Exploration of 19F-qNMR in Tapping the Functions of Intermediate GPCR-G Protein Complex

Abstract ID 92007 Poster Board 346 Understanding the roles of intermediate conformational states is pivotal to unraveling the activation processes of G protein-coupled receptors (GPCRs). However, the field is still struggling to define these sub-states with a sufficient resolution to study their individual functions. Using ¹⁹F-qNMR-guided conformational profile we demonstrate the feasibility of enriching the populations of discrete sub-states via conformation-biased mutants. These mutants adopt distinct distributions among five states that lie along the activation pathway of adenosine A2A receptor (A2AR), a class A GPCR. Among these mutants, the intermediate-state-trapped constructs make it possible for obtaining GPCR-G protein intermediate complexes, filling the gap that all resolved cryo-EM structures are fully activated end-state GPCR-G protein complexes. These mutants also allow us to study the functions of these individual conformational states and their complexes. In addition, the success of resolving structures of intermediate complexes provide a foundation to establish a ¹⁹F NMR-guided cryo-EM approach for complex structural elucidation, especially for the intermediate conformational complexes of plastic membrane proteins like GPCRs and ion channels. NIH: 1R01GM149659