Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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Item COMBINED CHEMICAL AND ENZYMATIC APPROACHES TO PROTEIN GLYCOSYLATION(2021) Prabhu, Sunaina Kiran; Wang, Lai-Xi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Glycosylation is a key post-translational modification of proteins and influences the structure and biological functions of proteins. Glycoproteins are significant in treating a variety of diseases and make up a large fraction of biotherapeutics. The carbohydrate structures on the proteins regulate biological activity and pharmacokinetic properties, thereby dictating the efficacy and cost of glycoprotein drugs. However, glycoproteins expressed in biological systems are heterogeneous in nature and impose a challenge to structure-function studies as well as design of potent therapeutics. Thus, developing tools to modulate the glycan structures on proteins is highly significant. In my thesis, we have explored biological and chemoenzymatic methods to generate homogeneously glycosylated therapeutic proteins. First, we designed a glycosylation machinery in Escherichia coli (E. coli) using an N-glycosyl transferase enzyme to transfer a sugar handle onto a model protein. The protein was then elaborated with a homogeneous glycoform using in vitro chemoenzymatic transglycosylation. Using this methodology, we produced a fully glycosylated human interferon alpha-2b that was biologically active and displayed significantly enhanced proteolytic stability. Next, we focused on expanding the toolbox of enzymes available to perform the chemoenzymatic glycan remodeling of proteins. Specifically, we compared the substrate specificities of the human α-L-fucosidase (FucA1) and two bacterial α-L-fucosidases (AlfC and BfFuc) with a panel of structurally well-defined core-fucosylated substrates. FucA1 was the only α-L-fucosidase to display hydrolytic activity towards full-length core-fucosylated glycopeptides and glycoproteins. Moreover, FucA1 showed low but apparent activity to remove core fucose from intact monoclonal antibodies. This finding reveals an opportunity to employ FucA1 to remove core fucose from therapeutic antibodies to improve their antibody-dependent cellular cytotoxicity. Finally, we explored modulation of core fucosylation of monoclonal antibodies through metabolic glycoengineering. We designed L-fucose analogs to potentially incorporate functionalized fucose into IgG-Fc glycan. We showed incorporation of a few fucose derivatives into antibodies and identified a concentration-dependent effect of some of the previously known analogs. While some of the novel compounds did not show effect, the study supplements the existing tools available for metabolic modulation of antibodies. In summary, these studies present feasible new approaches to produce therapeutic eukaryotic glycoproteins with desired, homogeneous glycosylation.Item BIOPHYSICAL STUDIES OF UBIQUITIN: FROM FOLDING TO PROTEIN ENGINEERING(2021) Camara, Christina M; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The signaling protein ubiquitin is known for its ubiquity — existing in nearly all cel- lular compartments, holding a prominent role in major cellular signaling pathways and serving as a model system for protein folding. Herein, we honor this stature by exploring several aspects of the ubiquitin system form biophysical, structural, and computational per- spectives. Our efforts begin from the standpoint of protein engineering, where we extend ubiquitin’s function by installing a transition–metal binding motif and elevate it to the sta- tus of a metalloprotein. In doing so, we introduce novel spectroscopic behaviors, reactive propensities, and the capability to form non–canonical polyubiquitin chains — with appli- cations that span from molecular nanotechnology to synthetic biology. We then shift to foundational investigations of ubiquitin’s fold. By characterizing local degrees of freedom, we demonstrate how conformational motions of ubiquitin’s C–terminus can be controlled by the cellular microenvironment. This response, in turn, can regulate molecular recog- nition within the ubiquitination cascade. Finally, we approach global aspects of ubiquitin folding — exploring how a motif containing the C–terminus and the β5 strand might assem- ble into ubiquitin’s β –grasp architecture — with general lessons for ubiquitin–like proteins and other systems with an apparent two–state folding mechanism.Item CHARACTERIZATION OF THE STRUCTURE AND BINDING OF BRANCHED K6/K48-LINKED AND BRANCHED K6/63-LINKED POLYUBIQUITIN CHAINS(2021) Abeykoon, Dulith Maduwantha Bandara; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin (Ub) is an important post-translational protein modifier in eukaryotes. Post-translational modification with Ub is an essential process for eukaryotic cellular signaling including protein degradation, DNA repair, antigen-peptide generation and endocytosis. This post-translational modification with Ub occurs through ubiquitination where Ub attach as monoUb or polyUb chains. Ub form polyUb chains by forming covalent linkages between the C-terminus of one Ub and any of seven lysines or the N-terminus of other Ubs. Polyubiquitin chains can form homogeneous, heterogeneous, linear or branched chains, leading to diversity in polyubiquitin chain signaling outcomes. This diversity in signaling is due to the variety of conformations that arise based on the linkage specificity of the polyUb chains. Recently, branched K6/K48-linked polyubiquitins were shown to enhance deubiquitinating activity of UCH37 in the presence of Rpn13. To better understand the underlying structural mechanisms, here we determined the NMR structures of branched K6/K48-linked triubiquitin (Ub3) and discovered a previously unobserved interdomain interface between each of the distal ubiquitins and the proximal domain. We performed NMR binding assays to study the interactions of branched K6/K48-linked Ub3 with hHR23a UBA2, Rap80 tUIM and UCH37/Rpn13 complex. Binding studies of branched K6/K48-linked Ub3 to the UBA2 domain of the proteasomal shuttle protein hHR23A resulted in negligible differences between branched K6/K48-linked Ub3 and related dimers (K6-Ub2 and K48-Ub2). Interestingly, introducing hydrophobic patch surface residue mutations led to stronger affinity with both distal domains suggesting a change in the binding mode. Stronger binding affinity for K6/K48-linked branched Ub3 was observed with Rap80 tUIM. Moreover, deubiquitinating enzyme UCH37 (with Rpn13) showed strong affinity for both K6-linked and K48-linked distal domains, thereby suggesting a functional impact of this interdomain interface towards enhanced deubiquitinating activity of UCH37. Moreover, mutation studies of the hydrophobic patch residues of the proximal ubiquitin have shown the importance of the hydrophobic patch surface to maintain the interdomain interface of this branched trimer and for interactions with binding partners. Finally, initial studies done with the regulatory domain of the DNA repair protein p53 (p53c) have shown that p53c is a promising candidate for ubiquitination via non-enzymatic ubiquitination method introduced by our lab.Item BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF NUSG PARALOG LOAP(2021) Elghondakly, Amr; Winkler, Wade; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The NusG family of transcription factors is the only universally conserved family of transcription elongation regulators in all three domains of life. NusG proteins exert ubiquitous genetic regulatory effects by reversibly binding RNA-polymerase (RNAP) during transcription elongation and modulate its function. A phylogenetic analysis of the NusG family of proteins identified several distinct subfamilies of NusG paralogs that are widespread amongst bacterial species. These different NusG paralogs are likely to exert regulatory control over distinct subsets of genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Additionally, the regulatory mechanisms that these transcription elongation factors employ are likely to differ between one another to allow for specific recruitment to target operons and prevent competition with the housekeeping NusG factor. The LoaP subfamily of NusG proteins is primarily encoded by Actinobacteria, Firmicutes and Spirochaetes. While regulons for the LoaP subfamily have only been identified in a few organisms, the loaP gene is oftentimes found adjacent to long operons encoding for biosynthesis of secondary metabolites suggesting a regulatory relationship with these pathways. In Bacillus velezensis, LoaP promotes transcription antitermination of two long biosynthetic operons which encode for two different polyketide antibiotics: difficidin and macrolactin. Intriguingly, the cis-determinants for LoaP antitermination include a small RNA hairpin (~26 nts) located within the 5’ leader region of target operons. LoaP associates with the RNA hairpin in vitro with nanomolar affinity and high specificity via basic residues that are highly conserved within the C-terminal KOW domain, in contrast to other well-characterized bacterial NusG proteins which do not exhibit RNA-binding activity. These data indicate that LoaP employs a distinct regulatory mechanism to achieve targeted regulation of large biosynthetic operons in bacteria. Furthermore, this discovery expands the repertoire of macromolecular interactions exhibited by bacterial NusG proteins during transcription elongation to include an RNA ligand. Crystallographic studies of LoaP-RNA complex are in progress, and recent results will be discussed.Item Structural and Biophysical Characterization of Homo Sapiens RIOK2 in Complex with Selective Prostate Cancer Inhibitors and its Transition State Complex(2021) Seraj, Nishat; LaRonde, Nicole; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Homo sapiens Riok2 (HsRiok2) is an atypical, Ser/Thr protein kinase that has been implicated in a number of cancers, including Prostate Cancer. More recently, evidence has shown that HsRiok2 plays a biological role in the upregulation of ERG-positive cancer cells, thus presenting itself as an attractive drug target. The first known prostate cancer inhibitor against HsRiok2 has been identified, as well as select derivative inhibitors and has been shown both in vitro and in vivo to target HsRiok2. Here, the first ever Homo sapiens Riok2 in complex with two known Prostate Cancer inhibitors at 1.75 Å resolution and 2.70 Å resolution is reported, respectively. To evaluate conformational changes upon inhibitor binding, the first ever Homo sapiens Riok2 transition state complex is reported, capturing a snapshot of the kinase poised for phosphoryl transfer, at 2.80 Å resolution. These structural insights reveal the influence of the phosphate-binding loop (P-loop) in HsRiok2’s dimerization and potentially a catalytically inactive state, mediated by the presence of the drug candidate. Its dimerization interface is prevented from interacting with the Pre-40S ribosome, preventing HsRiok2 from carrying out its function as an ATPase/Kinase to further mature the pre-40S particle. These findings provide the first blueprint for a structure-based drug design approach to facilitate the development of more selective prostate cancer inhibitors.Item EPIGENETICS TUNE CHROMATIN MECHANICS, A COMPUTATIONAL APPROACH(2021) Pitman, Mary; Papoian, Garegin A; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The base unit of DNA packaging in eukaryotes, the nucleosome, is adaptively modified for epigenetic control. Given the vast chemical space of chromatin and complexity of signaling and expression, much of our knowledge about genetic regulation comes from a biochemical or structural perspective. However, the architecture and function of chromatin also mechanically responds to non-equilibrium forces. Mechanical and biochemical properties are not independent of one another and the interplay of both of these material properties is an area of chromatin physics with many remaining questions. Therefore, I set out to determine how the material properties of chromatin are altered by biochemical variations of nucleosomes. All-atom molecular dynamics is employed coupled with new computational and theoretical tools. My findings and predictions were collaboratively validated and biologically contextualized through multiscale experimental methods. First, I computationally discover that epigenetic switches buried within the nucleosome core alter DNA accessibility and the recruitment of essential proteins for mitosis. Next, using new computational tools, I report that centromeric nucleosomes are more elastic than their canonical counterparts and that centromeric nucleosomes rigidify when seeded for kinetochore formation. We conclude that the material properties of variants and binding events correlate with modified loading of transcriptional machinery. Further, I present my theoretical approach called Minimal Cylinder Analysis (MCA) that uses strain fluctuations to determine the Young's modulus of nucleosomes from all-atom molecular dynamics simulations. I show and explain why MCA achieves quantitative agreement with experimental measurements. Finally, the elasticity of hybrid nucleosomes in cancer is measured from simulation, and I implicate this oncogenic variant in potential neocentromere formation. Together, these data link the physics of nucleosome variations to chromatin states' plasticity and biological ramifications.Item STRUCTURAL CHARACTERIZATION OF THE PRMT5-RIOK1-NUCLEOLIN COMPLEX(2021) Wang, Hongpeng; LaRonde-LeBlanc, Nicole NL; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Protein methylation is one of the most important protein post-translational modifications. Protein arginine methyltransferase 5 (PRMT5) catalyzes the mono- and symmetric dimethylation of arginine residues of a wide range of proteins that play critical roles in various cellular processes. It is demonstrated that PRMT5 directly interacts with commonly misregulated or mutated proteins in cancer and PRMT5 plays an important role in cancer as a potential oncogene. RioK1 is required for cytoplasmic maturation of the small (40S) subunit of ribosome. Increased ribosome biogenesis is a critical requirement for all cancers and RIO kinases play an important role in efficient ribosome biogenesis. It is also reported that RioK1 plays distinct roles in cancer-supportive processes such as proliferation and migration. The PRMT5-MEP50 core complex binds to pICln or RioK1 in a mutually exclusive fashion to alter the specificity of substrate recognition and subsequent recruitment. RioK1 serves as adaptor protein of PRMT5 to recruit Nucleolin for methylation. Despite the fact that structural information for each component of the PRMT5-RioK1-Nucleolin complex is available, little is known about the molecular details of the interaction between the components. In this project, we report our attempt at the structural characterization of the PRMT5-RioK1-Nucleolin complex. Firstly, we investigated the interaction of PRMT5 and RioK1 homolog in Chaetomium thermophilum and solved the structure of ctPRTM5. CtPRMT5 adopts a tetrameric overall structure and an elongated α helix in the N-terminal region compared to human PRMT5. Secondly, we investigated the effect of RioK1 knockdown on ovarian cancer cells. Our results show that RioK1 function is positively related to the migration rate of ovarian cancer cells. Finally, we pulled out endogenous PRMT5 complexes from HEK293F cells. We report here that RNA integrity is required for the interaction between RioK1 and Nucleolin. We find evidence that indicates a direct interaction between human PRMT5 and the 80S ribosome particle. We also identified a novel assembly between human PRMT5 and the nucleosome.Item PLASTICITY IN PROTEIN SEQUENCE-FUNCTION RELATIONSHIPS(2021) He, Chenlu; Beckett, Dorothy; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Allostery is defined as the functional regulation at one site in a protein by activity at a distant site. Because of the ubiquitous occurrence and diverse cellular roles of allosteric proteins, designing novel allosteric proteins is of great interest for applications in synthetic biological and disease treatment. However, the engineering of allostery is often hindered by our limited understanding of the protein sequence- function relationship, especially at residue positions that are distant from functional sites or evolutionarily nonconserved. In this dissertation, the sequence-function relationship was investigated in the Escherichia coli biotin protein ligase (BirA) system, which serves as both an essential metabolic enzyme and a transcription regulator. In its repressor function, binding to the vitamin biotin allosterically activates BirA dimerization and the resulting repression complex assembly on the biotin operator sequence. Although the allosteric regulation is conserved among bifunctional biotin protein ligases such as BirA, their sequences, even those of functional importance, are highly divergent. The in vitro characterization of BirA super repressor variants reveals that the sensitivity of transcription repression response to input biotin concentration can be altered solely through substitution-perturbed dimerization. These single amino acid substitutions are located at sites scattered throughout the protein structure including some that are distal from the BirA dimerization surface. Computational simulations indicate that the long-range effect of substitutions on dimerization results from rearrangement of a residue network that contributes to the allosteric activation in BirA. Several loops on the BirA dimerization surface were characterized for their roles in the corepressor-induced dimerization. The study of nonconserved amino acid positions spanning these surface loops reveals that a broad range of functional response in dimerization and transcription repression can be achieved by sequence variations at the nonconserved residues. Surprisingly, the substitution outcomes poorly correlate with amino acid chemistry or evolutionary frequencies, which deviates from canonical expectations based on conserved residues. Combined, these results illustrate the plastic nature of protein sequence-function relationship and provide insight into how this plasticity functions in the mechanism and evolution of allostery in BirA. Our deepened understanding of allostery in BirA and in general may facilitate the development of synthetic allosteric proteins in the future.Item THE UNIQUE PROPERTIES OF BRANCHED K11/K48-LINKED POLYUBIQUITIN, AND THE PROTEASOMAL RECOGNITION OF (POLY)UBIQUITIN AND UBIQUITIN-LIKE SIGNALS BY RPN1(2021) Boughton, Andrew; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Post-translational modification of substrates with ubiquitin (Ub) is an essential process across all eukaryotes, responsible for regulating a myriad of cellular pathways. Many aspects of ubiquitination are extensively studied, and its role in proteasomal degradation is of significant therapeutic interest. This degradation pathway relies upon the coordinated interplay among Ub, shuttle proteins containing Ub-like (UBL) domains, and the proteasome. Substrates are tagged with polymeric Ub (polyUb) and shuttle proteins; these signals are recognized by the proteasome, which subsequently degrades the substrate.The diversity of polyUb signaling reflects Ub’s ability to form a covalent linkage between the C-terminus of one Ub and any of seven lysines or the N-terminus of another Ub. Furthermore, polyUb may contain homogeneous, heterogeneous, unbranched, or branched linkages. Thus, polyUb can be assembled with numerous specific architectures, each of which may convey distinct signaling outcomes. Notably, branched K11/K48-linked polyUb was recently shown to enhance proteasomal degradation during mitosis. Here, we determined the crystal and NMR structures of branched K11/K48-linked Ub3 and discovered a previously unobserved interface between the distal Ubs. Additional techniques corroborated this interfacial effect, which we hypothesized to be influential in the physiological role of branched K11/K48-linked polyUb. Although initial probing of polyUb interactions – binding to the shuttle protein hHR23A; deubiquitination assays – resulted in negligible differences between branched K11/K48-linked Ub3 and related Ub2 moieties, stronger binding affinity for branched K11/K48-linked Ub3 was observed with proteasomal subunit Rpn1, thereby suggesting a functional impact of this interdomain interface and pinpointing the mechanistic site of enhanced degradation. We devoted further attention to Rpn1, the largest and least characterized proteasomal subunit. We confirmed that Rpn1 associates with UBL-containing proteins and polyUbs, while exhibiting a preference for shuttle protein Rad23. Moreover, our results suggested that Rpn1 contains multiple Ub/UBL-binding sites. These sites are shared among polyUb and Ub-like moieties, thereby ruling out the possibility of exclusive recognition sites for individual signals. Finally, we identified the location of a novel Ub/UBL-binding site in Rpn1, which exhibits relatively strong affinity for polyUb and Ub-like signals. Surprisingly, this site is situated in a region of Rpn1 previously surmised to be devoid of functionality.Item Impact of lipopolysaccharide administration on novel RNA biomarkers for systemic inflammation in swine(2020) Swain, Trevon Brandon; Dayie, Theodore K; Myers, Michael J; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In veterinary medicine, inflammation in swine is evaluated solely by clinical signs. This method is often unreliable when assessing large animal populations because of inconsistent interpretations of clinical observations between different clinicians. The lack of a validated swine animal model prevents an accurate measurement of inflammation and inhibits the development of new veterinary effective drugs for swine. This study examined whether changes in miRNA expression can predict the severity of the inflammatory response in swine after administration of lipopolysaccharide (LPS) from Escherichia coli (E.coli). Identification of a reliable biomarker from a systemic inflammatory response needs to be easily obtained, safe, and provide the lowest risk of discomfort to the subject. The correlation of the clinical signs with individual miRNA levels may establish a plasma biomarker that can determine the severity of inflammation in swine. The long-term goal is to determine the most powerful tool for analysis and biomarker discovery. Exploring the different methodologies and monitoring different miRNAs increases the likelihood for potential advancements in disease detection applications