Date of Award

December 2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Ivan V. Korendovych

Keywords

calmodulin, hepatocellular carcinoma, lanthanides, protein engineering, protein nmr

Subject Categories

Physical Sciences and Mathematics

Abstract

Enzymes are powerful tools that are capable of catalyzing reactions with high specificity and efficiency. Many naturally occurring enzymes are used as tools in industry and medicine, from food additives to pharmaceuticals to biofuel production. Protein engineering is used to make enzymes more readily available, to gain new or improved catalytic function, or optimize properties such as thermostability and enantioselectivity. In this thesis we will discuss the variety of ways proteins can be modified to one day be able to be put to use in industrial and clinical settings.

My goal has been to optimize proteins for industrial and clinical use by either a) understanding how the protein evolved so that the knowledge can be applied to other protein designs, b) finding new ways to express proteins so that they can be isolated and used more easily in industrial and clinical settings, and c) creating new proteins that can be used for industrial and clinical settings. In Chapter 2, through protein engineering, a new protein was made that selectively binds lanthanides over its natural metal, calcium, with high affinity. This provides us with important information into how metalloproteins evolve metal specificity, which can be applied to designing new metalloproteins. In Chapter 3, we investigate a possible application of this protein in therapeutics by attaching an antibody to the protein structure that is specific for Hepatocellular Carcinoma cells and using the protein to bind and deliver radioisotopes. This would allow us to not only create a new treatment option for this variety of cancer, but also allow us to optimize the treatment more effectively and easily through protein evolution. In Chapter 4, we discuss how changing the expression vector of an industrially relevant enzyme, formate dehydrogenase, allows it to be synthesized and secreted by Pichia pastoris and can be an important first step in creating a self-sustaining and environmentally safe method of producing methanol biofuel. Chapter 5 focuses on investigating a new method of evolving designed proteins that uses NMR by analyzing known mutations in an extensively evolved protein. This would one day allow potential enzymes to be optimized readily and cheaply for commercial use. Finally, Chapter 6 will focus on using amino acids as an alternative to traditional chiral ligands in organometallic synthesis. These new ligands would be much cheaper to make, easier to produce, and have the added benefit of forming fibrils that would make them easy to collect and attach to surfaces. These studies contribute to a better understanding of the variety of methods proteins can be optimized for use in our daily lives.

Access

Open Access

Download

Share

COinS