Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
Browse
3 results
Search Results
Item STRUCTURAL AND FUNCTIONAL STUDIES OF CYCLIC K48-LINKED DIUBIQUITIN(2016) Sundar, Adithya; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)K48-linked di-ubiquitin exists in a dynamic equilibrium between open and closed states. The structure of K48-Ub2 in the closed conformation features a hydrophobic interface formed between the two Ub domains. The same hydrophobic residues at the interface are involved in binding to ubiquitin-associated (UBA) domains. Cyclization of K48-Ub2 should limit the range of conformations available for such interactions. Interestingly, cyclic K48-linked Ub2 (cycUb2) has been found in vivo and can be isolated in vitro to study its structure and dynamics. In this study, a crystal structure of cycUb2 was obtained, and the dynamics of cycUb2 were characterized by solution NMR. The crystal structure of cycUb2, which is in agreement with solution NMR data, is closed with the hydrophobic patches of each Ub domain buried at the interface. Despite its structural constraints, cycUb2 was still able to interact with UBA domains, albeit with lower affinity.Item THE INTERPLAY OF SUBSTRATE, PROTEIN AND ITS COFACTOR IN CONTROLLING THE CATALYTIC PROPERTIES OF HUMAN IODOTYROSINE DEIODINASE(2014) Hu, Jimin; Rokita, Steven E; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human iodotyrosine deiodinase (hIYD) belongs to nitro-FMN reductase superfamily and is responsible for recycling iodine from the I-Tyr (monoiodotyrosine) and I2-Tyr (diiodotyrosine) formed as byproducts of thyroid hormone synthesis. Heterologous expression of hIYD lacking its membrane domain in E. coli provided large quantities of highly pure hIYD that allowed for its physical and biochemical studies. Its kinetic parameters, binding constants and crystal structure were characterized. Substrate was able to induced dramatic effects on FMN coordination in hIYD, including the zwitterionic region of I-Tyr interacts with the N3 and O4 of the FMN, the OH of Thr239 moved close to N5 of FMN (from 4.8 Å to 3.1Å) and allowed the formation of a hydrogen bond. The aromatic ring of I-Tyr additionally stacks above the FMN. Accumulation of the neutral semiquinone was observed during the reduction of hIYD in the presence of substrate analogue 3-fluoro-L-tyrosine (F-Tyr). In the absence of ligand, only the oxidized and two-electron reduced forms of FMN were observed. Among all of the interactions to FMN in hIYD, H-bonding at the N5 FMN was identified as most important to control the redox of flavin. Mutagenesis of Thr239 to Ala removed this H-bonding as confirmed by the crystal structure of hIYDT239A*F-Tyr. As a result, no semiquinone was detected during the titration of hIYDT239A in the presence of F-Tyr. The deiodination activity of T239A was also dramatically decreased (10-fold). The zwitterion region of the substrate was found critical for binding to enzyme. Modifications of the zwitterion region resulted in at least a 500-fold increase in KD. The catalytic activity was unlikely determined by the zwitterion region since all of the modified substrates exhibited similar initial rates as the native substrate under conditions in which their concentration equaled their KD. The pH dependence of hIYD binding indicated that the phenolic group of I2-Tyr is also critical for binding and the hIYD prefers binding to the phenolate form of substrate. All the results presented in this thesis support the current proposed catalytic mechanism of IYD involving a stepwise electron transfer process.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.