ADVANCED BIOANALYTICAL TECHNIQUES FOR CHARACTERIZING PROTEIN INTERACTIONS IN COMPLEX BIOLOGICAL SYSTEMS

Abstract

Structural biology aims to study three-dimensional structures of macromolecules to better understand their functions in physiological contexts. Specifically, detailed insights into protein interactions and dynamics is essential for understanding protein function, revealing the fundamentals of all types of cellular processes and diseases. Recent advancements in different bioanalytical techniques has drastically expanded applications to studying protein systems under these physiological conditions, but the inherent complexity of native biological samples consistently demands new techniques and strategies. This dissertation focuses on using recent advancements in hydrogen deuterium exchange mass spectrometry (HDX-MS) and cryogenic electron microscopy (cryoEM) to characterize protein interactions and dynamics in uniquely difficult protein samples.Conventional HDX-MS applications have largely been restricted to simplified, purified protein systems, with considerable effort focused on using HDX-MS for epitope mapping monoclonal antibodies (mAbs). However, in order to study the immunology of a vaccine-induced anti-toxin response, a close examination of the polyclonal antibody (pAb) response is needed. Epitope mapping of human polyclonal antibody (pAb) is inherently complex, and predicting vaccine-elicited serum antibodies binding is difficult, resulting in limited techniques for direct conformational epitope mapping of pAbs. Here, we demonstrated the utility of our subzero temperature ultra-high-pressure liquid chromatography hydrogen-deuterium exchange coupled mass spectrometry (UPLC-HDX-MS) platform for direct conformational epitope mapping of pAbs in anthrax vaccine adsorbed (AVA) vaccine-elicited serum antibodies to protective antigen (PA). Moreover, we modified the protein thermal depletion (PTD) technique to remove unbound antigens, thereby enhancing the sensitivity of HDX-MS-based epitope mapping in complex serum samples. By implementation of PTD-HDX-MS at 60°C, we successfully characterized conformational epitopes of PA in two human vaccine-elicited serum samples. HDX-MS experiments have made significant efforts to limit back-exchange, the unavoidable loss of varying degrees of deuteration occurring at later stages in the workflow, which reduces sensitivity to shifts in deuterium uptake. Regardless of the approach, whether bottom-up or top-down workflows, back-exchange remains one of the most persistent problems in HDX analysis and solutions to this shortcoming are often complicated. While attempts to reduce back-exchange have been successful, these methods typically require complex sub-zero cooling systems to reduce rates of exchange. Recently, our group developed an electrospray-assisted device capable of ultra-low volume sample extraction termed Spray-capillary. Here, we utilized Spray-capillary to inject low-volume deuterium-labeled sample plugs (1-100 nL), which were surrounded by background buffer, for MS analysis using pressure elution. We observed minimal back-exchange in the middle of the sample plug at room temperature, attributed to the low diffusion rate of the sample within the surrounding background buffers. We conclude with room temperature Spray-capillary time-course HDX-MS analysis on a standard protein system, showing that Spray-capillary facilitated HDX-MS analysis offers a simple alternative for protein characterization. A complementary technique in cryoEM has become an indispensable technique in structural biology, providing crucial information about conformational variability of purified protein samples. As such, we sought to exploit this for purposes of exploring vulnerabilities for antifolate inhibitor development. The folate biosynthesis pathway has long been a target for antibiotic inhibition by FDA-approved trimethoprim and sulfamethoxazole, due to its vital role in bacterial cell growth. Few enzymes in the folate pathway are well-studied, which includes dihydrofolate reductase (DHFR), the target of trimethoprim, pyrimethamine, and methotrexate. Recently, sulfamonides targeting dihydropteroate synthase (DHPS) have been shown to act synergistically with DHFR inhibitors, suggesting additional antifolates acting synergistically could potentially improve the current antibiotic profile. An interesting candidate in dihydroneopterin aldolase (DHNA) exists as an octamer in solution with eight active sites shown to be allosterically regulated in Mycobacterial tuberculosis DHNA. Multiple inhibitor scaffolds and crystal structures of DHNA from several microorganisms have been reported, but a lack of activity of DHNA inhibitors in cells suggests alternative approaches to classical substrate-mimetic strategies could be needed. To gain insights into DHNA enzyme dynamics or potential allostery during catalysis, we used cryoEM single particle analysis (SPA) to capture high-resolution features of DHNA in the presence of substrate and product. Here, we report the first cryoEM structures of Yersinia pestis DHNA and kinetic data supporting an allosteric mechanism. These contributions enhance our understanding of DHNA dynamics during catalysis and provide valuable insights into potential vulnerabilities for inhibitor development.