Bibliography: leaves 194-217. / This thesis investigates whether DNA and histones contain sufficient information to direct nucleosome cores into specific positions. The "in vitro" assembly of nucleosome cores promoted by poly(glutamic acid) has been optimized with respect to rate and yield. This was achieved by paying attention to the purity of the core constituents and in particular by the use of histones in their octameric form. The suitability of a number of octamer purification protocols, to produce pure undenatured histone octamers, has been investigated and the methodology improved. The particles assembled on random DNA have been found to be indistinguishable from native nucleosome cores by the following criteria: Their S value on sucrose gradient centrifugation, resistance to Micrococcal nuclease digestion, DNase I digestion patterns, DNase I digestion kinetics at the susceptible sites, electronmicroscopic appearance, hi stone content and electrophoretic mobility. Cores were also assembled on unique DNA, namely the intact h22 histone quintet of Psammechinus miliaris. Low resolution mapping, by indirect endlabelling of polycores assembled on the quintet, did not reveal any preferred sites of assembly. To investigate the core associated DNA at single base pair resolution, a series of fragments, excised from the H2A-Hl and the Hl-H4 spacer areas, were inserted into pGV403 plasmids. These plasmids can be strand specifically end-labelled with the Klenow fragment at the two different Tth 111 I excision sites utilised to isolate the propagated insert. On the free linearised DNA a complex digestion pattern is produced due to the sequence specificities of Micrococcal nuclease and DNase I. When cores are assembled on this DNA the digestion pattern is changed. This pattern reveals two preferential frames of assembly and indicates that in the remainder of the fragments cores are assembled, randomly, or in a number of overlapping frames. It is concluded that the DNA fragments investigated and the hi stone octamer contain enough structural information to influence the positions occupied by some nucleosome cores. The implications of these findings are discussed.

abstract: Non-alcoholic fatty liver disease occurs when triglycerides are stored in the liver leading to irreversible scarring and damage of liver tissue. Inside the liver, adipose triglyceride lipase is responsible for the breaking down of triglycerides and is regulated by the inhibitor g0/g1 switch gene 2 (G0S2). G0S2 is proposed to be one of the targets against drug design for non-alcoholic fatty liver disease, and more information is needed on the structure of this protein to aid in drug discovery. Here I describe the expression of G0S2 in an E. coli system as well as purification and biophysical characterization of a functional G0S2 in amounts viable for solution state Nuclear Magnetic Resonance (NMR) spectroscopy. Initial spectra of the isotopically labeled protein show well dispersed 15N resonance lines, clean 13C resonances, and dominant a-helices characteristics. These results show that a prepared G0S2 construct is suitable for solution NMR such that 20 amino acids are now assigned in the G0S2 portion of the protein, allowing for further NMR work with this protein for structural discovery. Further work with a large oligomeric complex of G0S2 with Maltose Binding Protein also shows promise for future cryo-EM work. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

Bibliography: pages 108-124. / Acetylation, one of the post-synthetic modifications of histones, weakens histone-DNA interactions and may play a regulatory role in gene control of eukaryotes. The literature available on histone acetylation as well as other post-synthetic modifications of histone has been reviewed. Histone acetylation is catalysed by an enzyme(s) which transfers acetyl groups from a donor molecule to histones. A crude histone transacetylase preparation was isolated from nuclei and the optimal conditions for the acetylation of histones were determined. This enzyme(s) was shown to be specific for histones with protamine displaced histone complex being the best substrate. Using this histone transacetylase preparation ³H-acetyl total histone was prepared in sufficient yield and with a high enough specific activity to enable sequential Edman degradation of the histone sub-fractions isolated from the total histone complex to be undertaken. Histones H3 and H4 were isolated from the acetylated total histone as they exhibited the highest degree of acetylation. Histone H4 peptides were generated by chymotryptic and tryptic digestion as the intact histone H4 polypeptide chain is blocked at its N-terminus. The Edman degradations of histone H3 and H4 showed that the acetylation sites that have been determined under in vitro conditions are the same as those undergoing acetylation in vivo. All of the acetylation was found in the N-terminal region of histones H3 and H4 with histone H4 showing a gradient of decreasing acetylation from the N- to the C-terminus, in contrast to histone H3 where the first two possible acetylation sites are acetylated to a minor degree only.

abstract: In this study, the stability of two protein homo-oligomers, the β clamp (homodimer) from E. coli and the Proliferation Cell Nuclear Antigen (PCNA) from the yeast cell, were characterized. These clamps open through one interface by another protein called clamp loader, which helps it to encircle the DNA template strand. The β clamp protein binds with DNA polymerase and helps it to slide through the template strand and prevents its dissociation from the template strand. The questions need to be to answered in this research are, whether subunit stoichiometry contributes to the stability of the clamp proteins and how does the clamp loader open up the clamp, does it have to exert force on the clamp or does it take advantage of the dynamic behavior of the interface? The x-ray crystallography structure of the β clamp suggests that there are oppositely charged amino acid pairs present at the interface of the dimer. They can form strong electrostatic interactions between them. However, for Proliferation Cell Nuclear Antigen (PCNA), there are no such charged amino acids present at its interface. High sodium chloride (NaCl) concentrations were used to disrupt the electrostatic interactions at the interface. The role of charged pairs in the clamp interface was characterized by measuring the apparent diffusion times (\tau_{app}) with fluorescence correlation spectroscopy (FCS). However, the dissociation of the Proliferation Cell Nuclear Antigen (PCNA) trimer does not depend on sodium chloride (NaCl) concentration. In the next part of my thesis, potassium glutamate (KGlu) and glycine betaine (GB) were used to investigate their effect on the stability of both clamp proteins. FCS experiments with labeled β clamp and Proliferation Cell Nuclear Antigen (PCNA) were performed containing different concentrations of potassium glutamate and glycine betaine in the solution, showed that the apparent diffusion time\ {(\tau}_{app}) increases with potassium glutamate and glycine betaine concentrations, which indicate clamps are forming higher-order oligomers. Solute molecules get excluded from the protein surface when the binding affinity of the protein surface for water molecules is more than solutes (potassium glutamate, and glycine betaine), which has a net stabilizing effect on the protein structure. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

Phenylalanine Hydroxylase (PheH, EC: 1.14.16.1) is a non-heme iron monooxygenase that converts L-Phe to L-Tyr using a tetrahydrobiopterin coenzyme and dioxygen. PheH is allosterically regulated by its substrate L-Phe. Binding of L-Phe to the allosteric site causes PheH to convert from a low-activity T-state to a high-activity R-state. Upon the T to R conversion, PheH experiences an increase in hydrophobicity, a shift in fluorescence emission peak maximum, increased hydrodynamic radius, and elongation of its quaternary structure. While PheH has been well characterized in terms of these global changes that occur upon allosteric activation, less is known about how the active site properties change upon this process. Herein, we report the use of cw-EPR and pulsed-EPR techniques (electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) methods) to probe the properties of the active site of PheH with respect to the primary coordination sphere of the non-heme iron center and the orientation of pterin coenzyme and substrate L-Phe. A suit of cw-EPR experiments using 17O enriched water and 1H-HYSCORE approaches were used to probe the water coordination of the iron center across mechanistic states of PheH and select mutants. The ESEEM technique was combined with 2H labelled L-Phe and H4pterin coenzyme to examine how the binding orientation of both L-Phe and H4pterin change upon allosteric activation. These results demonstrate that L-Phe allosteric activation causes the pterin coenzyme to move ~1.8 Å closer to the iron center. Furthermore, 2H-ESEEM samples prepared at non-activating concentrations of L-Phe suggest that its binding in the active site alone is not sufficient to induce the movement of the pterin coenzyme. 2H-ESEEM studies using site specifically deuterated L-Phe demonstrate that allosteric activation and binding of pterin cause the reorientation of L-Phe such that both the meta and ortho positions are similarly close to the iron center. Lastly, steady state kinetic measurements of pterin oxidation within the active site of apo-wtPheH and apo-PheH118-452 were measured to understand how the active site influences the reactivity of pterin with O2. These results will be explained in the context of the regulatory properties of PheH as well as the catalytic mechanism.

No description available.

Type II diabetes was identified as a major cause of adult-onset blindness. Patients with diabetes are at increased risk of developing corneal complications due to abrasions, lesions, and ulcers, because of an altered wound healing. Several surgical treatment modalities can correct the complications mentioned above, including corneal transplant. However, due to impaired wound healing displayed by diabetic tissue, surgical options come with an increased risk of complication. Purinoreceptors P2X7 and P2Y2 have been implicated in cell-cell communication, migration, cytoskeletal actin rearrangement. All of these are required for wound healing. Previous studies utilized irreversible inhibitors and siRNAs to block these purinoreceptors and showed that both cell movement and wound healing were affected to a significant degree. This thesis will attempt to determine the impact of inhibiting P2X7 and P2Y2 with their respective competitive inhibitors on cell movement during the wound healing process in Human Corneal Limbal Epithelial cell cultures.

No description available.

No description available.

Amyloidosis encompasses a group of diseases that are characterized by protein misfolding and irregular extracellular deposits formed by the buildup of non-native protein structures and amyloid fibrils in visceral organs and tissues. There are over 35 proteins having the potential to form amyloid deposits and fibrils in humans. Amyloid pathologies are hereditary or can develop sporadically and exist in local and systemic forms. In localized forms of the disease, amyloid deposits are limited to one tissue area or organ in the body, whereas the systemic types feature multiple sites of infiltration. There are many different systemic amyloid disease types, but this study focuses on a rare form of systemic amyloidosis known as beta2-microglobulin (ß2-m)-related amyloidosis. The ß2-m protein, it is a 99-residue protein, that makes up the light chain portion of the class 1 major histocompatibility complex (MHCI) which is present of the surface of all nucleated cells. ß2-m is usually cleared through the kidneys upon dissociation from MHCI. However, patients with end stage renal failure can develop hemodialysis-related systemic amyloidosis when dialysis fails to remove ß2-m from the circulation and elevating concentrations of the protein lead to formation of amyloid fibrils. Alternatively, a hereditary form of ß2-m amyloidosis was identified in 2012 and reported to be associated with the gene mutation encoding the ß2-m variant, D76N. The phenotypic expression in this type of ß2-m amyloidosis was very different from the dialysis-related disease associated with the wild-type protein. The present study focuses on a newly identified and unique amyloid-forming ß2-m variant protein associated with a clinical presentation unlike the dialysis-related or previously reported hereditary form of ß2-m amyloidosis. In 2019, a novel ß2-m variant was discovered at the Amyloidosis Center at Boston Medical Center. A 59-year-old woman of Portuguese descent with a family history of cardiac amyloidosis presented with amyloid deposits in her fat tissue along with indications of cardiac involvement; the combination of these features led to a diagnosis of systemic amyloidosis. DNA sequencing and mass spectroscopy revealed that the patient had a mutation in her ß2-m gene resulting in the replacement of leucine for proline at the 32nd residue (P32L) of the ß2-m protein. The aim of this research project was to understand the amyloid-forming nature of the P32L variant. To address this goal, we characterized the biophysical properties of P32L ß2-m. The structural stability of P32L was compared to results for wild-type ß2-m, the previously described D76N amyloidogenic protein, and a synthetic variant with an alternative amino acid replacement at residue 32, P32G. This latter protein was chosen as it has been previously studied and shown to have amyloidogenic properties in vitro. Using a pQE-1 plasmid optimized for expression in E. coli, N-terminal MKH6 tagged wild-type ß2-m, was produced and subsequently modified, to individually produce P32L, D76N, and P32G. Variant plasmids were transformed in Rosetta-gami 2 cells for expression and purification. Circular dichroism, chemical denaturation, and limited proteolysis were used to assess protein secondary structure and stability. Aggregation of variants was evaluated using Thioflavin T fluorescence and ultrastructural imaging of aggregates was accomplished with electron microscopy. Results displayed that wild-type and variant ß2-m proteins showed similar secondary structures, all mainly comprised of beta-sheets. Based on high percentages of beta-sheets and low amounts of unordered secondary structure, wild-type ß2-m was the most stable of the proteins and D76N was the least. Moreover, wild-type had the highest apparent melting temperature followed by P32L, P32G and D76N. In thermal unfolding and refolding experiments, wild-type exhibited an irreversible mechanism, while P32L was semi-reversible upon heating and cooling. Chemical denaturation showed that D76N required the lowest concentration of guanidine hydrochloride to denature and wild-type the highest amount. Aggregation of digestion products was apparent in both trypsin and thrombin reactions; electron microscopy demonstrated that trypsin treatment of P32L yielded pre-fibril oligomeric structures. Overall, our data suggest that P32L and all variants were less stable than the wild-type protein. D76N appeared to be the least stable of the variants; P32L and P32G exhibited similar characteristics, both more stable than D76N and less stable than wild-type. The amyloid-forming nature of P32L ß2-m may be a result of the peptidyl-prolyl bond at P32 presenting in a trans conformation. When P32 is in the thermodynamically favorable trans conformation, it has been shown to trigger amyloid formation. This case was heterozygous for the mutation and the mixture of variant and wild-type proteins may destabilize the P32 isomer.