Date of Award
Doctor of Philosophy (PhD)
Post-translational modification (PTM) of a protein refers to any chemical change that occurs to the protein after its ribosomal synthesis. The seemingly endless number of PTMs can endow proteins with new functionalities that are not present in the unmodified proteins. In order to study the functions of PTMs on a given protein, it is often necessary to have access to pure preparations of the modified protein and its analogues. Traditional biological methods frequently do not allow for the isolation of significant amounts of pure modified proteins, therefore chemical methods are often employed in this regard. Protein semi-synthesis is a chemical method that entails the melding of at least two protein fragments in which at least one fragment is isolated from a biotic source while another fragment is synthesized by chemical methods. This framework enables the installation of PTMs into the complete protein through chemical control over the synthesized fragment without the need to synthesize the entire protein molecule, which is often a practically impossible task. This thesis describes efforts employing the protein semi-synthesis technique known as expressed protein ligation (EPL) to the study of the cellular signaling protein Smad2. Smad2 is activated by phosphorylation, which is the best characterized reversible PTM. Once phosphorylated, Smad2 accumulates in the nucleus of cells where it helps to direct transcriptional changes that affect cell behavior. Techniques were developed to cage Smad2 by directly blocking the activating phosphates on Smad2 with bulky photoremovable groups. This affords the investigator control over the timing and localization of Smad2 activity by judicious application of light of the appropriate wavelength to remove the photocaging group. This approach can be employed to generate caged analogues of any phosphorylated protein. A parallel caging approach was developed that relies upon indirect blockade of Smad2 phospho-dependent activity through the installation of a caging group on the C-terminus of the protein. This approach was compatible with a fluorescence reporter that is fluorescent only when the photocaging group is removed, thus allowing for selective monitoring of the activated form of the protein. This protein was introduced into live cells and upon activation allowed for real time visualization of the active protein through fluorescence microscopy. The activity of Smad2 is dependent upon differential protein-protein interactions that the phosphorylated protein is able to participate in while the non-phosphorylated protein is not. In an effort to identify new binding partners that are sensitive to the phosphorylated state of Smad2, methods were developed to install stable phosphoanalogues (phosphonates) into Smad2. These analogues were used to identify a candidate Smad2-binding protein, PRMT5, that preferentially binds non-phosphorylated Smad2. Studies are ongoing to determine if this interaction has physiological relevance.
Hahn, Michael Eric, "Semi-Synthesis of the Transcription Factor SMAD2 Containing Caging Groups and Phosphoaminoacid Analogues" (2008). Student Theses and Dissertations. 192.