Project Summary G protein-coupled receptors (GPCRs) are important conduits to relay extracellular signals to downstream intracellular signal transduction pathways. In addition to being a central mode of intercellular communication the magnitude of the gene family and their structural diversity make GPCRs superb targets for therapeutics. Understanding the mechanism of hormone action on GPCRs and understanding how drugs modulate their behavior is an important fundamental endeavor but also an important mission for health scientists. The primary goal of this proposal is to study the mechanism of GPCR regulation of their primary signaling partners, G proteins. We will take a biochemical and biophysical approach to understand the mechanism of GPCR·G protein interactions and determine how agonist activation of GPCRs leads to G protein activation. We will utilize cutting-edge approaches including x-ray crystallography, cryo-electron microscopy, double electron-electron resonance spectroscopy and single molecule spectroscopy to study these receptor-G protein interactions. A critical component of these studies is obtaining sufficient quantities of purified preparations of G protein- coupled receptors and heterotrimeric G proteins, as well as other critical reagents such as nanobodies. In our studies, the use of chromatography and in particular, fast performance liquid chromatography (FPLC) is an essential approach to obtaining our purified preparations. This is an ongoing research grant, now in its 16th year and our instrumentation for liquid chromatography is becoming obsolete. In my laboratory more and more trainees requiring the use of our current FPLC instrumentation, which are now between 20 and 25 years old. The instruments have been experiencing significant downtime, requiring more and more costly service and repair. More importantly, parts are now starting to be no longer available. We propose to update our instrumentation for this critical component of our research on the biochemistry, biophysics and structural pharmacology of interaction between G protein and GPCRs.