Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
Browse
5 results
Search Results
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 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 MANIPULATING AND SIMPLIFYING THE INTERMOLECULAR INTERACTIONS IN LIQUID MIXTURES(2017) Gao, Ang; Weeks, John D; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Long ranged intermolecular interactions have significant influence on the structure of the liquid and present serious challenges for computer simulations. In particular, the long ranged tail of Coulomb interaction usually needs to be calculated using Ewald summation or related techniques in computer simulation, which can be too time consuming to be carried out for large systems. Local Molecular Field(LMF) theory has been developed to simplify long-ranged Coulomb and Van der Waals interactions for nonuniform liquids by approximating these long ranged interactions as effective static single-particle fields. Despite the success LMF theory made in describing the structure of nonuniform liquids, it is not appropriate to use LMF theory to describe the structure of uniform liquid mixtures, since the dynamically moving unbalanced forces produced in mixture can not be captured by the framework of LMF theory. In this thesis, we propose a new framework which approximates the unbalanced forces produced in a mixture as effective intermolecular interactions. This new framework can simplify the long ranged intermolecular interactions and produce a mimic system with short ranged solvent-solvent interactions, which is much easier to simulate or analyze. Based on this framework and other techniques introduced in this thesis, we have constructed a "Short Solvent Model", which has noticeable advantages compared to the explicit solvent model and implicit solvent model. This framework has also been used to simplify the interactions of phase-separating mixtures. The impact of using this framework on the diffusion dynamics of the solutes has also been discussed. Possible application of this framework and the Short Solvent Model to biopolymers folding problems is briefly discussed.Item SITE-SPECIFIC INTERACTION OF DOXORUBICIN IN THE IRON RESPONSIVE ELEMENT RNA: IMPLICATIONS IN CELLULAR IRON HOMEOSTASIS AND NON-IRON DEFICIENCY ANEMIA(2014) Alvarado, Luigi Jhon; Dayie, Theodore K; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A widely utilized chemotherapy drug, doxorubicin, has recently been shown to bind to a mammalian 5′ untranslated region Iron Responsive Element (IRE) RNA. In conjunction with the Iron Regulatory Protein (IRP), IRE RNA is involved in cellular iron homeostasis at the translational level. This tight RNA/protein complex prevents ribosomal assembly, hindering translation initiation of iron storage proteins, e.g. ferritin, under low cellular iron conditions. Conversely, iron overload is conducive to complex dissociation, allowing for up-regulation of the same proteins. However, this system is not entirely efficient. Some anemic patients receive adjuvant chelation therapies upon chronic blood transfusions to sequester excess labile iron. The use of doxorubicin to promote RNA/protein dissociation could potentially allow for downstream up-regulation of ferritin. In this work, I show how doxorubicin interacts with IRE RNA using multidimensional nuclear magnetic resonance, fluorescence spectroscopy, and electrophoretic mobility shift assays. All three approaches converge on the observation that the IRE/IRP complex formation is disrupted by doxorubicin. Obtaining further data on the RNA/protein/drug interactions may lead to unveiling a validated RNA target as a complementary treatment of iron overload disease, e.g. sickle cell anemia.Item Structural Studies of the Atypical Rio2 Kinase and N-Acetylglutamate Synthase(2011) Sagar, Vatsala; LaRonde, Nicole; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rio2 is required for small subunit ribosomal RNA (rRNA) maturation. It is specifically involved in site D cleavage of the 20S rRNA to produce the mature 18S rRNA. Loss of Rio2p is lethal and a decrease of Rio2p activity results in 20S accumulation in the cytoplasm. One of the goals of this thesis was to crystallize Rio2 from a eukaryotic organism in order to determine the structural differences between eukaryotic and archaeal versions. Another goal was to define the importance of individual domains. Yeast Rio2 was the first eukaryotic protein purified, but it formed only microcrystals. Human Rio2 was purified next, but the solubility was too low to set up crystal trays. Finally, Rio2 from Chaeotomium thermophilum was purified and crystals were obtained. The structure revealed a possibly inhibitory alpha helix blocking the active site. The role of the N-terminal winged helix domain of Rio2 in yeast was investigated and found not to be necessary for binding of Rio2p to the ribosome. The crystal structure of the first N-acetylglutamate synthase (NAGS) was also determined. The crystal structure of NAGS complexed with acetyl-CoA and with CoA plus N-acetylglutamate was determined at 2.5 and 2.6-Å resolution. Each NAGS consists of an N-terminal amino acid kinase domain (AAK) domain and a C-terminal N-acetlyltransferase (NAT) domain connected by three amino acids. The monomers form a six membered ring with a trimer of dimers symmetry. The AAK domains form two dimeric contacts with other AAK domains. Each AAK domain interacts with the NAGS domain of another monomer at the polar ends of the ring. The NAGS domain contains the active site. The AAK domain is believed to bind arginine and also helps to bind acetyl-CoA. Structural insights suggest a one step mechanism in which both substrates bind and the acetyl group is directly passed from acetyl-CoA to the alpha amino group of gluatamate. In addition, collaborative work on the structural characterization of the MphR(A) protein is reported.