Our long term goal is to understand how scaffold proteins contribute to the organization of signal transduction. This proposal focuses on post-synaptic glutamate signaling, which mediates excitatory neurotransmission in the mammalian brain. Glutamate receptors and many components of their downstream signaling pathways are organized into a multiprotein complex by the membrane-associated guanylate kinases (MAGUKs), a family of scaffold proteins that includes PSD-95. PSD-95 is central to the organization of glutamate signaling because it regulates receptor activity and localization and couples receptors to second messengers and substrates. Although the importance of MAGUKs in glutamate signaling is well established, little is known about the specific roles these proteins play. This proposal will investigate the molecular basis of PSD-95 function. Aim 1 will characterize the structure of PSD-95 to test the hypothesis that PSD- 95 regulates access to the protein binding sites through conformational changes. Fluorescence resonance energy transfer (FRET) will be used as a spectroscopic ruler to probe the structure of PSD-95. Single molecule analysis will characterize conformational dynamics, which are lost in ensemble measurements. Aim 2 will determine the interdependence of binding affinities for PSD-95 ligand proteins to test the hypothesis that the multiple binding sites on PSD-95 influence one another to select for signaling complexes that function together. FRET between PSD-95 and ligands will be used to quantitate binding constants. Analysis of a reconstituted system is needed to characterize this complex multiprotein assembly. Single molecule analysis will allow specific interactions to be followed in complex multiprotein mixtures. These studies will provide new inroads to understanding higher level organization of signal transduction and will provide a platform for the development of antagonists and agonists to specific components of the glutamate signaling pathways.