Biochemistry electronic theses and dissertations (MU)
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The items in this collection are the theses and dissertations written by students of the Division of Biochemistry. Some items may be viewed only by members of the University of Missouri System and/or University of Missouri-Columbia. Click on one of the browse buttons above for a complete listing of the works.
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Item Structural studies of flavoprotein inhibitors and inactivators(University of Missouri--Columbia, 2020) Campbell, Ashley Clare; Tanner, John J., 1961-[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI--COLUMBIA AT REQUEST OF AUTHOR.] Flavoenzymes have a versatile capacity to catalyze a wide range of reactions, using their flavin cofactor. In this thesis, work on two flavoenzymes is presented. The bifunctional enzyme SmPutA, from Sinorhizobium meliloti is composed of the proline dehydrogenase (PRODH) and glutamate semialdehyde dehydrogenase (GSALDH) domains. SmPutA was used as a surrogate for the human enzymes, to identify proline analogs that bind the active sites of PRODH and GSALDH, which are valuable chemical probes to study the roles of proline metabolism in cancer. Binders of the enzymes were searched for in crystallo resulting in the serendipitous identification of two distinct new mechanisms of covalent inactivation of PRODH. The first, presented in Chapter 1, thiazolidine-2-carboxylate (T2C) was characterized in the active site of PRODH. T2C was found in the active site of PRODH covalently bonded to the N5 of the flavin. Although the inactivation by T2C was slow, it led us to the discovery of two related inactivators of PRODH: tetrahydrothiophene-2-carboxylate (C2C) and 1,3-dithiolane-2-carboxylate (D2C), presented in Chapter 2. The inactivators D2C and C2C differed from T2C by only one atom, but surprisingly they inactivated the PRODH enzyme through an entirely different mechanism, which was triggered by light. D2C and C2C are decarboxylated in the active site and become bound to the N5 of the flavin via their C2 atoms. A mechanism starting with the light-generated triplet excited state of FAD is proposed. Additionally, non-covalent binders of GSALDH have been structurally characterized in the active site of GSALDH, in Chapter 3. Isomers of proline and hydroxyproline were able to bind the active site. Although the binding affinity is low, the contacts revealed in the structures could be leveraged when designing more potent inhibitors of GSALDH. Finally, Chapter 4 presents a series of crystal structures of the ornithine hydroxylase SidA, which reveal a dynamic flavin and active site.Item Merging technology development of trapped ion mobility spectrometry quadrupole time-of-flight mass spectrometry with biological investigation of Medicago truncatularoot exudates(University of Missouri--Columbia, 2020) Schroeder, Mark; Sumner, Lloyd W.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] "New commercial ion mobility instrumentation has the potential to improve current analytical techniques, but developments need to be applicable to real-world problems. Analytically challenging biological samples (i.e. root exudates) can be improved by this application of new ion mobility instrumentation. To this end, I have established an analytical reference library and developed new instrumentation methods (Bruker timsTOF Pro) to be applied specifically for specialized plant metabolites. I have also developed a method for collecting biological samples specifically for the new instrumentation. Utilizing this co-development strategy, new technology coupled with biological objectives, the capabilities of both have been improved" --From Chapter 1.Item Viral RNA structural equilibria and their interactions with host proteins(University of Missouri--Columbia, 2019) Brady, Samantha Elizabeth; Heng, Xiao[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI SYSTEM AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.Item RNA aptamers and HIV reverse transcriptase : molecular basis of their interactions and the evaluation of aptamer resistance(University of Missouri--Columbia, 2019) Nguyen, Phuong Dinh Minh; Burke-Aguero, Donald H.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI SYSTEM AT REQUEST OF AUTHOR.] Human Immunodeficiency virus (HIV) is a pandemic Lentivirus that causes acquired immune deficiency syndrome (AIDS) in infected individuals. One of the critical steps in HIV replication is the reverse transcription of viral RNA to generate viral DNA, which will be integrated into the host genome to generate materials for the production of new viruses. The enzyme responsible for this reverse transcription process is HIV reverse transcriptase (RT). Because of its importance in HIV life cycle, RT has been a common target for HIV inhibitors. In recent years, RNA aptamers have emerged as potential inhibitors against HIV RT. Although previous studies had shown that the inhibitory effect of RNA aptamers against HIV RT comes from their ability to compete with primer/template for binding to RT, the molecular details of RT-RNA aptamer interaction are still limited. The work described herein highlights advancements in identifying the functional features of a broad-spectrum RNA aptamer and elucidating the molecular details in the interaction of this aptamer and the target RT. Furthermore, this work explores the potential use of anti HIV aptamers in studying RT maturation by proteolytic processing.Item Biophysical characterization of the structure and flexibility of the E. Coli ribosome(University of Missouri--Columbia, 2019) Armbruster, Emily Doris; Cornish, Peter V.At the discovery of ribosomes by George Palade in 1955 in the first image of the subcellular environment, he described them as "a particulate component of small dimensions (100 to 150 [alpha]) and high density". Subsequently, the ribosome was shown to be the site of protein synthesis, or translation, and thus an essential macromolecular complex for all cells. Ribosomes can have variability from species to species, but the overall structure and function are conserved [66]. Ribosomes are named according to their sedimentation coefficients, a unit of density expressed in Svedbergs (abbreviated S). The three most studied and most prevalent ribosomes are the bacterial 70S, the eukaryotic 80S, and the 55S mitoribosome, which is present in the mitochondrion organelle. The bacterial ribosome serves as a target for many antibiotics and is a model system for investigating the structure and function of this "nanomachine". Despite variations in size, all ribosomes consist of a small and a large subunit that when bound together have an internal cavity that is divided into three sites, named A, P, or E site. The bacterial ribosome has a 30S small subunit, which consists of a 16S rRNA and 21 attached proteins, and a 50S large subunit that is made up of the 23S rRNA, 5S rRNA and 31 proteins. This dissertation discusses the 70S bacterial ribosome, other than when the 80S eukaryotic ribosome is specified.