UMD Theses and Dissertations
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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 EXPANDING THE TOOLKIT: STRUCTURE, DYNAMICS, AND DRUG INTERACTIONS OF THE “PRIMING LOOP” FROM HEPATITIS B VIRUS PRE-GENOMIC RNA BY SOLUTION NMR SPECTROSCOPY(2022) Olenginski, Lukasz Tyler; Dayie, Theodore K; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNAs are dynamic macromolecules that function as essential components of biological pathways that result in human disease, making them attractive therapeutic targets. Yet, RNA structural biology lags significantly behind that of proteins, limiting mechanistic understanding of RNA chemical biology. Fortunately, solution NMR spectroscopy can probe the structure, dynamics, and interactions of RNA in solution at atomic resolution, opening the door to their functional understanding. However, NMR analysis of RNA – with only four unique ribonucleotide building blocks – suffers from spectral crowding and broad linewidths, especially as RNAs grow in size. One effective strategy to overcome these challenges is to introduce NMR-active stable isotopes into RNA in an atom- and position-specific manner. Here, we outline the development of labeling technologies, their use in benefiting RNA dynamics measurements, and applications to study the structure, dynamics, and interactions of a conserved regulatory RNA stem-loop from hepatitis B virus that is critical for viral replication.Item ISOTOPIC LABELING STRATEGIES AND NMR METHODOLOGIES TO FACILITATE RNA STRUCTURAL AND DYNAMICS STUDIES: APPLICATIONS TO A LONG NON-CODING RNA FROM KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS(2020) Becette, Owen; Dayie, Kwaku T; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNAs are essential components of biological pathways that result in human disease making them attractive therapeutic targets. Currently, NMR spectroscopy is the only high-resolution technique capable of probing RNA interactions in solution. Although NMR spectroscopy is well-suited to characterize macromolecular interactions at atomic-level detail, the currently available isotopic labeling strategies and NMR methodologies are limited to relatively small RNAs (~ 30 nts, ~ 10 kDa). This size limitation is due to poor sensitivity and limited spectral resolution both of which worsen with increasing size. Here I present novel isotopic labeling schemes and NMR experiments to help expand the size limitations of NMR. These new technologies are then applied to characterize the structure and dynamics of a non-coding RNA from Kaposi’s sarcoma-associated herpesvirus (KSHV) that causes cancer in AIDS patients.Item THE DEVELOPMENT AND APPLICATION OF SITE-SELECTIVELY ISOTOPICALLY LABELED NUCLEOTIDES TO PROBLEMS IN NMR SPECTROSCOPY:STRUCTURAL INSIGHTS INTO THE EPSILON RNA AND TARGET COMPOUND INTERACTIONS(2017) Longhini, Andrew Paul; Dayie, Theodore K; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA plays a central role in a multitude of cellular processes. Understanding the complex interplay between its structure and function is a requisite for understanding these cellular roles mechanisms of action. Herein we describe technologies that we have developed to help better study RNA structure and function via NMR. Our development of site-selectively isotopically labeled pyrimidine and purine nucleotides has reduced spectral crowding, eliminated problems associated with scalar coupling, and led to novel assignment protocols. We have applied these labels to a 61-nucleotide viral RNA element, HBV-ε. This RNA is central Hepatitis B’s viral life cycle. Its NMR resonances have been assigned and initial structure calculations have begun to show how it interacts with compounds screened to bind to an internal six nucleotide bulge.Item Purple Pepper Plants, An Anthocyanin Powerhouse: Extraction, Separation and Characterization(2014) Taylor, Cassandra Lynn; Mignerey, Alie C; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of Dissertation: PURPLE PEPPER PLANTS, AN ANTHOCYANIN POWERHOUSE: EXTRACTION, SEPARATION AND CHARACTERIZATION Cassandra Lynn Taylor Doctor of Philosophy, 2014 Dissertation directed by: Professor Alice C. Mignerey Department of Chemistry and Biochemistry Most plants have multiple anthocyanins present that produce their color. In contrast, the foliage of the purple pepper plant (Capsicum annuum L.) contains high concentrations of a single anthocyanin delphinidin-3-p-coumaroylrutinoside-5-glucoside (Dp-3-p-coumrut-5-glc) in the foliage, making it very unique. This provides an excellent platform to extract the single anthocyanin at high concentrations. A food-grade extraction method was developed using 1% hydrochloric acid and 200 proof ethanol (1% HCl/EtOH) in order to remove the intact anthocyanin. A separation method using High Performance Liquid Chromatography (HPLC) was developed to identify Dp-3-p-coumrut-5-glc. The retention time was compared with the Blue Ribbon Iris, a known source of Dp-3-p-coumrut-5-glc. The HPLC results confirmed the presence of Dp-3-p-coumrut-5-glc in the pepper extract, but the chromatograms also demonstrated the presence of additional highly colored compounds. The extract was injected onto the HPLC and the major anthocyanin peak (Dp-3-p-coumrut-5-glc) was collected over the course of multiple injections. The collected fractions were dried down and re-solubilized in 1% HCl/methanol for analysis by mass spectrometry. A HPLC coupled to a photodiode array detector and an electrospray ionization tandem mass spectrometer (LC-PDA-ESI-MS/MS) was utilized to characterize Dp-3-p-coumrut-5-glc. The precursor compound was confirmed at m/z 919 with product ions at m/z 757, 465 and 303 by comparing against plant extracts of freeze-dried purple pepper foliage, Chinese eggplant and Chinese celery. The extract's structure was elucidated by Nuclear Magnetic Resonance (NMR) by analyzing both proton (1H) and carbon (13C) spectra. The 1H and 13C data matched very well with previous NMR data of Dp-3-p-coumrut-5-glc elucidated in eggplant peels. The major difference was that the trans isomer of Dp-3-p-coumrut-5-glc greatly dominated over the cis in the purple pepper extract.Item THE DYNAMICS OF DIUBIQUITIN REVEALED BY NMR: WHAT IS THE DRIVING FORCE BETWEEN THE OPEN AND CLOSED STATES?(2012) Lai, Ming-Yih; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The K48-linked polyubiquitin chains are important signals for proteasomal degradation and other biological processes. Their recognition of ubiquitin binding partners such as the UBA2 (ubiquitin associated domain 2) domain of hHR23a is via the canonical hydrophobic patch formed by L8, I44, and V70. In near physiological pH (pH 6.8), the K48-linked diubiquitin predominantly adopts the closed conformation in which the binding sites for ubiquitin-binding partners are buried in the inter-domain interface, and therefore are not available for binding. The K48-linked diubiquitin also can adopt an open conformation at acidic pH. However, the mechanism of the transition between the open and closed states is poorly understood. This study is aimed at elucidating the driving force for the exchange between the open and closed conformations of K48-linked diubiquitin. Using different mutations of H68 in diubiquitin and NMR methods, I found that the protonation state of the histidine side chain is crucial for controlling the equilibrium between open and closed conformations. I also found that H68 is essential for maintaining the integrity of the inter-domain interface. I concluded that there are at least four interactions involved in controlling the transitions between open and closed states. These are point-to-point repulsion (strongest), point-to-bulk repulsion (medium), bulk-to-bulk repulsion (weakest), and hydrophobic interaction. Based on these results, I also proposed a pre-open state model for K48-linked diubiquitin which assumes that the closed conformation of Ub2 opens by twisting instead of directly pulling two domains away from each other.Item Trapping Labile Adducts Formed Between an ortho-Quinone Methide and DNA(2012) McCrane, Michael Patrick; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exogenously generated electrophiles are capable of alkylating DNA. If not repaired, the resulting DNA adducts can lead to mutations and either cancer or cell death. Electrophilic ortho-quinone methides (o-QM) are reactive intermediates that alkylate DNA and are generated during xenobiotic metabolism of a variety of compounds including environmental toxins and therapeutic agents. Identifying the full alkylation profile of o-QM towards DNA would allow for the genotoxicity of o-QM precursors to be better understood. From model studies based on nucleosides, o-QMs react most readily, but reversibly with the strong nucleophiles 2'-deoxycytidine (dC) N3, 2'-deoxyguanosine (dG) N7, and 2'-deoxyadenosine (dA) N1 and less efficiently, but irreversibly with the weak nucleophiles dG N1, dG N2, and dA N6. The reverse reactions complicate analysis of their products in DNA, which requires enzymatic digestion and chromatographic separation. Selective oxidation by bis[(trifluoroacetoxy)iodo]benzene (BTI) can transform the reversible o-QM-DNA adducts into irreversible derivatives capable of surviving such analysis. To facilitate this analysis, a series of oxidized o-QM-dN adducts were synthesized as analytical standards. Initial oxidative trapping studies with an unsubstituted o-QM and dC demonstrated the necessity of an alkyl substituent para to the phenolic oxygen to block over-oxidation. A novel o-QM included a methyl group para to the phenolic oxygen that successfully blocked the over-oxidation allowing for generation of a stable MeQM-dC N3 oxidized product. Further oxidative trapping studies with MeQM and dG resulted in the formation of three stable MeQM-dG oxidized products (guanine N7, dG N1, and dG N2). Initial studies with duplex DNA optimized the enzymatic digestion and confirmed that the assay conditions were compatible with oxidative trapping. The low yielding MeQM alkylation of duplex DNA needs to be scaled up prior to the oxidative trapping studies. Alternative studies quantified the release of MeQM from DNA with the use of 2-mercaptoethanol as a nucleophilic trap. These studies revealed single stranded DNA as a superior carrier of MeQM than duplex DNA and, therefore, a better target DNA for the oxidative trapping studies due to increased yield of MeQM adducts. With the increased MeQM-DNA yield, the intrinsic selectivity and reactivity of MeQM towards DNA can be determined.Item Protein-Protein Docking Using Long Range Nuclear Magnetic Resonance Constraints(2010) Berlin, Konstantin; O'Leary, Dianne P; Fushman, David; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)One of the main methods for experimentally determining protein structure is nuclear magnetic resonance (NMR) spectroscopy. The advantage of using NMR compared to other methods is that the molecule may be studied in its natural state and environment. However, NMR is limited in its facility to analyze multi-domain molecules because of the scarcity of inter-atomic NMR constraints between the domains. In those cases it might be possible to dock the domains based on long range NMR constraints that are related to the molecule's overall structure. We present two computational methods for rigid docking based on long range NMR constraints. The first docking method is based on the overall alignment tensor of the complex. The docking algorithm is based on the minimization of the difference between the predicted and experimental alignment tensor. In order to efficiently dock the complex we introduce a new, computationally efficient method called PATI for predicting the molecular alignment tensor based on the three-dimensional structure of the molecule. The increase in speed compared to the currently best-known method (PALES) is achieved by re-expressing the problem as one of numerical integration, rather than a simple uniform sampling (as in the PALES method), and by using a convex hull rather than a detailed representation of the surface of a molecule. Using PATI, we derive a method called PATIDOCK for efficiently docking a two-domain complex based solely on the novel idea of using the difference between the experimental alignment tensor and the predicted alignment tensor computed by PATI. We show that the alignment tensor fundamentally contains enough information to accurately dock a two-domain complex, and that we can very quickly dock the two domains by pre-computing the right set of data. A second new docking method is based on a similar concept but using the rotational diffusion tensor. We derive a minimization algorithm for this docking method by separating the problem into two simpler minimization problems and approximating our energy function by a quadratic equation. These methods provide two new efficient procedures for protein docking computations.Item Molecular Interactions of Ubiquitin and Polyubiquitin with Ubiquitin Binding Domains(2007-10-22) Haririnia, Aydin; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin is a small protein that is covalently attached to proteins, either as a single ubiquitin moiety or as polyubiquitin chains. A cascade of enzymatic reactions is required for the isopeptide linkage between the C-terminus of ubiquitin and a lysine residue on a substrate protein or another ubiquitin. Attachment of ubiquitin or polyubiquitin, termed ubiquitination, mediates numerous cellular processes by acting as a versatile signal. The signal transmitted by the tag depends on the nature of the modification, which defines the specificity of the tag for different cellular machinery. This versatility is conferred by the variations in polyubiquitin tags, both in terms of length and lysine-linkage. Polyubiquitin chains can adopt a variety of different conformations based on these variations. The conformational and dynamic properties of the tag may optimize its binding to specific ubiquitin binding domains, therefore committing the target protein to distinct cellular outcomes. A combination of NMR methods are used to study the interaction of several ubiquitin binding domains with Lys48- and Lys63-linked di-ubiquitin, the simplest model of a polyubiquitin chain, to gain insights into polyubiquitin recognition. The di-ubiquitin binding interface with ubiquitin-interacting motifs (UIMs) and ubiquitin-associated domains (UBAs) are mapped. Structural models of the complexes are also presented. The results provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interface. The results also show that the UBA domain of Ede1 preferentially binds to Lys63-linked di-ubiquitin. Structural models of the UBA in complex with Lys48- and Lys63-linked di-ubiquitin are shown. Although ubiquitin is highly conserved in eukaryotes, it is promiscuous with regard to its binding partners, ranging from small molecules to UIM and UBA domains. This study examines the effects of point core leucine to serine mutations on UIM and UBA binding specificity. The results show that these mutations bestow ubiquitin with the ability to discriminate between ubiquitin-receptor proteins. Here, we solved the three-dimensional structure of the L69S Ub mutant in solution by NMR. These mutations have a profound effect on binding specificity while causing subtle changes in the protein's three-dimensional fold and reducing its stability. Modification of a specific lysine located on Ub's hydrophobic surface has been reported to inhibit proteasomal degradation and endocytosis. Here, the effects of mutation to tryptophan at this position are investigated within the context of binding to a proteasomal receptor protein, hHR23A, and an endocytic receptor protein, Ede1.