Evaluating And Exploiting The Substrate Selectivity Of Farnesyltransferase

Abstract

Protein prenylation is a post translational modification where an isoprenoid is attached to a CaaX sequence at the c terminus of a protein. Ultimately, after this modification and subsequent steps, prenylated proteins associate with the plasma membrane and are involved in many critical cellular functions. This dissertation describes an exploration of the amazing range of substrates of FTase, with both regards to the peptide and isoprenoid binding site, as well as how this selectivity can be expanded and leveraged for novel applications. The first chapter is a comprehensive literature review of the knowledge that got us to this point, discussing how the canonical rules of the composition of CaaX sequences was determined as how those rules were expanded, as well as the use of bioorthogonal isoprenoid probes and how they have been used to study prenylation. Chapter 2 describes the development of a MALDI-MS peptide library based assay to probe what amino acids are allowed in a pentapeptide CaaaX sequence, based on the recently discovered prenylatable sequence CMIIM. Upon success of this work, expansion to another sequence, CSLMQ, as well as a comparison between the specificity of rat and yeast FTase for these two sequences is discussed in chapter 3. Finally, chapter 4 focuses on the evaluation of a dual-bioorthogonal mutant FTase, an idea that combines two previous mutants, selectively transferring a bulky coumarin containing isoprenoid to an non-naturally recognized charged CaaX sequence. This dissertation expands our continuing understanding of potential substrates of FTase and their potential applications.