Abstract In this proposal we seek to optimize and apply HDX as an enabling technology for the functional analysis of complex biological systems, such as transcriptional complexes and G-protein coupled receptors. We will study nuclear receptor complexes, GPCRs, and the MAP kinase signaling pathway. These biological targets have been implicated in metabolic disorders, cancer, osteoporosis, immune regulation, and neurodegeneration. Although implicated in a wide range of disorders, our group and our collaborators have specific expertise in metabolic disorders, immune regulation, and Parkinson's disease. A better understanding of the mechanism of action of modulators of NRs, GPCRs and kinase signaling pathways will enable the development of more functionally selective and safer therapeutic agents. The technological aspects of this work are to optimize and apply hydrogen/deuterium exchange (HDX) to enable characterization of complex proteins such as GPCRs and large multi-protein complexes involved in the regulation of signal transduction pathways. Recently we have made several significant advances in HDX technology. Here we will build from this work and optimize methodology for the characterization of complex systems. These methods will improve on the current limits of dynamic range, protein size, and throughput. In addition, our automated system for data acquisition will be integrated with a relational database and a new suite of software tools will be further developed to facilitate seamless integration of HDX data acquisition with data management, analysis and display. We will apply this technology to the functional analysis of nuclear receptor transcriptional complexes, S1P family receptors, and the MAP kinase JNK3. We will also demonstrate the utility of HDX technology in the context of a fully integrated high throughput screening center. Combined, these studies will raise the impact of this enabling biophysical technology on structural studies of complex biological systems.