The μ-opioid receptor (μOR) is a key target for pain management, and understanding its molecular mechanisms can enhance therapeutic development. This study investigates how ligand-specific conformational changes in μOR translate into a range of intrinsic efficacies at the transducer level. Using double electron–electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), the researchers identified several conformations of the cytoplasmic face of the receptor that interconvert on different timescales. These include a pre-activated conformation capable of G-protein binding and a fully activated conformation that reduces GDP affinity within the ternary complex. Interaction with β-arrestin-1 appears less specific and occurs with lower affinity than G-protein binding. The study reveals that μOR activation by opioids like morphine and fentanyl can lead to adverse effects such as constipation, tolerance, and respiratory depression. Previous research suggested that G-protein-biased agonists, which preferentially activate G proteins, would reduce side effects. However, recent studies show that overly strong G-protein signaling (super-efficacy) is responsible for respiratory depression, and partial agonists with lower efficacy provide a safer profile. High-resolution structures of μOR in complex with G-proteins are available, but structures of μOR in complex with β-arrestin are not. The researchers used DEER and smFRET to investigate the structural underpinnings of μOR activation and G-protein signaling. They found that μOR conformational states exchange on fast and slow timescales, and these exchanges fine-tune the receptor's efficacy and signal bias. DEER experiments revealed conformational heterogeneity in μOR, with two active conformations of TM6. SmFRET experiments further characterized the dynamics of μOR, showing that low-efficacy G-protein-biased agonists do not stabilize the canonical 'active' conformation. Instead, the addition of G-proteins and β-arrestin-1 revealed that these ligands pre-activated the receptor, facilitating transducer binding. The study concludes that μOR functional selectivity and super-efficacy are based on the coexistence and differential population of inactive and active conformations, exchanging on fast or slow timescales. These findings highlight the importance of solution-state, biophysical studies for characterizing GPCR-ligand-transducer signaling and suggest potential approaches for designing therapeutic agents with fewer adverse effects.The μ-opioid receptor (μOR) is a key target for pain management, and understanding its molecular mechanisms can enhance therapeutic development. This study investigates how ligand-specific conformational changes in μOR translate into a range of intrinsic efficacies at the transducer level. Using double electron–electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), the researchers identified several conformations of the cytoplasmic face of the receptor that interconvert on different timescales. These include a pre-activated conformation capable of G-protein binding and a fully activated conformation that reduces GDP affinity within the ternary complex. Interaction with β-arrestin-1 appears less specific and occurs with lower affinity than G-protein binding. The study reveals that μOR activation by opioids like morphine and fentanyl can lead to adverse effects such as constipation, tolerance, and respiratory depression. Previous research suggested that G-protein-biased agonists, which preferentially activate G proteins, would reduce side effects. However, recent studies show that overly strong G-protein signaling (super-efficacy) is responsible for respiratory depression, and partial agonists with lower efficacy provide a safer profile. High-resolution structures of μOR in complex with G-proteins are available, but structures of μOR in complex with β-arrestin are not. The researchers used DEER and smFRET to investigate the structural underpinnings of μOR activation and G-protein signaling. They found that μOR conformational states exchange on fast and slow timescales, and these exchanges fine-tune the receptor's efficacy and signal bias. DEER experiments revealed conformational heterogeneity in μOR, with two active conformations of TM6. SmFRET experiments further characterized the dynamics of μOR, showing that low-efficacy G-protein-biased agonists do not stabilize the canonical 'active' conformation. Instead, the addition of G-proteins and β-arrestin-1 revealed that these ligands pre-activated the receptor, facilitating transducer binding. The study concludes that μOR functional selectivity and super-efficacy are based on the coexistence and differential population of inactive and active conformations, exchanging on fast or slow timescales. These findings highlight the importance of solution-state, biophysical studies for characterizing GPCR-ligand-transducer signaling and suggest potential approaches for designing therapeutic agents with fewer adverse effects.