Unnatural amino acid mutagenesis: Progress towards non-natural biocatalysis and investigations of protein thermodynamics via 19F NMR Public Deposited

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  • March 20, 2019
Creator
  • Rydeen, Amy
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Evolution created the complex and versatile protein landscape that underpins cellular function, through the combinatorial assembly of only 20 canonical amino acids. Contemporarily, protein engineers are expanding the capabilities of protein beyond the constraints of natural selection by tailoring them for applications in a ‘man-made’ world. For instance, proteins are finding widespread success as immuno-therapeutics and as industrial catalysts for the production small molecule pharmaceuticals. However, the pursuit of protein engineers is hindered by the limited chemical functionality imparted by the natural proteinogenic amino acids. In vivo unnatural amino acid mutagenesis technology, also known as genetic code expansion, has allowed the localized and/or widespread installment of non-canonical amino acids bearing non-natural functional groups. This simple yet powerful technology has supplemented the fundamental few with hundreds of unnatural amino acids (UAAs). Despite the numerous applications of UAAs that have been disclosed, there still exists significant potential for further exploration. This dissertation advances the applications of UAA mutagenesis to include the installation of UAAs for novel biocatalysis and the biophysical investigation of protein and protein complex stability via 19F NMR. We present the design, synthesis and in vivo incorporation of organocatalyst-inspired proline-modified UAAs. Free proline-elaborated UAAs perform aqueous aldol chemistry, however fail to transfer this activity to a protein scaffold. The groundwork discussed herein should assist future efforts towards the creation of protein catalysts with UAA-based reactivity. In addition, we utilized a 19F UAA to study the stability of a protein and weak protein complex in the presence of osmolytes and related biological small molecules via 19F NMR. The data demonstrate that although osmolytes are primarily characterized as protein stabilizing agents, related non-osmolytes stabilize a protein fold within the range of the most and least powerful osmolytes. On the other hand, osmolytes and non-osmolytes differ in their effect on the stability of a protein complex. Osmolytes had negligible influences on a charge mediated protein-protein interaction and non-osmolytes were significantly perturbing. This result implies that osmolytes were selected by nature for their compatibility with the electrostatic interactions between protein surfaces that are attributed to the regulation of cellular function and structure.
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  • In Copyright
Advisor
  • Pielak, Gary
  • Water, Marcey
  • Erie, Dorothy
  • Brustad, Eric
  • Redinbo, Matthew
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2018
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