The specific aim of this proposed work is to further characterize Tax*bZIP interactions and identify potential Tax inhibitors. To determine what Tax recognizes on the bZIP dimer, I will synthesize three different biased combinatorial libraries each comprising of millions of peptide dimers. The randomized region of the libraries will be flanked by residues from the ATF-2 bZIP element since it exhibits a significant enhancement in DNA binding in the presence of Tax. The libraries will be synthesized on poly(dimethylacrylamide) beads using a 'split and pool' procedure whereby each bead contains only a single peptide dimer species. A diamine linker (link) will be attached to the bead through a molecular spacer (sp). Two peptide chains will then be extended off each amine of the linker to create the peptide dimer. The first peptide dimer library to be synthesized and screened will be (RQKR-XXXXXX-LEKK)2-link-sp- [bead]. This library is designed to identify peptide spacer segments with high affinity for the Tax protein. The RQKR basic region and the LEKK zipper domains flanking the ATF-2 spacer segments will be conserved. The second and third libraries will be constructed of the peptide sequences (KVWVQS-XXXXXX)2-link-sp[bead] and (XXXXXX-KVWVQS-LE)2-link- sp[bead]. These libraries are designed to identify residues on the zipper and basic sides of the ATF-2 spacer segment which influence the affinity of Tax for the bZIP dimer. The Tax protein will be derivatized or radiolabeled to allow for rapid screening of the peptide dimer libraries. The peptide dimer (RQKR-KVWVQS-LEKK)2-link-sp-[bead] from ATF-2 will also be synthesized to provide an appropriate control The sequence of the high affinity binding peptide dimers will be identified by the automated Edman degradation. To determine if the binding peptide dimers are potential Tax inhibitors, they will be independently synthesized and screened in the presence of Tax and ATF-2 by quantitative electrophoretic mobility shift assays. The most interesting peptide sequence and dimer will be isotopically labeled so that structural features of the dimer may be determined in the presence of Tax by nuclear magnetic resonance (NMR). Dissociation constants will be determined using fluorescence spectroscopy respectively. From this data, intuitive three dimensional structures will be generated using the computer minimization program X-PLOR.
