PDZ (PSD-95, DLG, ZO-1)domains are modular protein units that play important roles in targeting and clustering of channels, receptors, cell adhesion proteins, and other signaling molecules at the cell specific sites of cell-cell contact. Organization around a PDZ-based scaffold allows stable localization of interacting proteins and enhances the rate and fidelity of signal transduction within the complex. In this dissertation, the biochemical and structural characteristics of several PDZ domain related proteins including 8-kDa dynein light chain (DLC8), neuronal nitric oxide synthase (nNOS) and glutamate receptor interacting protein (GRIP) were investigated. An efficient expression and purification scheme was developed for DLC8. Utilizing this protocol about 30 mg of DLC8 can be purified...[ Read more ]

PDZ (PSD-95, DLG, ZO-1)domains are modular protein units that play important roles in targeting and clustering of channels, receptors, cell adhesion proteins, and other signaling molecules at the cell specific sites of cell-cell contact. Organization around a PDZ-based scaffold allows stable localization of interacting proteins and enhances the rate and fidelity of signal transduction within the complex. In this dissertation, the biochemical and structural characteristics of several PDZ domain related proteins including 8-kDa dynein light chain (DLC8), neuronal nitric oxide synthase (nNOS) and glutamate receptor interacting protein (GRIP) were investigated. An efficient expression and purification scheme was developed for DLC8. Utilizing this protocol about 30 mg of DLC8 can be purified from one liter LB cell culture. DLC8 forms a symmetric stable homo-dimer in solution. The DLC8-binding domain of nNOS adopts a random coil in solution and a 17-residue fragment of nNOS (Met 228 to His 244) is sufficient for the DLC8 binding. The DLC8-binding region of myosin V was shown to be within a 20-residue peptide fragment of myosin V (Lys 1283 to Met 1302). Extensive mutational studies unravel the amino acids of DLC8 that play critical role in binding to nNOS, DIC, GKAP, and myosin V. Biochemical and structural studies indicated that the ligand binding site and the C-terminal peptide binding site of nNOS PDZ domain are independent of each other and the salt bridge between Arg 121 in the β-finger and Asp 62 in the βD strand of the nNOS PDZ induces and stabilizes the β-hairpin structure of the nNOS PDZ domain. Preformed β-finger structure from nNOS PDZ domain is necessary for the formation of nNOS/PSD-95 dimer. The inter-domain chaperoning effect observed in the PDZ domains of GRIP represents a previously uncharacterized function of PDZ domains. The work described in this dissertation provided mechanistic insights into protein/protein interactions in neuronal signaling events.