Project Summary This NOSI response is an instrumentation request for a Nicoya Surface Plasma Resonance (SPR) equipment that will supplement the parent award (5R01GM149659-02) and will foster the parent award research scope and have long-term impact on the research in my lab in the study of both ligand-receptor and receptor-transducer interactions. In the parent award, we created a series of conformation-biased mutants that can populate different conformational states, ranging from the inactive state S1 to the fully activated state S5, that lie along the GPCR activation process into different mutants, a result that has allowed us to study the function and structure of each individual conformation. Specifically, we can trap an intermediate state S4 into a single point mutant R291A and two conformational states S3 and S5* into a double mutant R291AR293A. These progresses allow us to study the roles of intermediate states S3 and S4 in the signaling, in reference to the fully activated state S5. Of note, S5* state is a pseudo-active state, in which two intermolecular salt bridges are knocked out due to the R to A mutagenesis, distinct from the real S5 state. Most recently, we also made progress in resolving the first intermediate GPCR- mini-Gasbg complex. The requested equipment acquisition will add a digital tool to study the interactions between the S3/S4 states and Gasbg without a specific labeling (e.g.,19F-labeling for NMR and isotopic-labeling for GDP and radioligand binding assays). The SPR will also help us to study the kinetics of nucleotide exchanges in the G proteins that are regulated by different conformational states. All noted experiments are in the scope of the proposed parent award. Furthermore, given our current work on multi-GPCRs, the equipment acquisition will significantly elevate our lab’s capacity to screen drugs for different physiological GPCRs and interrogate the interactions of receptors with different downstream transducers, including various G proteins, GRKs, and b-arrestins in the future.