Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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Item CHARACTERIZATION OF NON-CODING RNAS VIA NMR SPECTROSCOPY: ANALYSIS OF STRUCTURE, THERMAL STABILITY, AND DYNAMICS(2019) Nam, Hyeyeon; Dayie, Theodore K; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Non-coding RNAs are involved in various cellular processes and characterization of these RNAs may provide better insights into their functional roles. NMR spectroscopy is a powerful biophysical tool that can provide residue-specific information. Herein we examine an RNA triple helix at the 3' end of the lncRNA MALAT1, which may be a potential therapeutic target for cancer treatment. We investigate the local stability of the MALAT1 triple helix by analyzing the individual base-pair stability via NMR spectroscopy. In addition, we screened small molecules to identify the compounds that can selectively target the MALAT1 triple helix. In the second part, we studied the effect of dipolar couplings on the relaxation measurements of various non-coding RNAs using both computational and experimental measurements. The results suggest an increasing contribution of the dipolar coupling effect with the increasing size of the RNA.Item PROTEIN FOLD SWITCHING: INVESTIGATING THE MECHANISM OF αβ-PLAIT TO 3α FOLD INTERCONVERSION(2019) Solomon, Tsega Lily; Orban, John; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Naturally occurring metamorphic proteins have the ability to interconvert from one folded state to another through either a limited set of mutations or by way of a change in the local environment. However, the design of these types of proteins has remained challenging. This dissertation shows that it is possible to switch reversibly between two different but common folds employing only temperature changes. The study demonstrates that a latent 3α state can be unmasked from an αβ-plait topology with a single V90T amino acid substitution in a designed system, populating both forms simultaneously. The equilibrium between these two states exhibits temperature dependence, such that the 3α state is predominant (>90%) at 5°C, while the αβ-plait fold is the major species (>90%) at 30°C. The structure and dynamics of these two temperature-dependent topologies, as well as their energetics and kinetics of interconversion, are characterized utilizing NMR spectroscopy. Additional analysis show that the temperature-dependent characteristics of the 3α<->αβ-plait fold switch can be modulated by mutations. Stability studies through H-D exchange approach provide insight on the energetic basis for temperature induced 3α<->αβ-plait fold conversion. Further investigations demonstrated that interconversion between the 3α and αβ-plait states can be triggered by additional environmental factors including pressure, ligand binding, and redox state. This dissertation adds to the growing body of literature on protein fold metamorphism providing the first description of switching between two distinct monomeric protein folds using only temperature or pressure. Additionally, the studies of ligand- and redox-induced 3α<->αβ-plait fold switching emphasize the ability to mimic by design some of the mechanisms of fold interconversion that are found in naturally occurring metamorphic proteins. Given the high occurrence of the 3α and αβ-plait folds in the universe of known protein structures, the results suggest that such fold switching events may have occurred in the evolutionary expansion of function for natural versions of these topologies.Item ISOTHERMAL DNA DETECTION UTILIZING BICYCLIC AMPLIFICATION OF PADLOCK PROBES(2019) Zimmermann, Alessandra C.; Kahn, Jason D; White, Ian M; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)As healthcare worldwide changes to more patient-centric models, medical diagnostics need to adapt to being used in settings outside of the central lab. Current strategies to bring diagnostics to the patient’s bedside involve miniaturizing complicated amplification techniques, such as polymerase chain reaction, or building convoluted microfluidic assays that are difficult to operate. Ideally, a patient-centric diagnostic would require little instrumentation or training to operate, for which isothermal amplification techniques are ideal. Recent developments in catalytic DNA have enabled novel ways of iterating on amplification strategies to detect medically-relevant target sequences in systems that require little manipulation to operate. In this thesis we improve upon the body of research on DNAzymes, catalytic DNAs that can self-cleave in the presence of a cofactor, used in concert with amplification techniques. We create a one-pot, bicyclic amplification assay capable of detecting single-stranded oligonucleotides, with straightforward extensions to double-stranded targets, multiplexing, and integration into advanced detection platforms. The target is detected through its hybridization to a circle template, using the sequence specificity of DNA to splint the ligation of this ‘Template I,’ with minimal detection of off-target sequences. The circular Template I is copied through rolling circle amplification (RCA), with the amplicon containing a DNAzyme that will self-cleave in the presence of copper ions. This generates a second primer in situ that can be used to prime a second, pre-ligated, Template II to elevate the RCA amplification scheme from a linear method to a polynomial one. This Circle II template can then be used in a variety of detection modalities. The second amplicon can be used to cleave a hybridized FRET probe through the same copper ion cleavage mechanism as the primer generation, resulting in real-time fluorescence tracking. Alternatively, the RCA of the second circle can produce G-quadruplexes, which can be visualized with ABTS as a colorimetric endpoint that can be seen by eye, reducing the need for peripheral electronics. Finally, this thesis demonstrates the performance of the bicyclic RCA system in a phase-change system providing sequential mixing of components separated by wax layers, allowing the assay to proceed without any user interaction other than heating.Item Towards the synthesis of PNAG crosslinkers to identify protein binding partners(2019) Mrugalski, Kevin R; Poulin, Myles; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial biofilms are an area of major concern in the medical field due to natural drug resistance. Many pathogenetic species of bacteria that infect humans including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholera form biofilms and their associated infections are becoming harder to treat. Poly β-(1→6)-N-acetyl-D-glucosamine (PNAG) is a major component of biofilms across multiple species and has been found to play a key role in the early stages of the biofilm life-cycle. However, little information is known about what proteins interact with this important polysaccharide. Our goal is to create small PNAG analogues to covalently capture and identify PNAG binding partners in E. coli, an important model organism. PNAG analogues will contain photoaffinity groups, that when activated, covalently link associated proteins to the probe. Then, using a proteomics-mass spectrometry-based approach, we will identify PNAG binding partners. Here, we describe the efforts and challenges encountered synthesizing the final PNAG probes. New synthetic routes are proposed based on literature precedent that will enable synthesis of the desired compounds.Item EXPLORING ENDOGLYCOSIDASES FOR ANTIBODY GLYCOENGINEERING(2019) Tong, Xin; Wang, Lai-Xi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Monoclonal antibodies (mAbs) comprise a rapidly growing class of therapeutics with great potential in treating infections, inflammations, cancers, and autoimmunities. The glycosylation of an antibody determines its functional efficacy and structural integrity, but stringent control of the intrinsically heterogeneous N- glycans on an antibody remains a formidable challenge. Recent development of a chemoenzymatic glycosylation remodeling strategy using a family of carbohydrate- modifying enzymes called endoglycosidases is emerging as an attractive method for producing homogeneous antibody glycoforms. The success of this method depends on the discovery of efficient endoglycosidases and glycosynthase mutants. Here, we describe mutagenesis studies on several endoglycosidases and their applications in expanding the current chemoenzymatic glycoengineering strategy to different antibodies. Five projects related to this effort are described here. First, glycosynthase variants of Endo-S were generated by mutagenesis to provide enhanced transglycosylation activities and diminished tendency to hydrolyze the product. Second, mutational studies on another endoglycosidase, Endo-S2, identified novel glycosynthase variants with broader substrate specificity and higher catalytic efficiency than Endo-S mutants. This work also provided the first kinetic studies for Endo-S and Endo-S2 mutants, with important mechanistic implications. Third, the unique properties of Endo-S allowed us to develop an alternative glycan remodeling strategy to synthesize several antibody glycoforms that were not easily accessible using the conventional glycoengineering approaches. This new method can be applied in a facile, one-pot fashion to modify antibody glycosylation without the need for purifying intermediates or switching enzymes. Fourth, to further expand the toolbox for antibody glycoengineering, the substrate specificity of a newly discovered endoglycosidase Endo-CC was characterized. The highly flexible selectivity of this enzyme for protein framework substrates paved the way for glycoengineering of additional antibody isotypes, particularly IgE, as a single N-glycan was found to be indispensable for the biological functions. Finally, remodeling of IgE glycosylation with alternative glycan structures was achieved for the first time by endoglycosidases. These novel IgE glycoforms displayed distinct binding properties for IgE receptors and revealed important new aspects of the structure-function relationship of IgE antibody glycosylation. Together, these studies should facilitate the development of novel antibody-based therapeutics that are optimized in their glycosylation patterns.Item HIGH-THROUGHPUT SEQUENCING CHARACTERIZATION OF DNA CYCLIZATION, WITH APPLICATIONS TO DNA LOOPING(2019) Hustedt, Jason Matthew; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA flexibility is important both for fundamental biophysics and because DNA flexibility affects DNA packaging and regulation of gene expression through DNA looping. DNA flexibility has been studied with experiments ranging from biochemical ring closure or DNA looping experiments to AFM, crystallography, and tethered particle microscopy. Even so, the flexibility of DNA in vitro and in vivo remains controversial. In an attempt to resolve this controversy, we have developed a high- throughput, internally controlled, comparative ligation methodology using a library constructed of 1023 distinct DNA sequences ranging in length from 119 to 219 base pairs via ligation of pools of synthetic DNA of different lengths and PCR. The design incorporated barcoding for redundant identification of each molecule, allowing for a ligation reaction to be performed on the entire library in one reaction mixture. Two DNA concentrations were used in separate reactions to promote either unimolecular cyclization or bimolecular ligation and thereby explore a wide range of cyclization efficiencies (J factors). Half of each reaction mixture was treated with BAL-31 to destroy non-cyclized molecules. All products were linearized by restriction digestion and Illumina indices were added. The initial library and reaction mixtures were sequenced in a single Illumina MiSeq run. From roughly 15 million assembled reads, over 13 million were identified using software written to identify and sort our sequence library. Each molecule was counted for each condition. From our analysis we see no evidence of extreme bendability at short DNA lengths. At higher DNA concentrations where bimolecular products are produced more rapidly, we see oscillatory behavior as a function of length. In contrast, at lower concentrations where unimolecular products dominate, we observe no helical variation due to the ability for all molecules to cyclize given enough time. In order to determine J factors through cyclization, bimolecular products must also be counted. Given the constraints of this experiment, not all bimolecular products could be observed. Future experimentation can be performed to determine J factors across this size range, the results of which will improve coarse grain modeling of DNA. Extension of this methodology should be applicable to DNA loops anchored by proteins.Item Functionalized 3D DNA Crystals through Core-Shell and Layer-by-Layer Assembly(2019) McNeil, Ronald; Paukstelis, Paul; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A fundamental goal of DNA nanotechnology has been assembly of DNA crystals for use as molecular scaffolds to organize arrays of guest molecules. We use previously described 3D DNA crystals to demonstrate core-shell and layer-by-layer assembly of DNA crystals capable of accommodating tethered guest molecules within the crystals’ pervasive solvent channel network. We describe the first example of epitaxial biomacromolecular core-shell crystallization through assembly of the crystals in two or more discrete layers. The solvent channels also allow post-crystallization guest conjugation with layer-specific addressability. We present microfluidics techniques for core-shell crystal growth which unlock greater potential for finely tunable layer properties and assembling complex multifunctional crystals. We demonstrate assembly of these DNA crystals as nanoscale objects much smaller than previously observed. These techniques present new avenues for using DNA to create multifunctional micro- and nanoscale periodic biomaterials with tunable chemical and physical properties.Item An investigation of allosteric mechanisms in biotin protein ligases using integrated biophysical approaches(2019) Wang, Jingheng; Beckett, Dorothy; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Allostery is a biological process in which action, often ligand binding, at one site of the protein alters the function at another site. It provides a mechanism for modulating protein functions in a variety of cellular events ranging from signaling, metabolism, to transcription regulation. Despite the critical role of allostery in biology and intense research during the past few decades, the mechanism of long-range communication through the protein is still elusive. The Escherichia coli biotin protein ligase (BirA) is a bifunctional protein that catalyzes post-translational biotinylation and represses transcription initiation. It serves as a model system to investigate long-range allosteric communication, as binding of the effector molecule, bio-5’-AMP, promotes the repressor complex assembly by enhancing BirA homodimerization occurring at a surface 30Å away. Previous studies have established that disorder-to-order transitions of several loop segments on the ligand binding and dimerization surfaces contribute to BirA allostery. In this dissertation, integrated structural, functional, and computational approaches were used to investigate the molecular mechanisms of allosteric communication between these transitions. Double-mutant cycle analysis demonstrated reciprocal coupling between residues on two distant surfaces, and results of molecular dynamics simulations indicated that functional coupling occurs via modulation of structure and dynamics of surface loops undergo disorder-to-order transitions. Further structural and simulation-based network analyses revealed that these transitions are linked to formation of a residue network, and alanine substitutions of residues at network positions perturb both input (effector binding) and output (dimerization) of allostery. In addition, Force Distribution Analysis showed that perturbed loop folding is associated with redistribution of mechanical stress experienced by network residues. The combined results indicated a mechanism for BirA allosteric regulation in which disorder-to-order transitions and joint network formation enables long-range communication through the protein. Finally, results of functional measurements indicated a conserved allosteric regulation mechanism among Escherichia coli (Ec), Staphylococcus aureus (Sa), and Bacillus subtilis (Bs), as bio-5’-AMP binding to Sa and BsBirA induces homodimerization similar to that observed for EcBirA.Item STRUCTURAL AND FUNCTIONAL STUDIES OF TAILSPIKE PROTEINS FROM ESCHERICHIA COLI O157:H7 PHAGE CBA120(2019) Greenfield, Julia Yudeh; Herzberg, Osnat; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteriophage CBA120, a member of the Ackermannviridae family, was isolated, sequenced, shown to selectively infect the food pathogen E. coli O157:H7. The CBA120 genome encodes four separate tailspike proteins (TSPs), TSP1-4 corresponding to ORFs 210 through 213. TSPs bind and degrade or modify the lipopolysaccharide (LPS) on the bacterial cell surface as part of the adsorption apparatus of tailed bacteriophages. Electron microscopy revealed that phage CBA120 possesses a long contractible tail with distinct star-like structures attached to the baseplate of the tail. Sequence analysis of the four CBA120 TSPs suggests two distinct classes; one class comprising TSP1 and TSP3 corresponds to two-domain structures. The second class comprising TSP2 and TSP4 have longer amino acid sequences and contain additional N-terminal regions responsible for the assembly of the TSP star-like complex. Crystal structure of TSP1 at 1.8 Å resolution and TSP3 at 1.85 Å resolution revealed two-domain homotrimers displaying conserved N-terminal head domain structures and C-terminal receptor binding domains with an overall β-helical fold. Sequence analysis of the assembly regions of TSP2 and TSP4 reveal remote homology to gp10 of phage T4, which is involved in the assembly of the phage baseplate. The crystal structure of TSP2 reveals the head domain and receptor binding domain, but the N-terminal assembly region is structurally disordered. In contrast, the assembly region of TSP4 has been purified without the head and receptor domain; pull-down experiments showed that this region binds TSP1. Bacterial halo assays of TSPs 1-3 revealed that all three proteins produced circles of clearing corresponding to polysaccharide depolymerase activity. Site-directed mutagenesis coupled with the halo assay confirmed the identity of TSP3’s catalytic residues as Asp383 and Asp426, which, based on the structure, suggests that the enzyme employs an acid/base mechanism to degrade lipopolysaccharide. The structure-based putative active site of TSP2 suggests that the catalytic machinery comprises Asp506 and Asp571. It was also shown that TSP2, but not TSP1 or TSP3, diminishes CBA120’s ability to infect E. coli O157:H7. Thus, TSP2 is the phage specificity determinant that binds to the lipopolysaccharide of E. coli O157:H7, whereas the bacterial targets of TSP1 and TSP3 remain unknown.Item Small molecule inhibitors of cyclic di-AMP signaling(2018) OPOKU-TEMENG, CLEMENT; SINTIM, HERMAN O; JULIN, DOUGLAS; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Globally, it is estimated that more than 700,000 people die annually from infections caused by drug-resistant bacterial pathogens. Resistant strains of bacteria continue to be isolated in healthcare and community settings. At the same time, the antibiotic pipeline remains dry – exemplified by the paucity of new antibiotics introduced into clinical use. Consequently, antibiotic-resistant strains are rapidly spreading, and antibiotic-resistant infections persist. Additionally, the existing antibiotics target one of the common targets – DNA, RNA, protein and cell wall synthesis. There is an apparent need to identify antibacterial agents against novel targets to slow down the generation of resistance. Cyclic dinucleotides have emerged as central regulators of bacterial physiology. Particularly, cyclic di-AMP (c-di-AMP) regulates cell wall homeostasis, cell size, potassium ion transport, virulence and biofilm formation in various Gram-positive pathogens including Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes and Streptococcus pneumoniae. It has been demonstrated that under standard laboratory conditions, deletion of the diadenylate cyclase genes that encode c-di-AMP synthesizing enzymes (diadenylate cyclase, DAC) was lethal in human pathogens like S. aureus and L. monocytogenes. Hence, DACs have been suggested as potential antibiotic targets. Thus far, the effect of c-di-AMP on bacterial physiology has been studied using genetic approaches whereby the key players of the second messenger signaling are deleted, inactivated or overexpressed to create conditions of varying intracellular c-di-AMP levels. However, these approaches are not amenable to drug development. Cell permeable small molecule modulator or c-di-AMP levels are required to validate the druggability of c-di-AMP signaling. This dissertation reports the identification of different small molecules that potently inhibit c-di-AMP synthesis. The cell permeable inhibitors possess the ability to decrease the intracellular concentration of c-di-AMP. Furthermore, the antibacterial activities of the cell permeable c-di-AMP synthesis inhibitors have been characterized. Efforts towards the development of antibiotics have also been discussed.