes” that sense changes in local structure, dynamics, and environments. By distributing NMR probes throughout the receptor, one can obtain a global view of GPCR conformational dynamics at atomic resolution. With advances in stable-isotope labeling approaches, inroads have been made into NMR studies with more challenging proteins, including GPCRs.

This review surveys stable-isotope labeling approaches for NMR studies of GPCRs, emphasizing methods that utilize NMR-observable nuclei other than 19F, i.e., 13C, 15N, 2H, and 1H. 19F-NMR complements experiments with these nuclei, as reviewed elsewhere [4,5]. Examples from the literature are presented that illustrate a range of expression systems for producing GPCRs and various methods for incorporating NMR probes, including stable-isotope labeling via chemical modification and via biosynthetic approaches. We discuss how advances in stable-isotope labeling and production of GPCRs have led to a more complete view of their functions by providing insights from NMR into GPCR-drug interactions, interactions with partner proteins, and impacts of the cellular environment on GPCR structure and conformational dynamics.