T cells play a central role in the adaptive antigen-specific immune response to invading pathogens and cancer while largely avoiding self-reactivity (autoimmunity). These cellular functions are mediated by interaction of the T-cell receptor (TCR) with peptide-MHC (pMHC) complexes which leads to signaling through the TCR-CD3 signaling complex. A great body of evidence has been generated to understand the molecular mechanisms of TCR-activation and evidence supports that the TCR undergoes conformational change upon recognition of pMHC. Still, it remains an enigma how molecular and structural events in TCR-recognition translate into differences in intracellular signaling. The central hypothesis of this application is that conformational changes induced in the TCR upon ligand binding can be transmitted across the membrane to expose cytoplasmic domains in the CD3??? chains, and that differences in conformational changes are responsible for quantitative and/or qualitative differences in T-cell signaling. To test our hypothesis we will combine X-ray crystallography, nuclear magnetic resonance (NMR) and a novel fluorescence energy transfer (FRET) assay to analyze the changes in the overall structural organization and conformation of TCR when binding to different pMHC. We expect that these studies will provide insight into the molecular mechanism of how ligand induced conformational changes at the pMHC-TCR interface translocate to the CD3 signaling complex to influence T-cell activation outcomes with a sensitivity and resolution that have not been possible before. An increased understanding of the structural biophysics of protein-protein interactions and of the propensity of structures to undergo conformational change will be of critical importance, particularly in the case of receptors involved in cell signaling. Furthermore, such biophysical studies will provide fundamental insights into protein structure and dynamics, explain how these features are used for specific signaling purposes and how the proteins function in distinct cellular environments. These results are expected to be of interest for the scientific community interested in receptor signaling. In addition, this basic knowledge will eventually allow us to design polypeptides or other agents that can be used to monitor and manipulate cell signaling events to guide the design of therapeutics and vaccines for cancer and autoimmune disease which afflict thousands of people. Proposal narrative We expect that our studies will provide insight into the molecular mechanism of how ligand-induced conformational changes in the T cell receptor influence T-cell activation outcomes with a sensitivity and resolution that has not been possible before. This information will prove useful in both understanding how receptor-mediated signaling is initiated and from a therapeutic point of view to modulate signaling through the T cell receptor pharmacologically to either increase the sensitivity of T-cells in patients with cancer or HIV or decrease the sensitivity in patients with autoimmune diseases (multiple sclerosis or diabetes).