Engineering Small Molecule-Dependent Control of Protein Localization and Stability

Biological systems comprise networks of interacting proteins, which in turn perform a multitude of functions as the workhorses of the cell. Proteins can serve as enzymes, hormones, receptors, antibodies, transport proteins, storage proteins, motor proteins, or signaling proteins. It would be of great value to deconvolute the contributions of each and every protein within complex biological networks. This thesis describes tools for conditionally regulating biological systems by targeting the protein level, affecting the function of a protein by controlling its localization and stability. The first chapter gives a general introduction to methods of regulation at the deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein levels. It details our search for a modified version of the ternary complex comprising the FK506 binding protein (FKBP), the small molecule rapamycin, and the FKBP-rapamycin binding domain (FRB) that could be used to regulate the expression of proteins of interest through a localization-based strategy. Rapamycin and several synthetic derivatives are analyzed by biological assays and quantitative mass spectrometry to determine their ability to bind the protein kinase mammalian target of rapamycin and its FRB domain. Both wild-type and mutant versions of the FRB proteins are found to have inherent instability that can be stabilized, or rescued, upon addition of a rapamycin derivative. This discovery of ligand-dependent stability leads into the second chapter. The second chapter discusses the development of a specific tool for regulation of genes in the budding yeast Saccharomyces cerevisiae. This technology involves engineering a destabilized version of the protein Escherichia coli dihydrofolate reductase that confers instability to a protein of interest. With this system, the protein fusion is unstable and degraded in the absence of the ligand trimethoprim and stabilized and active in its presence. Destabilizing domain systems that have been developed for use in a mammalian context are examined in yeast. This chapter addresses issues of vector-based versus genomic expression of fluorescent proteins for achieving optimal fluorescence profiles, engineering dual-promoter dicistronic DNA constructs for two-color screening, and designing random and directed libraries based on the protein: ligand pair Escherichia coli dihydrofolate reductase and trimethoprim. Several iterations of development ultimately lead to a successful destabilizing domain technology in yeast.