| The coiled coil is a widespread structural motif found in a variety of biologically important proteins. Coiled coils can be homomeric or heteromeric; they can be two-, three-, or four-stranded, and they can align with their helices in a parallel or an antiparallel relative orientation. Our goal is to dissect the protein sequence features that lead to helix orientation preference in coiled-coil proteins. To this end, we have developed a random selection method in which antiparallel coiled coils may be selected from a pool of randomized proteins using a readily selectable function, sequence-specific DNA binding.;Naturally occurring basic region-leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a parallel leucine zipper dimerization domain (coiled coil) and an N-terminal basic region that directly contacts DNA. The design of a functional antiparallel bZip protein poses several challenges. For example, direct substitution of an antiparallel coiled coil for the leucine zipper of GCN4 results in a molecule with basic regions on opposite sides of the coiled coil. Thus, in an antiparallel bZip protein, the basic region for one partner in the complex must be placed C-terminal to the leucine zipper. As the basic region is N-terminal to the leucine zipper in all naturally occurring bZip proteins, our first challenge was to design a bZip peptide with a C-terminal basic region.;We designed a series of GCN4 variants in which the GCN4 basic region is placed C-terminal to the leucine zipper. This design allowed us to test experimentally whether or not there is a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the leucine zipper dimerization domain. In addition, we have examined the importance of the helix N-cap on basic region folding and DNA binding.;The final chapter in this document outlines our progress to date on the design of a functional antiparallel bZip protein using the basic region of GCN4 and a well-characterized model antiparallel coiled coil. This work has led to the development of an in vivo transcription interference assay for selecting antiparallel coiled coils from a randomized pool of proteins. |
