The focus of the research presented in this thesis is on the investigation of human tyrosylprotein sulfotransferases TPSTs) using a novel mass spectrometry-based TPST assay. Two isozymes of TPST were identified to be present in the trans-Golgi network and were found to be membrane-bound. This has rendered their characterization a challenge compared to the well-studied cytosolic sulfotransferases. Tyrosine sulfation, a protein post-translational modification TPST catalyzes, is known to be essential for efficient protein-ligand binding involved in diverse biological functions. This has made studies on the molecular enzymology of TPSTs of particular interest. In the work presented herein, a quantitative LC/ESI-MS-based TPST assay was developed and applied to study its steady state kinetics. G protein-coupled CC-chemokine receptor 8 CCR8) peptides that have three tyrosine residues in series were chosen as substrates. This assay is the only method that can directly monitor individual sulfation of tyrosine residues in series and differentiate between mono- and multi-sulfated products, a feature radioactive labeling assays cannot provide. Hence, we were able to compare the kinetic properties of TPST-1, TPST-2, and an equal mixture of TPST-1 and -2 for both mono- and disulfation reactions of the CCR8 substrates. Our results show that the Km,appfor the monosulfated substrate was five-fold less than the nonsulfated substrate for both isozymes and its mixture. The development of this method is the initial step in the investigation of kinetic parameters of the sequential tyrosine sulfation of chemokine receptors by TPSTs. The MS-based TPST assay was used further to investigate the catalytic mechanism of TPST-2 using CCR8 substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling, experiments, our data strongly suggest a rapid equilibrium random two-site ping-pong model for TPST-2 catalysis. In this mechanistic model, the enzyme allows independent binding of substrates to two distinct sites and involves the formation of a sulfated enzyme covalent intermediate. Some insights on the important amino acid residues at the catalytic site of TPST-2 and its covalent intermediate are also presented. To our knowledge, this is the first detailed study of the reaction kinetics and mechanism reported for human TPST-2 or any other Golgi-resident sulfotransferase.

During spermatogenesis, one of the most drastic examples of chromatin remodelling takes place. In many organisms this coincides with drastic changes in chromatin composition, as histones are replaced by s perm nuclear basic proteins SNBPs) of the protamine type P-type). Due to their smaller size and higher charge, protamines compact sperm chromatin more efficiently. However, many organisms do not undergo this composition change and instead either retain histones similar to those in somatic cells in their sperm H-type) or gain protamine-like proteins PL-type), often in addition to histone. Fish and amphibian models are used in this thesis because they include genera with SNBPs representative of each of the three main types and provide a unique opportunity to study chromatin compaction. I focused on species that contain a partial or complete complement of histones in the sperm. Chapter 1 of this thesis is a review of the SNBP evolution, distribution and roles in chromatin compaction. In Chapter 2, the complete cDNA sequence of Xenopus laevis sperm specific proteins SP1 and SP2 is determined. Structural and functional analyses show that SP1/SP2 proteins are related to proteins of the histone H1 family, particularly to vertebrate histone H1x and are members of the protamine- like-I PL-I) group of SNBPs. In H-type organisms that retain histones in their sperm, a remodelling of chromatin and a reduction in nuclear volume still occur during spermiogenesis. However, the factors that lead to the condensation of chromatin in these organisms are unknown and are addressed in Chapter 3. Ictalurus punctatus is determined to have sperm chromatin of the H-type, which is maximally compacted and organized into a highly repetitive structure indicative of uniformly condensed chromatin. Several histone variants and post- translational modifications PTMs) are examined as a preliminary survey of factors potentially responsible for this compaction. Of the PTMs present in catfish testes, the most significant were histone H3 trimethylated at lysine 27, which is a well known marker of facultative heterochromatin, and histone H4 phosphorylated at serine 1, which has been documented to affect nuclear size and may help stabilize chromatin compaction in mice and yeast. A second extreme remodelling of the paternal pronucleus occurs following fertilization in order to convert the highly compacted, transcriptionally inert chromatin of the sperm into a substrate that is recognizable by the transcription and replication machinery of the zygote. Nucleoplasmin, a nuclear chaperone, participates in this remodelling in amphibians by displacing the specialized P-type and PL-type proteins from the sperm chromatin and by the transfer of H2A/H2B dimers. Nucleoplasmin was originally isolated from Xenopus PL-type) and belongs to the nucleophosmin/nucleoplasmin NPM) family of proteins, which have diverse functions in the cell Reviewed in Chapter 4). The existence of H-type sperm raises uncertainty about the need for a nucleoplasmin-mediated removal process in these organisms. In Chapter 5, the presence of nucleoplasmin in Rana catesbeiana H-type) and Bufo marinus P-type) is assessed. The amphibian nucleoplasmins are shown to phylogenetically group with mammalian NPM2 proteins and the implications suggested by the presence of nucleoplasmin in organisms of all three SNBP-types are discussed.

The analysis of protein residues recovered from archaeological artifacts provides a unique opportunity to reveal new information about past societies. However, many scientists are currently unwilling to accept protein-based results due to problems in method development and a basic lack of agreement regarding the ability of proteins to bind to, and preserve within, artifacts such as pottery. In this paper, I address these challenges by conducting a two-phase experiment. First, I quantitatively evaluate the tendency of proteins to sorb to ceramic matrices by using total organic carbon analysis and spectrophotometric assays to analyze samples of experimentally cooked ceramic. I then test a series of solvent and physical parameters in order to develop an optimized method for extracting and preparing protein residues for identification via mass spectrometry. Results demonstrate that protein strongly sorbs to ceramic and is not easily removed, despite repeated washing, unless an appropriate extraction strategy is used. This has implications for the future of paleodietary, conservation ecology and forensic research in that it suggests the potential for recovery of aged or even ancient proteins from ceramic matrices.

This dissertation describes three studies on the human NAD-dependent isocitrate dehydrogenase IDH), a heterotetrameric mitochondrial enzyme with 2alpha:1beta:1gamma subunit ratio. The three subunits share 40-52% identity in amino acid sequence and each includes a tyrosine in a comparable position: alphaY126, betaY137 and gammaY135. In the first study to examine the role of the corresponding tyrosines of each of the subunits of human NAD-IDH, the tyrosines were mutated one subunit at a time) to Ser, Phe or Glu. Enzymes were expressed with one mutant and two wild-type subunits. The results of characterization of the mutant enzymes suggest that betaY137 is involved in NAD binding and allosteric activation by ADP. The alphaY126 is required for catalytic activity and likely acts as a general acid in the reaction. The gammaY135 is also required for catalytic activity and may be involved in proper folding of the enzyme. The corresponding tyrosines in the three dissimilar subunits of NAD-IDH thus have distinctive functions. In the second study, we describe two families with retinitis pigmentosa, a hereditary neurodegeneration of rod and cone photoreceptors in the retina. Affected family members were homozygous for loss-of-function mutations in IDH3B, encoding the beta-subunit of NAD-specific isocitrate dehydrogenase NAD-IDH, or IDH3),which is believed to catalyze the oxidation of isocitrate to alpha-ketoglutarate in the citric acid cycle. Cells from affected individuals had a substantial reduction of NAD-IDH activity, with about a 300-fold increase in the Km for NAD. NADP-specific isocitrate dehydrogenase NADP-IDH, or IDH2), an enzyme that catalyzes the same reaction, was normal in affected individuals, and they had no health problems associated with the enzyme deficiency except for retinitis pigmentosa. These findings support the hypothesis that mitochondrial NADP-IDH, rather than NAD-IDH, serves as the main catalyst for this reaction in the citric acid cycle outside the retina, and that the retina has a particular requirement for NAD-IDH. In the third study, we separately expressed in bacteria and purified the alpha and gamma subunits of NAD-IDH. We studied the characteristics of the alphagamma complex and compared them to the properties of the complete wild-type enzyme as well as a previously studied complete mutant enzyme of gamma subunit gammaR97Q) which had wild-type alpha and beta subunits. Our results indicate that the alpha and gamma subunits alone are inactive. The kinetic properties of the wild-type alphagamma complex indicate that these two subunits are sufficient for efficient binding isocitrate, Mn 2＋, NAD and ADP, but the Vmax suggests that all three types of subunits are required for maximum activity. The gammaR97Q whole enzyme mutant had a high Km for NAD and a loss of allosteric activation due to ADP. Since the wild-type alphagamma complex exhibited Km values comparable to those of the wild-type, it is possible that the mutant gammaR97Q subunit interacts improperly with the other two wild-type subunits. The mutant complex of alphagammaR97Q was entirely inactive which confirms the possibility of improper interaction of mutated gamma with the other two subunits. It also proves that the activity of the whole enzyme mutant aR97Q can be attributed to the wild-type alpha and beta subunits which are present.

Advancements in technology have made it possible to print and visualize thousands of sequences of DNA on a glass slide (DNA Microarray). Specially designed DNA Microarrays with different oligonucleotide sequences were printed. These sequences do not self cross-hybridize and extend over the entire thermodynamic range of the Tm (melting temperature). KS＋ plasmid was used as model sequences. These labeled probes were hybridized onto our designed microarray slides and slowly melted by heating or by washing with stringent buffers. The image of the slide was taken at each time point to give a movie of different spots disappearing at different rates. This movie acted as a “Hybridization Profile” of the plasmids. Different DNA structures were designed and attached to silicon surface. Atomic force microscopy was used to confirm the attachment of DNA structures to the silicon surface. In the future, microarrays with several thousands of designed oligonucleotides will be printed on silicon surface and then used to generate different “Hybridization Profile” of the entire genome of closely related organisms like E. Coli and Anthrax using electrical methods.

The work described in this dissertation is divided into three sections. In the first section three surface modifications are used to produce MALDI targets having reduced surface-protein binding affinity with a goal of increasing peptide/protein MALDI ion signals and lowering the limits of detection LODs) for proteins and peptides. The second section discusses a bioselective MALDI target, produced via radio frequency rf) plasma deposited ethylenediamine EDA), for on-target separation of complex protein mixtures. The third section develops a new approach for characterization of rf plasma-deposited bulk polymers by using MALDI MS. Previous studies in our group have shown that the analyte signal in a MALDI MS experiment is strongly influenced by the binding interactions between the target surface and the analyte. Specifically, the analyte signal increases with decreasing surface-analyte binding affinity, which has been attributed to more unbound analyte being available for incorporation within the MALDI matrix. In the presented studies MALDI targets are modified with polyethylene glycol PEG)-like structures via chemical grafting of PEG onto polyurethane PU) film and rf plasma polymerization of ethylene oxide vinyl ether EO 2) and tetraglyme. It is shown that there are enhancements in the protein MALDI ion signals on these modified targets and that the LOD for target proteins is decreased by a factor of 2-10 in comparison with the conventional stainless steel MALDI target. On-probe affinity capture OPAC) MALDI MS, developed in our group, has shown that functional group modified MALDI targets can be used to rapidly and selectively isolate target analytes from complex samples. For applications involving analysis of complex peptide/protein mixtures, fractionation of the mixture on the basis of component pI can reduce MALDI ion suppression effects leading to efficient ionization of larger numbers of mixture components. In the present studies a MALDI target is modified by rf plasma deposition of polymerized EDA to yield an OPAC target suitable for capture of proteins with low pI expected to be negatively charged at neutral pH). In subsequent MALDI MS analyses of both control and biological mixtures after fractionation on the OPAC target it is observed that a significant number of additional peptide/protein ion signals are detected. The results of these studies, along with studies of the effects of the density of the primary amine functionality on the bio-selective MALDI ion signals, are presented. The complex nature of the polymer films resulting from plasma polymerization makes it very difficult to characterize their molecular structures. The presented study is the first to use MALDI MS for characterization of rf plasma-deposited bulk polymers and for investigation of the rf plasma polymerization process. It is shown that the mass spectra of the soluble fraction of allyl alcohol, EO2 and ethylene glycol butyl vinyl ether – plasma polymers contain clear polymer series. Furthermore, it is found that the peaks of the EO 2-plasma polymer series shift to higher molecular weight distribution with decreasing plasma duty cycle. In contrast to predictions based on conventional radical polymerization, the mass spectra of all three plasma polymers exhibit the same repeat unit of 44 Da, for which the most likely structure would be -CH2CH2O)-.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied, ubiquitous glycolytic enzyme that also exhibits several non-glycolytic activities, including DNA repair, membrane fusion, apoptotic signaling, tRNA transport and microtubule bundling. GAPDH binds telomere repeats, and in human A549 non small-cell lung adenocarcinoma cells, overexpression of GAPDH protects telomeres from chemotherapeutic agents. The maintenance of telomeric DNA in A549 cells appears to play a role in chemotherapeutic resistance. This study addresses the binding of GAPDH to human telomeric DNA and determines the extent to which GAPDH maintains the viability of A549 cells treated with the chemotherapeutic agents gemcitabine (GMZ) and doxorubicin (DOX). Molecular analysis of the GAPDH-telomere interaction shows GAPDH recognizes G5, G6 and T1 nucleotides of the telomeric-DNA repeat (5′-TTAGGG-3′), and binding to the single strand (ss) – telomeric DNA oligonucleotide (5′-(AGGGTT) 3-3′ with high affinity (Kd ＝ 45 nM). Furthermore, the DNA-binding site on GAPDH comprises both the enzyme catalytic site and NAD＋-binding site. The stoichiometry of binding is 2:1 (DNA:GAPDH) and GAPDH forms a high-molecular weight complex when bound to a synthetic ss-telomeric DNA oligonucleotide. Protection of telomeres by GAPDH in A549 cells increases cell viability following treatment with the chemotherapeutic agents GMZ and DOX. The data from these experiments combined with high-resolution crystal data allowed for construction of a putative model of GAPDH binding to ss-telomeric DNA.

Cellular signaling pathways transmit and process signals from receptors to activate gene expression programs that regulate development, cellular life/death decisions, or the coordinated activation of the immune response. While biochemical and molecular biological studies have identified a large number of signaling proteins with diverse adaptor and/or enzymatic functions, recent work has revealed that relatively few transcriptional effector proteins are responsible for specific gene expression programs in response to a large number of stimuli. This begs the question: what mechanisms ensure stimulus-specific gene expression? It has been shown that signaling events are highly dynamic, suggesting that further progress in the understanding of cellular signaling requires quantitative studies that include the temporal dimension. My graduate work has focused on the dynamic regulation of the transcription factor Nuclear Factor kappaB (NF-kappaB) in response to specific cellular stimuli, the mechanisms that encode stimulus-specific signaling dynamics, their potential functional roles, and the utility of integrated computational and experimental studies in unraveling complex regulatory networks.

Abuse of unregulated substances by young adults has been a great concern of the US and international community. Salvia divinorum has captured the interest of researchers and the general public because of its profound psychopharmacological properties. The active component of the plant, salvinorin A (1), has a potent affinity to the kappa-opioid receptor in CNS. This dissertation describes the biosynthesis of 1 through stable isotope labeling and determination of the enzymes involved in the pathway. Our data shows that 1 is formed in the glandular trichomes via the deoxyxylulose phosphate (DOXP) pathway. Incorporation of 13C isotopes was achieved through in vitro sterile culture of microshoots of S. divinorum. The synthesis of salvinorin A and B acids served as biosynthesis precursors for the characterization of carboxy methyltransferase (CMT), the enzyme catalyzing the methylation of carboxylic groups. The cell free enzyme assay, with the Escherichia coli expressed recombinant CMT, suggested substrate specificity of the CMT selectively methylating divinatorins A and C, and hardwickiic acid, the early intermediates of the pathway. A search for further biosynthesis precursors of 1 led to the isolation of a new neoclerodane diterpenoid, salvinorins J (10) as the first example of a neoclerodane hemiacetal (lactol) susceptible to mutarotation. A leaf surface extraction method was used for the first time on S. divinorum affording a chlorophyll-free extract containing predominantly neoclerodane diterpenoids.

Non-protein coding ribonucleic acids ncRNAs) are essential in biochemical processes in the cell including translation, protein regulation, and RNA modification. Of the various types of ncRNAs, transfer RNAs tRNAs) have proven difficult to analyze and monitor individually in a complex mixture. Analysis of individual tRNAs provides insight into changes in ncRNA expression levels based on experimental and environmental changes. The goal of this research is to optimize both the isotopic labeling method and the liquid chromatography tandem mass spectrometry approaches to quantify changes in non-coding RNAs. Quantification of RNA is achieved through endonuclease digestion and isotopic labeling of the 3-terminus of the digestion products. The accuracy and precision of the relative quantification is dependent upon the efficiency of 18O incorporation and its integrity upon analysis. I have investigated the effect of pH, temperature, and enzyme presence on the removal of the 18O label as it is replaced with 16O over time using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry MALDI-MS). Optimized conditions for sample handling and storage, which minimize back-exchange, should improve the accuracy and precision of results from this method. Previous studies on the relative quantification of isotopically labeled RNAs have been performed utilizing MALDI-MS. I have employed the isotopic labeling method for use on a liquid chromatography tandem mass spectrometry LC-MS/MS) platform； conditions that provide accurate and reproducible quantification of oligonucleotides have been investigated. The shift from a MALDI-MS to a LC-MS platform provides the ability for tandem mass spectral sequencing of RNAs and the analysis of more complex RNA mixtures. The analysis of individual isoacceptors within the global tRNA pool is one specific biological system that these methods are developed to address. To monitor changes in expression levels on individual tRNA isoacceptors, the characterization of quantifiable signature digestion products on LC-MS/MS was investigated using total tRNA isolated from Escherichia coli. These verified quantifiable signature digestion products were then used to monitor changes in tRNA abundances from Escherichia coli cells grown under two different nutrient conditions. The methods described in this dissertation allow for the simultaneous identification and quantification of tRNAs under various environmental and experimental conditions.