Project Summary G protein coupled receptors (GPCRs) are important transmembrane signaling proteins, which are activated by a multitude of extracellular ligands ranging from small molecules to entire proteins. Active GPCRs couple to different intracellular transducer proteins, such as G proteins and arrestins, thereby triggering diverse cellular responses. Due to their fundamental involvement in a multitude of signal transduction processes, many diseases have their origin in a malfunctioning GPCR, and approximately 35% of all FDA-approved drugs directly target a GPCR. Receptor promiscuity towards ligands and transducer proteins entails that drugs designed to alleviate symptoms are often associated with a range of possible side effects. A detailed molecular understanding of ligand binding, receptor activation and signal transfer is thus pivotal in order to facilitate design of receptor-specific and highly efficacious drugs with enhanced selectivity for downstream signaling pathways. To fulfil their role as allosteric and highly promiscuous regulatory proteins, GPCRs rely on a high degree of structural flexibility, which allows large scale conformational changes thus facilitating specific recognition by binding partners of distinct shape and size. An important and currently unexplored question is to what extent the motions of individual receptor segments are coupled, as opposed to moving independently. This new paradigm suggests that therapeutic drugs could be designed that modulate only a selected subset of conformational changes, while leaving other parts of the receptor unaffected. Identifying networks of coupled segments will define new targets for drug research and facilitate design of functionally selective therapeutics with fewer side effects. We will be testing this hypothesis investigating two prototypical GPCRs of very different function – the light receptor rhodopsin (Rho) and the peptide-activated type 1 angiotensin II receptor (AT1R) - using newly developed site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopic techniques. In Aim (I) we will determine the number and topology of different receptor conformations. For this purpose we will shift the conformational equilibria using a variety of different ligands and the application of hydrostatic pressure to map the conformational landscape of both GPCRs using Double Electron-Electron Resonance (DEER) spectroscopy. In Aim (II) we will develop an improved time-resolved EPR method (“TRED”) to determine activation energies and coupling between segmental motions. TRED will be capable of resolving conformational changes with microsecond time resolution and Angstrom spatial sensitivity. In Aim (III) we will determine segmental coupling during Rho and AT1R activation, triggered by flash illumination and pressure jump, respectively. Comparison of the two receptors will allow us to identify coupling networks and transition states of activation, which are either conserved ...