More than 90% of human genes undergo a processing step called splicing, whereby non-coding introns are removed from initial transcripts and coding exons are ligated together to yield mature messenger RNA. Roughly 50% of human genetic diseases correspond to aberrant splicing. Splicing is catalyzed by an RNA/protein machine called the spliceosome. RNA components of the spliceosome are at least partly responsible for splicing catalysis. In addition, in vitro analyses implicate an essential and very highly conserved protein, Prp8, in orchestrating key steps in spliceosome assembly and possibly catalysis. Interestingly, mutant alleles of Prp8 are the cause of retinitis pigmentosa, an inherited form of retinal degeneration. A key goal is elucidation of the precise role of Prp8 in the spliceosome by high resolution structural analysis. The large size of Prp8 and its insolubility hinder progress in this regard. Instead, structural understanding of Prp8 can be gained by investigating domains in isolation; however there is only limited information as to what domain boundaries are and few hints about the functional relevance of putative domains. Here we have further defined the previously proposed domain IV in Prp8, and identified the domain IV core. Structural determination of the domain IV core reveals an RNase H fold, which could not be predicted based on primary sequence alone. RNase H recognizes A-form nucleic acid duplexes, which strongly suggests the domain IV core interacts with double-stranded RNA in the context of the spliceosome. Characterizing the binding preferences of the domain IV revealed the highest affinity is for a 4-helix junction structure adopted by the very RNAs at the spliceosome active site. Our characterization of the protein/RNA binding interface by complementary footprinting techniques currently provides the best model of how RNA interacts with an essential protein component at the heart of the spliceosome.

Compound I of myeloperoxidase is capable of both one-electron oxidation and two-electron oxidation reactions. Halides and pseudohalides are the substrates for the two-electron oxidation and other compounds including a large variety of aromatic alcohols and amines can be oxidized via the single electron oxidation pathway. To investigate the catalytic mechanism of myeloperoxidase four structures of complexes of myeloperoxidase were solved. Two of them are complexes with hydroxamic acids and the other two are complexes with nitrite. Hydroxamic acids (salicylhydroxamic acid and benzylhydroxamic acid) can function as structural analogues for the aromatic alcohol and amine substrates of myeloperoxidase. The crystal structures of complexes of MPO with both hydroxamic acids have been solved at 1.85 Å resolution and their binding to myeloperoxidase is compared. The models show similar binding of their hydroxamic acid moieties but different orientations of their aromatic rings. The absence of the hydroxyl group covalently bound to the benzyl group in benzylhydroxamic acid creates an environment that does not permit the same favorable interactions with MPO when compared to salicylhydroxamic acid. These findings could explain the three orders of magnitude difference in the value of the dissociation constants of the two complexes. Nitrite has been shown to bind myeloperoxidase and also to reduce Compound I and Compound II. Crystal structures of the complex between myeloperoxidase and nitrite confirmed the binding of nitrite to the native enzyme both in the distal cavity and the chloride-binding site. The binding in the distal cavity occurred to the heme iron in the nitro mode. In the MPO-cyanide-nitrite ternary complex, nitrite had been shown to bind only at the chloride-binding site. No secondary site for nitrite binding had been seen in the distal cavity when cyanide was liganded to the iron. Overall, this study is the first to show from a crystallographic point of view a comparison in the mode of binding of the two hydroxamic acids to a mammalian peroxidase and also the binding of nitrite to a heme peroxidase.

Thesis (Ph. D.)--University of Delaware, 2006. / Principal faculty advisor: Tatyana Polenova, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 152-164).

Thesis (Ph. D.)--University of Chicago, Dept. of Astronomy and Astrophysics, August 2000. / Includes bibliographical references. Also available on the Internet.

Ph.D. / Biochemistry and Molecular Biology / The human copper chaperone to superoxide dismutase (hCCS) is a zinc and copper containing protein that delivers copper to the active site of the cytoplasmic protein superoxide dismutase (SOD). hCCS is a three domain protein with three possible copper binding sites: Domain I is called the Atx-like domain and contains the copper binding motif MXCXXC; Domain II is the SOD-like domain and includes the slightly altered histidine rich copper binding site seen in SOD; and Domain III is a short C-terminal tail that has the copper binding motif of CXC. Studies of the WT protein using EXAFS showed that the protein contained a binuclear copper-sulfur cluster. Initially, it was unknown whether this cluster was formed between domain I and domain II of the protein or formed intermolecularly between two hCCS monomers. Further studies, on the cysteine to serine mutants of the residues in the Domain I and Domain II motifs, showed that while the Domain I motif is capable of binding Cu(I), it is Domain III that is the site of the copper cluster and the cluster is formed between two hCCS monomers. Additional studies with cysteine to alanine mutants of the residues in the copper binding motifs of Domain I and Domain III showed that Domain III is not only the site of the copper cluster but also the site of transfer of copper from hCCS to SOD and also a imerization interface for hCCS. While Domain I can bind copper and may play a role in regulation of activity, it is Domain III that contains the activity of hCCS.

<p><font face="Arial"> <p align="left">In this study, the capabilities of Neutron Radiography (NRad) and -Tomography (NTomo), as well as X-ray Radiography (XRad) to investigate chlorides and chlorides corrosion effects within steel reinforced laboratory concrete samples are practically explored. Capabilities of Scanning Electron Microscopy (SEM), Prompt Gamma Neutron Activation Analysis (PGNAA), Particle Induced X-ray Emission (PIXE), Small-Angle Neutron Scattering (SANS), Neutron Diffraction (NDIFF) and X-ray Diffraction (XDIFF) analytical techniques are also explored through review of literature.</p> </font></p>

Rare earths and copper systems were studied using X-ray absorption spectroscopy (XAS) and resonant inelastic X-ray scattering (RIXS). The use of monochromased synchrotron radiation and improved energy resolution for RIXS made possible to obtain valuable information on the electronic structure in 4f, 5f and 3d systems. Experimental results for rare-earths (Ho, Gd, Cm,U, Np, Pu) were analyzed by atomic multiplet theory based on the Hartree-Fock calculations. The inelastic scattering structures in RIXS spectra at 5d edge of actinides found to be sensitive to actinide oxidation states in different systems. Comparison of experimental and calculated Cm 5d RIXS spectra gave direct information about valency of the 248-curium isotope in oxide. Scientific understanding of processes that control chemical changes of radioactive species from spent fuel is improved by studying interactions of actinide ions (U, Np, Pu) with corroded iron surfaces. RIXS measurements at the actinide 5d edge indicate the reduction of U(VI), Np (V) and Pu (VI) to U(IV), Np(IV) and Pu (IV) by presence of iron ions. This thesis is also addressed to the study of changes in the electronic structure of copper films during interaction with synthetic groundwater solutions. The surface modifications induced by chemical reactions of oxidized 100Å Cu films with Cl−, SO42− and HCO3– ions in aqueous solutions with various concentrations were studied in-situ using XAS. It was shown that the pH value, the concentration of Cl− ion and presence of HCO3– ion in the solutions strongly affect the speed of the corrosion reaction. The Cu 2p RIXS was used to distinguish between the species present on the copper surface while in contact with groundwater solution.

Human immunodeficiency virus type 1 (HIV-1), the pathogen responsible for the onset of acquired immuno-deficiency syndrome (AIDS) in humans has reached pandemic proportions. To this day, no cure is available for infection with this virus and the only treatment option for this chronic infection is the life-long adherence to anti-retroviral therapy. Efforts in the quest to control the worldwide AIDS pandemic include the search for an effective anti-HIV-1 vaccine. Providing hope in this endeavor are a few monoclonal antibodies possessing broad neutralizing characteristics (bnmAbs) that have been isolated from the sera of rare patients that have a delayed progression to AIDS. In this thesis, one of these bnmAbs, 2F5 is extensively characterized at the atomic level to better understand its binding and neutralization mechanism. In total, 27 crystal structures of the 2F5 Fab’ in complex with various peptides representing its linear gp41 membrane proximal external region (MPER) epitope are presented. Furthermore, expression of the 2F5 Fab in a bacterial system allowed to design mutants of the 2F5 Fab and therefore investigate the implication of specific domains of 2F5 in mediating binding and neutralization. Atomic level characterization of this immune complex revealed a somewhat promiscuous recognition of 2F5 for its 664DKW666 epitope as long as the following characteristics were conserved: the aspartate’s negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. Moreover, it was demonstrated that 2F5 has an elongated and flexible complementary determining region 3 loop of the heavy chain (CDR H3), which is required for neutralization and is involved in secondary binding interactions other than to its core linear epitope. These contributions will significantly help in guiding the structure-based design of an HIV-1 vaccine looking to elicit 2F5-like antibody responses.

Chronic arsenic poisoning due to arsenic contamination of groundwater is a serious public health problem in Bangladesh and neighboring countries. Severe health effects associated with chronic exposure to arsenic include melanosis and several kinds of cancer. It is now generally agreed that the arsenic contamination of groundwater in Bangladesh is of geological origin. Arsenic naturally present in aquifers may be mobilized into drinking water by microbial action.<p> The formation of a novel arsenic-selenium compound: seleno-bis (S-glutathionyl) arsinium ion, [(GS)2AsSe]-, and its subsequent excretion in rabbit bile has been demonstrated previously. This molecular basis for the in vivo antagonism between arsenic and selenium was discovered using X-ray absorption spectroscopy. There is growing evidence that, in Bangladeshi people who are suffering long term chronic lowlevel arsenic poisoning, this antagonism is causing a selenium deficiency. Administering selenium supplements might provide a simple but highly effective treatment of the Bangladeshi arsenic poisoning.<p> In order to examine the disposition of [(GS)2AsSe]-, a set of rabbits were intravenously injected with selenite, arsenite or both. Whole blood, red blood cell and plasma samples were collected at different time intervals within 2hrs after injection and cecotrope samples 24hr after injection. Samples were examined using X-ray absorption spectroscopy and both arsenic and selenium K-near edge spectra were recorded.<p> iii Speciation of arsenic and selenium will be discussed in this thesis. Results indicate that [(GS)2AsSe]- is formed in blood very rapidly after injection of both arsenite and selenite, and then is removed from blood stream within 2hrs post injection. Results also show that [(GS)2AsSe]- is assembled in red blood cells, with no [(GS)2AsSe]- detected in plasma samples. [(GS)2AsSe]- is also found in cecotrope samples after injection of both arsenite and selenite.<p> The results of this study in rabbits will contribute to the understanding of chronic arsenic poisoning in humans.