The study of protein-protein interactions in essential to our understanding of cell biology and our development of therapeutics for human diseases. Bioreactive unnatural amino acids (Uaas), which can selectively crosslink with specific nucleophilic canonical amino acids, have the potential to change the way we study protein-protein interactions. Bioreactive Uaas can allow us to study and manipulate protein-protein interactions without fear of drastically altering native protein structure, function, or binding capabilities. By engineering a bioreactive Uaa into a protein in order to target another protein – or proteins – of interest, we make a relatively small mutation to the protein, but endow the mutant protein with an incredibly powerful novel capability to crosslink the protein(s) of interest. The potential of bioreactive Uaas is great, but as yet has not been proven. The work described in this thesis attempts to showcase some of the potential of bioreactive Uaas as both tools to develop potential biotherapeutics and as tools to study and identify novel protein-protein interaction.
Chapter 1 describes efforts to elucidate how chemical crosslinking affects the biologics of receptor-ligand interactions. Using human growth hormone as a model system, we investigate whether a native ligand can be engineered to successfully bind and crosslink with its receptor, and how this crosslinking affects the downstream signaling of the receptor-ligand pair.
Chapter 2 is a review of current literature pertaining to the use of chemical crosslinking, including bioreactive unnatural amino acids, in the development of peptide and protein therapeutics. We compile known examples in the literature of peptide inhibitors that have been developed to crosslink proteins of interest and surmise their therapeutic potential. We also introduce the relatively novel concept of covalent protein therapeutics, particularly proteins containing bioreactive unnatural amino acids which would crosslink the proteins to their binding partners and effect a therapeutic outcome.
Chapter 3 details the major efforts of this doctoral degree in the study of the interactome of mammalian histidine kinases. Through the use of various bioreactive unnatural amino acids, we generate a library of mutants for the proteins NDPKA and NDPKB. We investigate whether these mutants crosslink proteins in mammalian cells and mammalian cell lysates, and, upon finding mutants with crosslinking potential, we utilize different purification technique to prepare samples for analysis via mass spectrometry. The proteins identified through mass spectrometry analysis have the potential to be substrates or binding partners of the mutant proteins.
