| In eukaryotic signal transduction pathways, modular protein-protein recognition domains are used to assemble multi-protein signaling complexes. PDZ domains; frequently found in signaling proteins, are one of the protein-protein recognition modules that play a central role in protein targeting and protein complex assembly. These domains recognize specific C-terminal, and on occasion internal, peptide motifs of a parnter protein. The specific focus of this work has been to design and develop novel peptide ligands to serve as modifiers of PDZ domain binding activity, a pursuit both fundamental and relevant to contemporary drug design efforts. Structure-based rational drug design was employed initially where the 'consensus sequence' of a peptide lead was subjected to further development via cyclization using non-peptidic bridging elements. Later, a 'semi-combinatorial' design approach was employed to modify the peptides via side chain diversification through the introduction of organic functionalities in a library format. In addition, N-terminal myristylation, FITC tagging and biotinylation processes were employed to facilitate cell permeability, cell visualization and ELISA identification, respectively.; Major protein domain studied was the PDZ3 of neuronal protein PSD-95, with several other PDZ domains. The in vitro binding thermodynamics were analyzed using Isothermal titration calorimetry (ITC), which can determine all the thermodynamic parameters; these are Kd, DeltaG, DeltaH, DeltaS, DeltaC p and the stoichiometry of binding. In this study, the experimental approach went beyond the ITC conditions that are typically employed in a standard binding study. These involved osmotic stress studies, heat capacity studies, macromolecular crowding studies and heat of buffer ionization studies to evaluate the role of the water, the hydrophobic effect, compartmentalization effect and proton exchange effects associated with the binding process, respectively. In addition to these studies, an ELISA was also developed in order to identify potent binding peptides for PDZ3 synthesized from combinatorial parallel libraries. Using both rational and combinatorial approaches, peptides with binding improvements of over one order of magnitude than unmodified peptides were identified. Binding behaviors for selected ligands associated with solvent and protein effects, were revealed in great detail, including an investigation into the thermodynamic value of preparing constrained, cyclic ligands from linear peptide ligands. These results not only shed light on the nature of PDZ domain interactions, but will have a broader impact on the design strategies of making modified peptide ligands from bioactive consensus peptides to yield higher affinity ligands. |
