In partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Biology
In the
School of Biological Sciences
Arshay Grant
Will defend her dissertation
Structural and Biophysical Characterization of Bifunctional Polymyxin Resistance Enzyme, ArnA, Reveals Multiple Oligomeric States
16 April 2026
10 AM
Howey N210
Virtual link:
https://teams.microsoft.com/meet/2335961128545?p=E1rgsrRVtJOuqooknb
Thesis Advisor:
Ingeborg Schmidt-Krey, Ph.D.
School of Biological Sciences
Georgia Institute of Technology
Committee Members:
Vinayak Agarwal, Ph.D.
School of Biological Sciences
School of Chemistry and Biochemistry
Georgia Institute of Technology
Aditi Das, Ph.D.
School of Chemistry and Biochemistry
Georgia Institute of Technology
JC Gumbart, Ph.D.
School of Physics
Georgia Institute of Technology
Raquel Lieberman, Ph.D.
School of Chemistry and Biochemistry
Georgia Institute of Technology
ABSTRACT: According to the World Health Organization, antimicrobial resistance is projected to cause up to 10 million deaths annually by year 2050. A common resistance mechanism used by bacteria involves modification of the outer membrane, particular through lipid A remodeling. ArnA, a bifunctional enzyme, plays a key role in the biosynthesis of 4-amino-4-deoxy-L-arabinose (L-Ara4N), a modification of lipid A that contributes to resistance against polymyxins.
Although ArnA has been structurally characterized and is thought to exist as a hexamer, with two functional domains coordinating lipid A modification, it remains unclear whether this is its only biologically relevant oligomeric states. The potential for ArnA to form smaller oligomeric assemblies has not been thoroughly investigated. Understanding the oligomerization mechanism may provide new opportunities for therapeutic development, especially given the absence of commercially available inhibitors targeting ArnA. Therefore, this thesis aimed to characterize the oligomeric states of ArnA using a combination of biochemical, biophysical, and structural techniques.
Chapter 1 provides evidence that ArnA exists in two oligomeric states in solution. Size exclusion chromatography revealed two distinct elution peaks, suggesting the presence of two ArnA species. This observation motivated further investigation using additional biochemical and biophysical methods to characterize these species.
Chapter 2 demonstrates that ArnA exists in multiple oligomeric states and support a concentration-dependent dependent, stepwise assembly model in which monomers associate sequentially to form higher-order oligomers. A combination of biochemical and biophysical techniques was used to identify and characterize these oligomeric species.
Chapter 3 revealed a pseudohexameric assembly exhibiting conformational variability, as well as additional unexpected assemblies, including an isolated domain pair. These findings are particularly significant as they uncover previously unrecognized structural variability in ArnA. Single particle cryo-electron microscopy was used to resolved and characterize these assemblies.
Overall, this thesis provides the first experimental evidence for ArnA assembly beyond previously reported structural data. This work is impactful as it is the first to characterize multiple and new oligomeric species of ArnA, providing first experimental evidence for concentration-dependent assembly. Furthermore, this work highlights the importance of combining techniques to investigate dynamic protein assemblies that can exist across a range of oligomeric species.