This thesis addresses several questions regarding the structure details of the proteins involved in sigma 54 dependent gene transcription initiation both in E.coli and Aquifex aeolicus. In order to place each topic in its appropriate context, a general introduction is provided in the first chapter, and supplemented with additional, more detailed introductions in each subsequent chapter. The second chapter reflects the central project of this thesis, the determination of the -24 DNA binding domain of sigma 54 from E.coli using Nuclear Magnetic Resonance spectroscopy. The structure, which is a three-helix fold, was compared with the same domain in the sigma 54 from A. aeolicus. DNA binding affinity with the -24 promoter element was also determined by using native gel.

In the third chapter, I describe the previous biochemical studies of histidine kinase sensor protein 4 from Aquifex aeolicus. Then I present my work towards the structural studies of PAS domain of Hksp4 using X-ray crystallography. Diffraction data with 2.8 Angstrom resolution was analyzed, which showed the completeness of the data is below 90%. Molecular replacement was used to build the initial electron density map with no success due to the low sequence identities between the target protein and the models. Experimental phasing was also tried with no success in growing crystals.

In the fourth chapter, I work on the structure determination of NtrC from Salmonella typhimurium, which is among the very first few proteins studied in the sigma 54 dependent gene transcription initiation process. The initial attempt was to use NMR. Beryllofluride was used to activate the RC domains of NtrC. Without the DNA binding domain, RC domains can not be activated by beryllofluride like the full length. Then X-ray crystallography was used for the full-length NtrC to determine the structure of NtrC in the activated state. However, the studies were complicated by low solubility and protein aggregation problems. No crystals were formed in the activated state.

Applications of laser-polarized xenon nuclear magnetic resonance (NMR) spectroscopy and imaging have grown in number due to the exceptional sensitivity of xenon to its local environment. When paired with microfluidic technology, xenon-based molecular sensors (XBMS) have the potential to be used in a broad range of applications from medical devices to geochemistry. Detection of XBMS on a microfluidic chip requires remote detection NMR methodology, where the encoding and detection of aqueous xenon is separated in both space and time. In order to give a clear sense for the state of XBMS with respect to both microfluidics in NMR and magnetic resonance (MR) contrast agents, this thesis provides the background of NMR relevant for performing remote detection NMR experiments, a history of laser-polarized xenon NMR spectroscopy, traditional proton-based magnetic resonance (MR) contrast agents, and the development of XBMS as MR contrast agents.

The primary XBMS used in this thesis is a water soluble version of the organic cryptophane-A molecule (cryA). Xenon had been shown to associate with cryA in water with high affinity and relatively fast exchange and cryA can be indirectly detected in low concentrations in solution. A variable previously unexploited in laser-polarized xenon NMR experiments using XBMS was temperature. Temperature is an important parameter in XBMS experiments for generating MR contrast as the affinity for xenon with all soluble versions of cryptophane-A had been shown to increase with increasing temperature and also the exchange of xenon in and out of the cryA was previously shown to increase at higher temperatures. In experiments described in this thesis, temperature was used to increase contrast in both spectroscopic and imaging studies. At 37 C, the threshold for detecting the presence of a negatively charged cryA in solution was decreased to 10 nM - two orders of magnitude lower than what was previously detectable for a positively charged cryA at 25 C.

In order to detect the presence of XBMS with an optimized coil separated in space from the location of the sensors (remote detection), a radio frequency (RF) probe tuned to the resonance of xenon was built that was compatible with aqueous xenon flow through 1/16 inch tubing. An efficient means of dissolving xenon into water close to the encoding region was developed leading to the detection of the first aqueous xenon remote detection travel curves. Furthermore, polystyrene beads grafted with 1.3 mM cryA were loaded into 1/16 inch tubing in the encoding region. By applying a saturation pulse at the cryA resonance frequency in the encoding region, the presence of cryA was remotely detected by monitoring the subsequent decrease in intensity at the aqueous xenon resonance frequency.

Following the remote detection of XBMS in this macroscopic environment, a platform was developed for the remote detection of XMBS on a microfluidic chip. An RF probe tuned to both H-1 and Xe-129 was designed and fabricated for remote detection experiments at the microfluidic scale. This probe was used to detect aqueous xenon flowing through a microfluidic chip via the time-of-flight dimension. Furthermore, water proton flow through a microfluidic chip was detected in remotely in both spectroscopic and imaging experiments.

Finally, a novel class of XBMS is discussed in detail. Instead of detecting XBMS by their unique resonance frequency in solution, when paired with a paramagnetic center XBMS have the potential to be used as T1 relaxation agents. The theory of T1 contrast agents is presented and the simulation developed to model this new type of xenon-based contrast agent is described in detail. The results of the simulation are tabulated and the early work with the first agent synthesized for this purpose is presented. The incorporation of a XBMS T1 relaxation agent with a microfluidic device could lead to the development of chemically sensitive, portable, low-field devices.

This dissertation addresses the mechanism of σ54 activation by the AAA+ ATPase transcriptional activators. The first chapter provides a general introduction to σ54‑mediated bacterial transcription initiation by outlining the existing structural and biochemical data on σ54 and its activators. The majority of our structural information comes from high resolution structures of individual σ54 and transcriptional activator domains, with the rest coming from low resolution structures that determine relative positions of the domains. The structural and biochemical properties of these domains, viewed in the context of the mechanisms of related motor proteins, led to our hypothesis of σ54 activation. In this dissertation, I propose that the transcriptional activator’s hexameric ATPase domain uses conserved loops, known to contact σ54, to pull on and thread the σ54 N‑terminal activator interacting domain through its central pore. In this model, through processive rounds of ATP hydrolysis, the transcriptional activator applies enough force to generate a conformational change in the σ54‑RNA polymerase holoenzyme that allows it to melt DNA thus initiating transcription. The primary goal of my work has been the study of the activation mechanism using a variety of techniques, including traditional NMR‑based structure determination, in vivo and in vitro biochemistry, and single molecule optical tweezers experiments.

The second chapter outlines the central work of this dissertation, the structural characterization of the region of σ54 responsible for contacting the activator and necessary for initiating transcription. We show that the activator interacting domain (AID) is intrinsically disordered, but becomes ordered when bound to the transcriptional activator, NtrC1, in its ATP state. In particular, we show that two predicted helices in the sequence are sufficient for activator binding with native‑like affinity, and that the first helix in particular represents the primary region of contact. We applied TROSY‑based NMR techniques to the structure determination of this high molecular weight complex, but most signals were broad due to dynamics or disorder, preventing a high resolution structure determination of the AID bound to the activator. This indicates that the AID does not bind the activator in a single conformation, but rather in multiple conformations each experiencing a different chemical environment. This may reflect an activation mechanism involving dynamic changes to the σ54‑activator interaction site.

The third chapter takes an in vivo biochemical approach to study the σ54 activation mechanism by characterizing the functionality of σ54 with insertions and deletions near the activator interacting domain. These insertions distinguish between two competing mechanisms: whether the σ54 AID only binds the surface of the activator or, as we propose, is threaded through its central pore. We find that a flexible linker region between the sites of activator and RNA polymerase binding is essential for fully functional σ54. While the length of the linker does not matter, the insertion of a small, stably folded domain between these two interaction sites is detrimental to σ54 activity in vivo. This is consistent with the threading of the AID and linker by the transcriptional activator, which would not be inhibited by the addition of extra unstructured residues but would be stalled by the addition of a folded domain in the linker.

The fourth chapter outlines single molecule optical tweezers experiments to characterize the effect of force on a domain of σ54. In particular, we examine the possibility that the σ54 core binding domain (CBD), responsible for contacting core RNA polymerase, acts as a conformational fracture point that undergoes rearrangements when force is applied. The NMR structure of the CBD revealed two folded subdomains with a hydrophobic interface that could plausibly be disrupted by the force of the activator threading the N‑terminus. By applying force with optical tweezers to either end of the CBD alone, we observed a separate unfolding event likely corresponding to unfolding of its seventh helix, but there was no evidence that the less stable of the two CBD subdomains unfolds before the other. Future experiments to conclusively test the force‑dependent activation of σ54 may require reconstituting and pulling on the full RNA polymerase holoenzyme complex, including core RNAP, promoter DNA, and full length σ54.

The fifth chapter steps back from the study of the σ54 activation by the transcriptional activators to examine the regulation of the transcriptional activators themselves. Existing evidence shows that phosphorylation of the regulatory receiver domain of the transcriptional activator NtrC changes the population of its active and inactive states, with most states resembling the active conformation after phosphorylation. The activated receiver domain promotes the oligomerization of the ATPase domain, which in turn can activate σ54. In this chapter, I studied an NtrC receiver domain from the piezophilic bacteria, Shewanella violacea, which turns on σ54‑dependent gene expression in response to high pressure. We hypothesized that pressure alone might drive the conformational change normally associated with phosphorylation, thereby activating S.v. NtrC independent of its normal, two‑component signaling pathway. Using high pressure NMR spectroscopy, we showed that increased pressure alone does not increase the population of the S.v. NtrC receiver domain’s active state. Future work must consider the pressure sensing behavior of the other components in the NtrC signaling pathway

The work presented in this thesis describes structural investigations of proteins implicated in diseases of protein aggregation, and a protein involved in the maturation of the nitrogenase enzyme complex. Each experimental chapter is full self-contained.

Protein aggregation into plaques has been identified as a central event in many human diseases such as Alzheimer's disease, type II diabetes, and the spongiform encephalopathies. Generally, the protein plaques display a common morphology consisting of long unbranched proteinaceous fibrils commonly referred to as amyloid. The intrinsically insoluble nature of proteins contained within amyloid fibrils has greatly impeded high resolution structural studies utilizing single crystal X-ray diffraction and high-resolution liquid NMR spectroscopy. Amide hydrogen exchange is often used to probe hydrogen bonding in proteins and does not strictly require soluble protein for study. In the first chapter of the thesis, I present an amide hydrogen exchange study conducted on an amyloid forming fragment of the protein implicated in the spongiform encephalopathies, PrP(89-143). These experiments identify the strongly hydrogen bonded fibrillar core.

The next section of the thesis contains work conducted on ABeta, the amyloid forming protein implicated in Alzheimer's disease. Due to increasing lines of evidence that the ABeta fibrils themselves do not cause neurodegeneration, much effort has been directed into studying the monomeric and pre-fibrillar oligomeric states of ABeta. This work is greatly impeded by both ABeta's strong propensity to aggregate as well as the inherent heterogeneity of the molecules in solution. In order to circumvent these issues the experiments presented herein are interpreted with respect to models of the structural ensemble obtained via molecular dynamics calculations, thereby yielding a model of the structural ensemble that is validated to an extent unavailable to either technique alone.

The final section of this thesis describes work conducted on the gamma-subunit of nitrogenase, the enzyme complex responsible for the entry of nitrogen into the biosphere. The gamma-subunit's proposed role in the cell is to deliver the cofactor present at the active site of nitrogenase that is required for activity. I present biochemical experiments that demonstrate that the N-terminal domain of the gamma-subunit is responsible for mediating the interaction between the gamma-subunit and apo-nitrogenase, the solution structure of N-terminal domain of the gamma-subunit, and NMR experiments that characterize the cofactor binding site on the C-terminal domain of the gamma-subunit.

This thesis addresses a diversity of questions regarding the structural details of sigma54 transcriptional activation, and the function of sigma54 activation in the hyperthermophile Aquifex aeolicus. In order to place each topic in its appropriate context, a general introduction is provided in the first chapter, and supplemented with additional, more detailed introductions in each subsequent chapter. The second chapter reflects the central project of this thesis, the determination of the structure of the DNA-binding domain of an NtrC-like sigma54 transcriptional activator protein in Aquifex aeolicus, in complex with its high-affinity DNA binding site. Although this project was attempted by NMR, its structure was ultimately solved by X-ray crystallography. This structure, which shows slight DNA-bending, is compared to the recent structure of the homologous Fis protein in complex with DNA.

In the third chapter, I describe a cross-comparison of DNA-binding domains from Aquifex aeolicus, including new structures of the DNA-binding domains of NtrC1 and NtrC2, and comparisons to NtrC4, ZraR and NtrC from Salmonella enterica serovar typhimurium, and Fis from Escherichia coli. These structures of DNA-binding domains from a single family enable a detailed comparison of structural changes that tune protein function. A trend is noted, in which larger dimerization interfaces in the DNA-binding domains appear to correlate with smaller dimerization interfaces in the other domains of the protein. The additional structure of the full-length NtrC1 protein is presented, but the DNA-binding domains were missing from the density, providing evidence for the flexibility of the linker between the central and DNA-binding domains. Together with structural information about the CD linker regions at the N-termini of the DNA-binding domains, I conclude that the CD linker regions are generally unstructured in the inactive state, functioning as tethers to bring the RC domains of NtrC into appropriate local concentrations for activity.

In the fourth chapter, I depart from sigma54 transcriptional activators, and discuss NMR studies on the sigma54 factor. The Darst lab at Rockefeller University has produced a crystal structure of the full-length intact sigma54 factor in complex with DNA. However, poor data quality prevents regions of the molecule from being traced unambiguously through the density. By applying new methods in NMR including TROSY spectroscopy and specific isotopic labeling, I attempted to resolve small regions of structure in this ambiguous region. However, these studies were complicated by protein aggregation.

The fifth and final chapter takes a step back from structural perspective and compiles and discusses our current understanding of sigma54 regulatory networks in Aquifex aeolicus. This analysis of genes with bioinformatics and biochemical techniques led me to discover that NtrC3, an unstudied sigma54 transcriptional activator, binds upstream of the dhsU gene, which is responsible for sulfur metabolism. NtrC3 also appears to be associated with a heme-binding, soluble histidine kinase, HksP4. In order to explore this two-component regulatory system further, the HksP4 protein was prepared and its gas-binding properties were studied in collaboration with Dr. Brian Smith of the Marletta lab. We find that the heme binds O2, NO, and CO gases in the reduced ferrous iron state, but did not observe any autophosphorylation activity in any state. I conclude that an additional element, such as an additional activator, or more physiological conditions, would be necessary for activity.

Glycopolymers have served as useful tools for studying glycobiology for over two decades. A significant body of literature has demonstrated that these macromolecules are capable of predictably stimulating lectin receptors, suggesting a potential for clinical applications ranging from adjuvants to immunosuppressants. However, commonly-used scaffolds exhibit notable limitations, including unnatural backbones and glycosidic linkages, which limit their therapeutic potential. In a recent publication, we demonstrated that N-carboxyanhydride (NCA) polymerization is amenable to glycosylated monomers, allowing for the synthesis of true glycopolypeptides. In this dissertation, I demonstrate the applicability of the platform to a variety of glycan structures. In particular, I focus on potentially immunogenic carbohydrate motifs with the ultimate goal of developing synthetic C-type lectin receptor (CLR) agonists designed to activate antigen-presenting cells (APCs) for cancer immunotherapy.

Designing such immunostimulatory glycopeptides requires a general understanding of the cells and pathways important to generating an anticancer immune response. In Chapter 1, I provide an overview of APCs and their associated immune receptors, including CLRs. I include in this section a brief summary of reported pattern recognition receptor agonists, including clinically-approved synthetic ligands, and note the lack of well-defined CLR agonists. I then discuss the relationship between cancer and the immune system with the goal of rationalizing our strategy of activating APCs to induce a robust anticancer response. To provide further context, I conclude with a summary of modern and historical approaches to cancer immunotherapy.

Our initial NCA polymerization publication focused on an N-acetylgalactosamine-serine monomer; synthesis of the desired immune-activating glycopolypeptides required that I first establish the generalizability of the platform to other carbohydrates. In Chapter 2, I present work showing that di- and trisaccharide-serine NCAs can be polymerized using transition metal initiators to give glycopolypeptides of controllable length and glycosylation density. In particular, I focus on mannosylated and glucosylated structures that could potentially bind and activate CLRs on antigen-presenting cells, setting the stage for my intended therapeutic application.

In Chapter 3, I demonstrate for the first time that NCA-derived glycopolypeptides can be rationally designed to activate desired CLRs. Specifically, glucosylated and mannobiosylated structures stimulate murine monocyte-derived cells and dendritic cells in vitro through the CLRs Dectin-1 and Dectin-2, respectively. Activation was dependent on the carbohydrate structures used and consistent with previous literature describing glycan preferences of these receptors. Chapter 4 expands on these promising results by taking the mannobiosylated glycopolypeptides, which activate the dendritic cell receptor Dectin-2, into murine models of pancreatic cancer. I first show that these materials are able to inhibit tumor growth in vivo. Furthermore, conjugation of these Dectin-2 agonists to a costimulatory Toll-like receptor agonist greatly enhances cytokine release in cell culture and tumor growth inhibition in vivo. Alternatively, attachment of these glycopolypeptides to tumor-targeting antibodies similarly enhances their immunostimulatory effects. These observations provide promising evidence that activation of APCs via NCA-derived glycopolypeptides may indeed be a viable strategy for cancer immunotherapy.

Finally, in Chapter 5 I describe some ongoing challenges with the NCA polymerization technology. As a platform, it would be desirable to have access to glycopolypeptides with multiple functional handles to maximize the available conjugation possibilities. Here, I describe my efforts to improve on our previously-reported dual-end-labeling technology. Through additional characterization and optimization of functionalized NCA initiators, I uncover some flaws with our initial strategy and propose potential solutions. This work points to the need for new initiators for the NCA polymerization of glycopolypeptides. Advancements in this area will allow us to better access multicomponent glycopolypeptide-based conjugates in the future.

Technology to profile nucleic acid materials has been greatly advanced over the past decade. Nowadays, tools such as real-time quantitative PCR (qPCR) and next generation sequencing (NGS) are standard workhorses for biomedical laboratory. However, in contrast to great advance in nucleic acid science, assays used to quantify proteins and antibodies immune markers remain much stagnant. Traditional assays immunoassays such as enzyme linked immunosorbent assays (ELISA) and western blots, invented almost half a century ago, are still widely used. These assays face limitations including low sensitivity, low specificity, limited multiplex power and requirement of large sample volume. To this end, I have thought to create a methodology that allows researchers to leverage advanced nucleic acid tools to profile protein and antibody immune markers. In this thesis, I will provide an overview on current landscapes of nucleic acid-based assays for protein and antibody immune markers, and discuss how the new nucleic acid-based assay that I developed differentiates and complements other nucleic acid tools. Then, I will describe in details about our new nucleic acid assays including the creation of protein-DNA conjugates and use of these reagents to transform protein/antibody identities into quantifiable nucleic acid signals. Furthermore, I will describe potential impact of such methods in research and clinical settings. Finally, I will discuss potential development that is required to make this assays widely deployable in research and clinical communities.