Any Way You Want It: Pushing the Limits of Chemical Protein Synthesis

Abstract

Chemical protein synthesis, via solid-phase peptide synthesis and chemoselective ligation of peptides, is a powerful approach for preparing peptides and proteins. These techniques enable complete atomic control over protein composition with both mechanistic and practical applications for biochemistry. The foundation of this dissertation is formed by two ambitious chemical protein synthesis projects: DapA (Chapter 2) and Dpo4 (Chapter 5). DapA is a 312-residue protein whose folding depends on the well-studied chaperone GroEL/ES. The successful synthesis of DapA (in both L- and D- chirality) was used to demonstrate cross-chiral folding by GroEL/ES-a fundamental biological insight and potential tool for future mirror-image synthetic biology research. However, the record-breaking synthesis of DapA was an arduous process requiring tremendous human and technical resources. The lessons from this project were then applied to the synthesis of our next target, Dpo4 (352 residues). Dpo4 is one of the shortest DNA/RNA polymerases, providing an accessible synthetic tool to amplify DNA/RNA for future synthetic biology studies. A new concept termed DOPPEL (Diversity-based Optimization of Peptide Properties to Enhance Ligations) was used to simplify this synthesis. Furthermore, various synthesis strategies and general advice for completing ! ! iv mega-synthesis projects in the future are detailed in this chapter. In both the DapA and Dpo4 projects, one of the most prominent challenges was the handling of poorly soluble peptide segments. These poorly soluble peptides can lead to dramatic yield losses and additional complexities. In the third major synthesis project of this thesis, GroES, we overcame an even greater insolubility challenge. This 97-residue protein could not be synthesized due to extreme solubility challenges with its C-terminal half. In response to this challenge, a new chemical tool was developed to link a solubilizing peptide (Helping Hand) to the C-terminal half of the protein. Key to this approach is a new synthetic building block, Fmoc-Ddae-OH, which is easy to synthesize, incorporate, and cleanly remove once the solubilizing function is complete. Overall, this dissertation pushes the limits of chemical peptide and protein synthesis, and provides exciting directions for the next wave of biochemists looking to use chemical protein synthesis to study interesting problems and engineering challenges.