Engineering Allostery in Tryptophan Synthase
Open Access
- Author:
- D'Amico, Rebecca
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 07, 2021
- Committee Members:
- David Boehr, Co-Chair & Dissertation Advisor
Wayne Curtis, Outside Unit & Field Member
Scott Showalter, Co-Chair & Dissertation Advisor
Timothy Meredith, Major Field Member
Wendy Hanna-Rose, Program Head/Chair
C Okafor, Major Field Member - Keywords:
- Enzymes
Allostery
NMR
Tryptophan Synthase
Allosteric Networks
Amino Acid Networks
Protein Engineering - Abstract:
- Allostery has been studied for decades, and more recently, it has been proposed that all proteins may exhibit allosteric behavior. Networks of communicating amino acids have been proposed as the mechanism to propagate allosteric signals throughout proteins. These allosteric networks are critical to protein function, regulation, and protein-protein communication. The tryptophan synthase (TS) system is a well-studied model system for protein-protein communication and allosteric regulation, due to the high level of synchronization exhibited between the alpha subunit (αTS) and beta subunit (βTS) to channel the indole intermediate. αTS and βTS also display a marked decrease in catalytic activity in the absence of the other, owing to this high level of cooperativity. Allosteric networks were previously identified for αTS and were implicated in catalytic function. In this thesis, I demonstrate that perturbation of a surface-exposed, αTS network residue in vivo led to changes in E. coli growth rates, indicating that these allosteric communication networks play a role in the function of TS in vivo. Further study on the gain-of-function mutation revealed that changing αTS dynamics propagates throughout the protein to the α/β binding interface, which leads to the widening of the indole channel. This change in channel structure may allow for more effective channeling and a more catalytically efficient enzyme. Solution-state NMR studies on the full TS complex further implicated the tight coordination between the subunits. Several substrates that bind to αTS or βTS resulted in chemical shift changes in residues associated with the other subunit, indicating that perturbing one subunit affects the entire complex. These results imply that allosteric networks play a critical role in maintaining protein function and communication.