Using small molecules to regulate protein localization and degradation

The complex network of interactions and reactions of proteins with DNA, RNA, and other biomolecules requires tight regulation to maintain the homeostasis of life. To understand the role of each protein in these complicated systems, biologists perturb one variable related to a protein and study the effects. A few of these variables are location within a cell, interactions with other proteins and biomolecules, and concentration. Unfortunately, proteins have typically been studied on an individual basis due to restrictive methodology. This dissertation focuses on developing methodologies that can be used to study the majority of the proteome. Using fusion proteins, a protein of interest can be perturbed by attaching a small molecule-regulated protein.; Chapter Two focuses on developing a light-sensitive molecule that can bring two FKBP fusion proteins together. This system can be used to relocate proteins within the cell or to bring two proteins together within a cell. The synthesis of a small library of azobenzene-containing molecules is reported. The isomerization of azobenzenes between the cis and trans isomers alters the steric environment of the molecules. The extent of isomerization of these compounds has been measured by NMR and HPLC. One of these compounds was isomerized alone and in the presence of a protein to show that the isomerization is not inhibited by a potential binding partner. Fluorescence polarization was used to study the binding interactions of these photosensitive molecules with their binding partners. However, limitations of this assay prevented the characterization of the possible binding events.; Chapter Three focuses on a system for controlling the degradation of a protein of interest. In this project, several permutations of bacterial DHFR are tested for stability in the presence of trimethoprim, a molecule orthogonal to mammalian metabolism, and degradation in the absence of the small molecule. DHFRs with varying N-terminal amino acids and linker regions were studied in mouse cells by Western blot. DHFR proteins containing a flexible 45-amino acid linker including lysines accessible to the ubiquitylation machinery display the desired small molecule regulation. Future work will determine if this degradation occurs when additional proteins are attached to the destabilizing DHFR.