Utilization of Unnatural Amino Acids to Modulate Protein Structure and Function

• Main Author: Halonski, John
• Format: Others
• Language: English
• Published: W&M ScholarWorks 2018
• Subjects: Biochemistry
• Online Access: https://scholarworks.wm.edu/etd/1530192786; https://scholarworks.wm.edu/cgi/viewcontent.cgi?article=1322&context=etd

Proteins are capable of an astounding array of functions using only the 20 canonical amino acids; however, the ability to add new functional groups to the genetic code through the utilization of unnatural amino acids (UAAs) has greatly expanded our ability to study and manipulate proteins. By expanding the diversity of functional groups within proteins, a wide variety of applications in industry as well as in fields such as diagnostics, biochemistry, and materials science are now possible. These applications have further been expanded through the development and optimization of bioorthogonal reactions which can occur under physiological conditions with a high degree of specificity, allowing modulation of the structure and function of proteins within their natural state. Several applications of UAA technology involving bioorthogonal reactions will be explored in this thesis. Optimization of a previously developed bioorthogonal Glaser-Hay reaction between a protein and a fluorophore will be discussed. A further application of the Glaser-Hay reaction involving natural product synthesis will also be explored. The utilization of UAA technology to form trivalent conjugates containing multiple functionalities will be described. Furthermore, the development and optimization of organic reactions leading to the formation of trivalent structures will be explored with the intention of translating these reactions to biological systems. The ability to site-specifically immobilize a hyperthermophilic carboxylesterase enzyme onto a stabilizing resin will also be discussed and the benefits of protein immobilization will be demonstrated. Finally, the synthesis and development of novel TMS and aldehyde UAAs will be described and their applications will be explored. The applications highlighted in each chapter demonstrate some of the numerous possibilities that can be explored through modulation of the building blocks of proteins.