UC Berkeley

Proteins carry out an enormity of complex diverse cellular functions: they are involved in innumerable cellular processes including but not limited to cell signaling, ion and small molecule transport, cell structure, and cellular homeostasis and stress responses. These diverse and complex functionalities are enabled by twenty α-amino acid building blocks strung together in precisely controlled sequences in an mRNA templated manner by the ribosome. In this sense, proteins are biopolymers constructed of α-amino acid monomers. Proteins can adopt a multitude of different three-dimensional conformations that confer functionality, from intrinsically disordered proteins to megadalton scale multi-subunit proteins. Harnessing the properties of proteins consisting solely of natural α-amino acids has already been transformative in protein therapeutics and materials. However, what architectures and functionalities could we access by moving beyond α-amino acids? Much progress has been made in this area using genetic code expansion, solid phase synthesis, bioconjugation and protein semisynthesis methods. From these studies, we know that non-α-amide backbones in proteins confer value-added properties like enhanced proteolytic stability, reduced immunogenicity, and novel folding patterns inaccessible to α-amide backbones. However, there remain several bottlenecks that limit scalable production of such valuable proteins, particularly those that are backbone-modified (non-α-amide backbones).In Chapter 1, I will detail the existing body of literature that focuses on introducingnon-α-amide backbones into proteins and peptides. I will discuss the relative merits and limitations to these methods, which include genetic code expansion in vivo and in vitro, post-translational backbone modification enabled by enzymes involved in natural products biosynthesis, enzymatic bioconjugation, and intein-mediated strategies.In Chapter 2, I will discuss the development of a strategy used to incorporate γ-amino acids into proteins post-translationally by leveraging proximity-guided acyl shift chemistry, which circumvents some of the bottlenecks to genetic code expansion described in Chapter 1. Efforts toward β-amino acid incorporation using this method will also be described.In Chapter 3, I will introduce a new strategy, Backbone Expansion via Enzymatic Side Chain Transfer (BEST) that will allow access to proteins with completely nonproteinogenic material inserted into the backbone in internal regions of proteins. This method utilizes genetic code expansion and chemoenzymatic modification to afford backbone-modified proteins. Chapter 3 encompasses unpublished work scoping compatible protein systems for BEST, which enabled successful demonstration of BEST in Chapter 4.In Chapter 4, I will detail successful demonstration of the BEST strategy introduced in Chapter 3. This chapter focuses on modification of a small, stable protein GB1 as a model system. Here, I demonstrate the post-translational insertion of an abiotic PEG chain into the protein backbone and profile the resulting biophysical ramifications of this modification via circular dichroism and hydrogen-deuterium exchange mass spectrometry. We anticipate that this method will be used to generate protein-polymer hybrids that combine the sequence definition and predictable folding properties of proteins with the stability and material properties of polymers.