| This dissertation describes the characterization of miniature DNA-binding proteins designed to be high affinity and high specificity ligands. Avian pancreatic polypeptide was used as the scaffold for protein grafting to preorganize binding epitopes and optimize their function using in vitro selections.; Chapter 1 describes an attempt to generate miniature proteins that bind DNA containing the nonnatural self pair, PICS:PICS. PICS:PICS presents a uniquely hydrophobic surface in the major groove of DNA, distinct from native base pairs. In addition to enhancing our knowledge of protein•DNA interactions, PICS:PICS-binding proteins have utility as tools in research and biotechnology, as well as being potential therapeutics. Towards this end, phage display was used to select for miniature proteins based on p007 that recognize DNA containing PICS:PICS pair to produce a complex that is functionally orthogonal to protein•DNA complexes found in Nature. The selections succeeded in generating miniature proteins that bound DNA with nanomolar affinity, but little specificity, meriting a detailed study of miniature proteins that bind natural DNA.; Chapter 2 describes the further characterization of the GCN4 mimic, p007, which binds DNA containing the half-site CRE (hsCRE) sequence ATGAC, with nanomolar affinity and high specificity. A complete alanine scanning mutagenesis study of the miniature protein p007 was performed to determine the contributions of individual residues to specific DNA affinity, nonspecific DNA affinity, and protein structure. As expected, variants containing alanine in place of known DNA-binding residues possess severely diminished DNA affinity and specificity. We also find that variants containing alanine in place of residues within the hydrophobic core also possess diminished DNA affinity and specificity. In addition, the importance of most of the residues selected by phage display was confirmed by diminished affinity and specificity of the corresponding alanine containing variants. While all variants were less structured than p007 as determined by circular dichroism spectroscopy, there was little correlation between structure and DNA-binding affinity or specificity. Finally, we find that no alanine substitutions improve upon the hsCRE specificity of p007, indicating that every residue makes a direct or indirect contribution to specificity. |
