| Description |
The chemical synthesis of peptides and proteins, through solid-phase peptide synthesis and chemoselective ligation reactions, has opened up access to many interesting biochemical applications. Synthetic peptides can be made with site-specific modifications such as fluorescein or specific post-translational modifications for binding or structural studies, while synthetic proteins can be made in the unnatural D-chirality amino acids for use in mirror-image phage display. This dissertation describes several applications of these techniques in the context of diverse projects, as well as the development of new tools to streamline chemical protein synthesis. One of the most commonly encountered problems in the synthesis of proteins and peptides is insolubility of peptide segments in aqueous conditions. Chapter 2 describes the development of a second-generation semipermanent solubilizing tag that addresses this issue. This tool, Ddap, is demonstrated in the synthesis of the challenging Shiga toxin subunit B protein (StxB), a promising target for mirror-image phage display. The chemical synthesis of proteins also requires chemoselective ligations; however, these ligations can be prohibitively slow and require high concentrations of reacting components. Chapter 3 describes the development of traceless click-assisted native chemical ligation (CAN), a technique that can greatly accelerate ligation kinetics and improve yield. Chapter 4 describes the synthesis and application of different cyclic peptides in the context of the AAA+ ATPase Vps4. Here, cryo-EM structures of Vps4 iv bound to a cyclic substrate-peptide demonstrate a general model for how AAA+ ATPases can translocate substrates from internal loops as well as termini. |
