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.