The goal of this Proposal is to design, prepare, and analyze a variety of ligands as molecular probes in the study of protein-protein interactions between macromolecules involved in signal transduction. Specifically, these ligands will target selected members of the recently discovered PDZ domain family. PDZ domains have been identified in a number of different proteins that are involved in the assembly of receptors at cellular membranes, as well as proteins that participate in the formation of the extracellular matrix. While clearly playing a significant role in certain cellular processes, there is much that is not known about the in vivo consequences of PDZ domain- mediated interactions. From a biochemical point of view, there is a near-total absence of an understanding of the thermodynamics that governs the basic molecular recognition process involving PDZ domains. In the initial work of this Proposal, PDZ domains and binding peptide ligands will be studied using isothermal titration calorimetry (ITC), a powerful method that provides a thorough thermodynamic profile for binding (enthalpy, entropy, free energy, and dissociation constants). In addition, X-ray crystallographic structural determinations will be made of the PDZ-ligand complexes, allowing for a correlation of the thermodynamics of binding to specific molecular interactions. Novel ligands will then be designed based on this data, as well as existing structural information, with the ultimate goal of preparing cell-permeable, selective and potent PDZ domain binding ligands. These compounds could then be used in cellular studies and serve as molecular probes to decipher the biological functions of PDZ-mediated binding events. From a therapeutic perspective, recent reports demonstrate a strong link between certain PDZ-bearing proteins and oncogenic activity, and suggest that PDZ may represent a completely new target for anti-cancer drug design. The central focus, then, in this Proposal will be on two PDZ proteins that may reflect the fundamental binding preferences of all other PDZ domains: the Class I PDZ domains from PSD95, and the Class II PDZ domain from hCASK. An interdisciplinary approach towards characterizing PDZ domain binding will be conducted according to the following plan: (1) prepare PDZ proteins and peptide ligands, then study binding with ITC; (2) employ X-ray crystallography to evaluate the protein- bound conformations of the PDZ-binding ligands; (3) using structure-based and combinatorial chemistry methods, design and synthesize a variety of novel peptide analog ligands to identify binding probes with improved PDZ domain affinity and/or selectivity; and (4) devise supporting binding and cellular assays that will lead to more rapid evaluation of PDZ ligand affinity and the effects these compounds have on the biological function of cells that actively utilize PDZ-mediated interactions.