Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
Browse
109 results
Search Results
Item USING DNA LOOPING PROTEINS TO ENHANCE HOMOLOGY DIRECTED REPAIR IN VIVO FOLLOWING A CAS9 INDUCED DOUBLE STRAND BREAK(2024) Ferencz, Ian Theodore; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genome engineering methods that start with a CRISPR/Cas9 targeted genomic DNA double strand break proceed through cellular DNA repair mechanisms after the induction of the break. Imprecise nonhomologous end-joining (NHEJ) is useful for knockouts, but precise homology-directed repair (HDR) is necessary for gain of function changes. NHEJ tends to be more efficient, so directing the cell to knock in a precise sequence via HDR is an active area of research. The system we have designed uses a bivalent protein to recruit HDR donor DNA to the site of a specific DNA double strand break induced by a Cas9/sgRNA nuclease. Previously described leucine zipper dual-binding (LZD) proteins areused because they are small and stable. The system was designed to reduce the effort needed for screening, shorten the time required for the repair process, and/or decrease the amount of donor DNA needed, reducing potential off-target effects. We developed a model system in Saccharomyces cerevisiae to measure gene disruption and HDR frequencies in yeast that contain combinations of non-replicating donor DNA plasmid or linear DNA, expression plasmids of four LZD variants, and a plasmid expressing Cas9 and an sgRNA targeting either the AGC1 or ADE2 genes. The donor DNA includes a gene coding for G418 resistance in yeast. It also includes an INV-2 site recognized by the C-terminal DNA binding domain of LZDs adjacent to suboptimal regions of homology to the target gene. The N-terminal DNA binding domain of the LZDs recognizes an endogenous CREB site near the target gene. The desired recombinants are scored by their inability to grow on acetate as a sole carbon source (for AGC1) or their red color (ADE2), accompanied by resistance to G418. We believe that LZD enhancement can become a simple and valuable adjunct to any other method of improving the efficiency of HDR, in any system. We were able to show that the inclusion of LZD73 in recombination experiments increased the number of colonies presenting with the desired phenotype and genotype nearly eight-fold in the absence of a designed DNA break. We also provide evidence suggesting that the presence of LZD73 has a slight positive effect on the efficiency of Cas9 targeting. Desired recombinants were recovered after Cas9/sgRNA cleavage in an experiment where there was no apparent recombination in the absence of LZD73. Future work on this project includes optimization of the homologous sequences to improve background recombination so a more quantitative measure of the improvement observed in the presence of LZD proteins. This work can be transferred laterally to enhance other recombination-based methods in other organisms: the LZD proteins could be analogous to an adjuvant that increases overall efficiency.Item DECIPHERING THE MOLECULAR MECHANISM BEHIND THE SARS-COV-2 FUSION DOMAIN(2024) Birtles, Daniel; Lee, Jinwoo; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)SARS-CoV-2 is an extremely infectious virus, yet despite a plethora of research the viral lifecycle is still not well understood, particularly the process of membrane fusion. The traditional means by which viral glycoproteins facilitate fusion is that of the six-helix bundle, within which a short, conserved sequence known as the fusion domain (FD) initiates the process as it embeds within and perturbs the target cell membrane, in turn lowering the energetic barrier necessary to coalesce two opposing membranes. Furthermore, the highly conserved coronavirus FD is found to be available on the SARS-CoV-2 spike protein surface, which along with its integral role within the viral lifecycle makes it an ideal therapeutic target. However, limited knowledge of the exact molecular mechanism by which the SARS-COV-2 FD conducts its role within the fusion process has prevented the production of antiviral treatments. Here we describe the elucidation of key molecular details regarding how the SARS-CoV-2 FD initiates the process of membrane fusion. Firstly, the FD was found to contain a unique assembly of fusogenic regions, known as a fusion peptide (FP) and fusion loop (FL), which operate in synergy to elicit efficient fusion. This was followed by the discovery of a preference for the FD to fuse within conditions akin to the late endosomal membrane, with both pH and lipid composition significantly impacting fusion. It was found that the endosomal resident anionic lipid BMP imparts a negative impact on lipid packing within the membrane, which positively correlates with fusion. The unique mechanism by which the coronavirus FD initiates fusion was cemented when we uncovered the importance of several positively charged residues towards the FDs function. This also led to unearthing a mutant of the FD (K825A), which if found to have naturally occurred within the full spike protein, has the potential to produce a more virulent strain of SARS-CoV-2.Item Structural Investigation into RioK1 for Cancer Therapeutics(2024) Hunter, Daniel Arthur; Weber, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cancer was the second leading cause of death in the United States in 2020.1 Cancer shares many similarities with healthy cells, making it a difficult therapeutic target.2 Current developments of cancer therapeutics are governed by targeting key proteins responsible for distinct features in each type of cancer phenotype. (e.g. decreased apoptosis, metastasis, immortalization, etc.). However, finding a rational therapeutic target, engineering a lead compound, and lead compound optimization is time-consuming and expensive. With the use of high-throughput screens and structure-based drug design it is possible to design lead compounds in a more efficient manner. Techniques such as x-ray crystallography and cryo-electron microscopy are used to observe how compounds interact with the target protein at atomic resolution, which helps facilitate optimization.2-4 Kinases in particular, have benefitted greatly from these techniques.5 Kinases play key roles in signal transduction and its regulation in many cellular pathways. The catalytic active site is highly conserved among many kinase families, so designing drugs targeting a single enzyme’s catalytic site could have potential off target effects as many kinases could be inhibited. Strategies to target kinases therefore use distinct features of each kinase that take both conserved and nonconserved residues into consideration as well as for active and inactive forms of the kinase being targeted, so tailormade therapeutic solutions are derived, as with the case of reading open frame kinase 1 (RioK1).2, 4, 5 RioK1 was identified as a key enzyme in both lung and colorectal cancer, cancer subtypes with some of the most severe prognoses.6 In a study done by Kiburu et al., toyocamycin was demonstrated to bind tightly to RioK1 from archaeoglobus fulgidus (afRioK1), thereby discovering the first scaffold for RioK1.7 Toyocamycin is an adenosine analog, commonly used as inhibitor, and thus making this drug scaffold non-specific with off-target effects other than for RioK1.8-12 To address this issue, computer aided drug design was used to find toyocamycin-like compounds that have improved selectivity for afRioK1 inhibition. A series of these compounds were identified via screening approaches and then co-crystallized with afRioK1 with the goal of elucidating useful structure-activity relationship data for next stage drug-design. Furthermore, one of the most newly studied interactions of RioK1 is with protein arginine methyltransferase type 5 (PRMT5), so understanding the details of this interaction provides yet another means to develop afRioK1 inhibition strategies as part of an approach to block cancer progression.Item Characterization of a novel Escherichia coli exopolysaccharide and its biosynthesis by NfrB(2024) Fernando, Sashika Hansini Lakmali; Poulin, Myles B; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biofilms are made from an association of bacterial cells and extracellular products dominated by a plethora of exopolysaccharides. Accumulating evidence have demonstrated that the bacterial second messenger cyclic-di-guanosine monophosphate (c-di-GMP) promotes the synthesis of these exopolysaccharides through direct allosteric activation of glycosyltransferase enzymes. The Escherichia coli inner membrane protein NfrB, which together with the outer membrane protein NfrA acts as a receptor system for phage N4, contains a N-terminal glycosyltransferase domain and C-terminal c-di-GMP binding domain. Recent research revealed that NfrB is a novel, c-di- GMP controlled glycosyltransferase that is proposed to synthesize a N-acetylmannosamine containing polysaccharide product, though the exact structure and function of this remains unknown. Nfr polysaccharide production impedes bacterial motility, which suggests a possible role of the Nfr proteins in bacterial biofilm formation. Here, we carry out in-vivo synthesis of novelNfr polysaccharide followed by its structural characterization. Preliminary data from MALDI- TOF mass spectrometry and Solid State 13C NMR spectroscopy indicated that the Nfr polysaccharide is mainly a homo polymer of poly-?-(1®4)-N-acetylmannosamine, bound to an aglycone. In addition, we report efforts to develop of a Nfr polysaccharide binding and detection tool, through the mutation of YbcH, a putative Nfr polysaccharide hydrolase enzyme. These studies advance the understanding of Nfr polysaccharide biosynthesis and could offer potential new targets for the development of antibiofilm and antibacterial therapies.Item Spatiotemporal proteomic approaches for investigating patterning during embryonic development(2024) Pade, Leena Rajendra; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Characterization of molecular events as embryonic cells give rise to tissues and organs raises a potential to better understand normal development and design remedies for diseases. In this work, I integrated bioanalytical chemistry with neurodevelopmental biology to uncover mechanisms underlying tissue induction in a developing embryo. Specifically, I developed ultrasensitive proteomic approaches to study the remodeling of the proteome as embryonic cells differentiate in space and time to induce tissue formation. This dissertation discusses the design and development of proteomic strategies to deepen proteomic coverage from limited embryonic tissues. A novel sample preparation workflow and detection strategy was developed to address the challenge of interference from abundant proteins such as yolk in Xenopus tissues which in turn boosts the sensitivity of detecting low abundant proteins from complex limited amounts of tissues. The refined analytical workflow was implemented to study the development of critical signaling centers and stem cell populations and the tissues they induce to form in developing embryos.Item UNRAVELING THE ROLE OF LASSA VIRUS TRANSMEMBRANE DOMAIN IN VIRAL FUSION MECHANISM(2024) Keating, Patrick Marcellus; Lee, Jinwoo; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa and recently isolated cases worldwide, no current therapeutics or vaccines pose LASV as a significant global public health threat. One of the key steps in LASV infection is the delivery of its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Investigating how the TM participates in membrane fusion will provide new insights into the LASV fusion mechanism and uncover a new therapeutic target sight to combat the dangerous infection. Here, we describe our protocols for expressing and purifying the isolated TM and our GP2 constructs which we use to probe the relationship between the structure of the TM and its influence on the function of GP2.We express TM as a fusion protein with a Hisx9 tag and a TrpLE tag using E. coli bacterial cells. We purify the TM using Nickel affinity chromatography and enzymatic cleavage to remove the tags. Since the TM is prone to aggregation, we must use a strong denaturant, trichloroacetic acid (TCA), to remove the TM from the resin. The isolated TM is then buffer exchanged to a detergent solution for structural studies. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, our structural studies revealed a pH-dependent structural change resulting in an N-terminal extension of the alpha helix in the postfusion state. To test the importance of this structural change, we used the GP2 construct to perform a modified lipid mixing fusion assay. Our results from the fusion assay and a combined mutational study revealed that this structural change is important for the fusion efficiency of GP2. Loss of this extension resulted in lower fusion activity. To further understand these structural changes and to probe the TM’s environmental interactions, we turned to fluorine NMR. This method gives us a unique and highly sensitive probe to monitor changes in the structure and membrane environment. We describe our incorporation protocol of fluorine into the TM and our method for incorporating the TM into a lipid bilayer system. We describe preliminary results showing sensitive changes in the structure of the TM and the implications this method has to enhance our understanding of the LASV membrane fusion mechanism.Item New method for kinetic isotope effect measurements(2023) Kljaic, Teodora; Poulin, Myles B; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Kinetic isotope effect (KIE) measurements are a powerful tool to interrogate the microscopic steps in enzyme catalyzed reactions and can provide detailed information about transition state structures. However, the application of KIE measurements to study enzymatic reactions is not widely applied due to the tedious and complex analytical workflows required to measure KIEs with sufficient precision. In this thesis I described the development of a novel competitive KIE measurement method using MALDI-TOF-MS and the investigation of the transition state of glycosyltransferase enzyme BshA from B. subtilis. We developed a method for the direct measurement of competitive KIEs using a whole molecule matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). This approach enabled quantitative measurements of both relative isotope abundance of an analyte and fractional conversion F in single measurements without the need for purification prior to analysis. The application of this MALDI-TOF MS approach has demonstrated the precision of KIE measurements comparable to those obtained using competitive radioisotope labelling, and NMR based approaches while requiring smaller amounts of stable isotope labelled substrates. Using two chemoenzymatic approaches, we then synthesized 5 substrates for the application of our method to investigate the transition state of BshA: UDP-GlcNAc (3.1), [1''-13C]UDP-GlcNAc (3.2), [2''-13C]UDP-GlcNAc (3.3), [13C6]UDP-GlcNAc (3.4) and [2''-2H]UDP-GlcNAc (3.5). Finally, we have begun to work on the synthesis of [1''-18O]UDP-GlcNAc and describe an approach to prepare this substrate that is currently underway in the lab. Application of the quantitative whole molecule MALDI-TOF MS approach enabled us to determine multiple competitive KIEs for the enzymatic reaction catalyzed by BshA. While previous studies suggested a front-face SNi (DNAN) TS for the conjugation of UDP-GlcNAc and L-malate, our KIE results show that a stepwise mechanism resulting in the formation of a discrete, though likely short lived, oxocarbenium ion intermediate is more likely. Our method be applied to study other glycosyltransferases whose mechanisms still remain to be elucidated and to design TS based inhibitors for enzymes involved in different bacterial infections. Future work on automation of this method would simplify the KIE measurement process and increase reproducibility making the measurement of KIEs for TS analysis a more experimentally accessible technique for the broader enzymology research community.Item DISCOVERYING THE ROLES OF SOLUBLE DHH-DHHA1 TYPE PHOSPHODIESTERASES IN RNA DEGRADATION AND CYCLIC DINUCLEOTIDE SIGNALING(2023) Myers, Tanner Matthew; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The synthesis and degradation of RNA is a fundamental and essential process for all life forms. It is imperative that cells utilize multiple mechanisms to modulate the lifetimes of RNA molecules to ultimately control protein synthesis, to maintain homeostasis or adapt to environmental challenges. One mechanism to directly alter the stability or half-life of an RNA is through the direct enzymatic activity of ribonucleases (RNases). Bacterial organisms encode for many different RNases that possess distinct functions in RNA metabolism. It is through the actions of multiple cellular RNases that a long RNA polymer corresponding to an mRNA, rRNA, tRNA, or sRNA can be fully degraded back into nucleotide monophosphate precursors. The nucleoside monophosphate precursors are recognized by kinases that ultimately recycle these molecules for use in the synthesis of other long RNA polymers. The processing of RNA polymers by endo- and exoribonucleases generates nucleoside monophosphates as well as short RNA oligonucleotides ranging from 2-6 nucleotides in length. Previously, a subset of enzymes broadly referred to as “nanoRNases” were found to process these short RNA fragments. Oligoribonuclease (Orn), NanoRNase A (NrnA), NanoRNase B (NrnB), and NanoRNase C (NrnC) were previously ascribed the function of indiscriminately processing nanoRNA substrates. However, recent analyses of the evolutionarily related DnaQ-fold containing proteins Orn and NrnC have provided compelling evidence that some nanoRNase protein families possess distinct dinucleotide substrate length preferences (Kim et al., 2019; Lormand et al., 2021). To determine whether all nanoRNases are diribonucleotide-specific enzymes, we utilized a combination of in vitro and in vivo assays to conclusively elucidate the substrate specificity and intracellular roles of the DHH-DHHA1 family proteins NrnA and NrnB. Through an in vitro biochemical survey of many NrnA and NrnB protein homologs, including from organisms of varying degrees of relatedness, we have determined that there are many functional dissimilarities contained within the DHH-DHHA1 protein family. Furthermore, we have conducted a rigorous investigation into the biological and biochemical functions of NrnA and NrnB in the Gram-positive model organism Bacillus subtilis. These analyses have shown that B. subtilis NrnA and NrnB are not redundant in biochemical activities or intracellular functions, as previously believed. In fact, we have found that B. subtilis NrnA is a 5’-3’ exoribonuclease that degrades short RNAs 2-4 nucleotides in length during vegetative growth, while B. subtilis NrnB is specifically expressed within the developing forespore and functions as a 3’-5’ exoribonuclease that processes short RNA in addition to longer RNA substrates (>40-mers). Our collective data provide a strong basis for the subdivision of the DHH-DHHA1 protein family on the basis on their diverse substrate preferences and intracellular functions.Item CHEMOENZYMATIC SYNTHESIS OF FUCOSYLATED OLIGOSACCHARIDES AND ANTIBODY GLYCOFORMS FOR ELUCIDATING BIOLOGICAL FUNCTIONS(2023) Lunde, Grace Henry; Wang, Lai-Xi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fucosylation is critical for molecular recognition events in the immune system, microbial interactions, and cancer metastasis. Terminal fucosylation (α1,2, α1,3, and α1,4) is characteristic of the histo-blood group antigens (ABO, H, and Lewis) and is expressed on many N- and O-glycans and most human milk oligosaccharides (HMOs). Core fucosylation (α1,6) exists primarily on the sixth position of the core GlcNAc of N-glycans and plays a profound role in modulating the biological functions of IgG antibodies. Due to their diverse biological properties, structurally well-defined fucosylated oligosaccharides and glycoconjugates are highly demanded for detailed structure-function relationship studies and translational applications. Three main approaches exist to meet this end: metabolic engineering, traditional chemical synthesis, and chemoenzymatic synthesis. Metabolic engineering has unmatched scalability but can suffer from limitations in many factors, including the availability of various sugar nucleotides, genetic instability, and inherent microheterogeneity due to the non-template-driven multiple-step assembly of glycans. Traditional chemical synthesis is ideal for producing a diverse array of pure targets due to its flexibility but may require prohibitively tedious multistep protocols. Alternatively, chemoenzymatic synthesis harnesses the precision of traditional chemical synthesis and marries it to the usually regio- and stereo-selective enzymatic transformations. Glycosyltransferases (GTs), the natural enzymes for synthesizing glycosidic bonds, are most widely applied in chemoenzymatic routes yet some glycosidases, which naturally hydrolyze glycosidic bonds, can be used in transglycosylation mode for chemoenzymatic synthesis. Generally, glycosidases can be easier to express, have a more relaxed acceptor substrate specificity, and utilize simpler synthetic donor substrates than GTs. However, glycosidases possess an inherent propensity toward product hydrolysis. This has led to the design of mutant glycosidases, known as glycosynthases and glycoligases, which have diminished hydrolysis and enhanced transglycosylation activity. A glycosynthase is a catalytic nucleophile mutant that inverts the anomeric configuration of the donor substrate (α→β or β→α) in glycosylation and a glycoligase is a mutant at the general acid/base residue that retains the anomeric configuration (α→α or β→β) in its catalytic glycosylation. Both require an activated glycosyl donor substrate with a suitable leaving group at the anomeric position. Readily synthesized glycosyl fluorides are frequently used. Given their superior stability in aqueous conditions, α-glycosyl fluorides are preferred over β-glycosyl fluorides. Therefore, the glycoligase approach is favored in the synthesis of α-glycosidic bonds, like the α-fucosides found in mammalian systems. Our group has successfully applied the glycoligase strategy for robust synthesis of core fucosylated (α1,6) N-glycans and glycoproteins. For my first project, I aimed to expand our fucoligase toolbox and designed an α1,3/4-fucoligase (AfcB E746A) for the synthesis of α1,3 and α1,4-fucosylated oligosaccharides. AfcB E746A very efficiently catalyzed the synthesis of the Lewis X (LeX) and A (LeA) trisaccharides and two HMOs: 3-fucosyllactose (3FL) and lacto-N-fucopentaose (LNFP) III with only slight excess (1.5 eq.) of the αFucF donor substrate. I concluded that AfcB E746A prefers to synthesize α1,3- over α1,4-fucosidic bonds, demonstrates a unique specificity for acceptors with a reducing end GlcNAc over glucose, and seems to require a free terminal Gal in its acceptor substrate. In my second project, I aimed to characterize the enzyme activity of two of our lab’s fucoligases, AfcB E746A and BfFucH E277G, in the synthesis of novel difucosylated tetrasaccharides. I concluded that AfcB E746A requires a free terminal galactose in the acceptor substrate given that AfcB E746A cannot efficiently utilize BfFucH E277G’s monofucosylated products. Alternatively, BfFucH E277G utilizes AfcB E746A’s monofucosylated products. BfFucH E277G synthesized several difucosylated tetrasaccharides, which were characterized by mass spectroscopy and one- and two-dimensional NMR analysis. I concluded that BfFucH E277G catalyzes the synthesis of an α1,3-fucosidic bond on the terminal galactose of 3FL and LeA to yield two novel HMO/Lewis antigen-like structures: 3’3-lactodifucotetraose (LDFT) and 3’-fucosyl-Lewis A (3’-FLeA), respectively. This study further elucidates BfFucH E277G’s unique acceptor substrate driven regioselectivity. In my first two projects, I meticulously characterized the transfucosylation activity of AfcB E746A and BfFucH E277G to provide insight into how these fucoligases may be integrated into synthetic schemes. Enzyme-catalyzed synthesis is an indispensable synthetic strategy given its reliability in substrate specificity and stereo- and regioselectivity. Pivoting from the study of fucoligases, in my third project I prepared highly pure and structurally well-defined IgG antibody glycoforms by chemoenzymatic remodeling with enzymes discovered and designed by our group. The objective of this work was to demonstrate our lab’s expertise in the chemoenzymatic remodeling of the IgG Fc N297 glycan. Furthermore, these antibodies will be applied in future experiments to more confidently characterize the effect of antibody core (α1,6) fucosylation on their binding affinity for Fcγ receptors (FcγRs) and demonstrate how the allosteric effect of immune complex formation contributes to this phenomenon. Core fucosylation of the N297 glycan reduces antibody-dependent cellular cytotoxicity (ADCC) by decreasing the antibody’s affinity for the FcγIIIa receptor. In fact, by removing core fucose, binding can be enhanced up to 50-fold, leading to improved ADCC and enhanced therapeutic efficacy. However, the current data is considerably disparate with enhancements ranging from 3- to 53-fold. These discrepancies may be attributed to glycoform heterogeneity and contamination with afucosylated glycoforms. Additionally, some studies have reported that, in the presence of core fucosylation, sialylation negatively impacts ADCC. This is accompanied by only a modest decrease in FcγRIIIa binding affinity which cannot solely account for the large difference in ADCC. This phenomenon may be explained by conformational allosteric cooperativity where a conformational change in Fab, upon antigen binding, is transmitted to the Fc to alter its affinity for the FcγRs. We hypothesize this phenomenon explains the discrepancy between FcγRIIIa binding affinity and the degree of ADCC activated by the sialylated and core fucosylated IgG antibodies. These detailed studies on the IgG-FcγRIIIa interaction are important for basic research and translational science. Exceedingly pure glycoforms are required for these experiments. My final project demonstrates the Wang group’s exceptional position in the field of basic antibody research and the indispensable nature of our work for the improved therapeutic application of monoclonal antibodies.Item Structural and Biophysical Explorations of Protein Degradation Tags(2023) Bonn, Steven Michael; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin is a 76-amino acid, well-folded and highly stable protein that is highly conserved across all eukaryotes. It is a post-translational modifier of other proteins through itsattachment via an isopeptide bond to a substrate lysine sidechain. Multiple ubiquitin units can be stacked to form a polyubiquitin chain, with chain topologies and cellular outcomes varying based on which of ubiquitin’s lysine residues they are attached to. The most common and well-studied outcome of polyubiquitin attachment is degradation by the proteasome, a massive barrel-shaped protease complex responsible for general protein quality control as well as cell cycle progression. Proteasomes have been discovered in archaea and bacteria, and are controlled by the small archaeal modifier protein (SAMP) and the disordered prokaryotic ubiquitin-like protein (Pup), respectively. Recently, a second bacterial proteasome operon was discovered with a new putative signaling protein, ubiquitin bacterial (UBact). Here, the first investigation of the UBact proteasomal operon is presented. Using nuclear magnetic resonance (NMR) spectroscopy and a variety of biophysical techniques, UBact is demonstrated to be disordered in solution and interact with its putative proteasomal receptor. This sets the groundwork for further studies of the UBact system. Additionally, NMR is used to explore the activity and directionality of various deubiquitinase enzymes responsible for breaking down polyubiquitin chains, and for exploring small molecule binding to ubiquitin chains themselves. This likewise provides a groundwork for further studies of the ubiquitin system, whose dysregulation is responsible for many diseases and is an area of intense therapeutic development.