Kinetic and structural implications of using noncanonical amino acids and purification tags to study proteins

In biomolecular research, various tools are employed to enhance efficiency and enable study of proteins. These tools facilitate the isolation of specific proteins from a multitude of contaminants, introduce light-interacting functional groups to proteins, or immobilize enzymes to prevent their loss during chemical processes. However, implementing certain tools becomes challenging when they need to be integrated into proteins without disrupting the natural system. In this thesis, I present two case studies investigating the assumptions commonly made by researchers when modifying proteins.

The first case study focuses on human purine nucleoside phosphorylase (PNP), a protein I use to examine the implications of incorporating an unnatural amino acid, para-cyanophenylalanine, into two positions within the active site. The results demonstrate the enzyme tolerates the incorporation of para-cyanophenylalanine with minimal structural or kinetic perturbations at one sequence position and slightly more harm at another position. Additionally, this study provides the first comparative analysis of two common PNP enzyme sequences, revealing no significant structural or kinetic differences, challenging the long-held misunderstanding of a disease-causing variant at residue 51 and instead both sequences can now be considered WT.

The second case study investigates formate dehydrogenase by introducing a series of hexahistidine purification tags on the enzyme. Steady-state kinetic data and structural analysis from molecular dynamics simulations reveal different purification tagging strategies may have significant implications for certain experiments, contradicting the assumption that purification tags are inconsequential.

Collectively, the findings presented in this thesis highlight that while some protein modifications are well-tolerated, others introduce subtle changes that can have consequences. To prevent misinterpretation of data when employing common tools like purification tagging or unnatural amino acid incorporation, this work offers considerations and analysis methods for researchers to implement.