Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3486. Adviser: David M. Kranz. Includes bibliographical references (leaves 98-113) Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3489. Adviser: Susan A. Martinis. Includes bibliographical references (leaves 144-155) Available on microfilm from Pro Quest Information and Learning.

Thesis (M.S.)--Lehigh University, 2009. / Advisers: Laurie B. Joseph; Ned D. Heindel.

<p> Alzheimer's Disease (AD) is a progressive neurodegenerative disease characterized by the formation of insoluble neurotoxic &beta;-amyloid (A&beta;) plaques and loss of cognitive function. Plaques have been shown to co-precipitate with both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Interestingly, there is a dramatic increase in BuChE activity relative to AChE in AD patients. Neuroblastoma cells were used to determine the effect of di-n-butyl 2-chlorophenyl phosphate (DBPP), an irreversible inhibitor of BuChE, on formation of A&beta;. Cells cultured in 10 &mu;M DBPP accumulated significant amounts of the compound without an effect on cell proliferation, membrane integrity, or induction of apoptosis. The intracellular level of BuChE activity was reduced and there was a decrease in amyloid precursor protein (APP) levels. In contrast, there was a concomitant increase in the levels of both A&beta;40 and A&beta;42 peptides. The implication is that irreversible inhibition of BuChE activity may increase the rate of A&beta; formation.</p>

The catalytic mechanism of thymidylate synthase (TS) was investigated using X-ray crystallography: four structures that yield new information about the early stages of TS action are reported. TS catalyzes the production of thymidylate (TMP), one of the four nucleotide bases of DNA, from the substrate, deoxyuridylate and cofactor, methylenetetrahydrofolate (MTF). Knowledge about the TS mechanism is important for both the medical and basic sciences. TS is the sole de novo source of TMP and it is thus a target for anti-proliferative drugs aimed at addressing cancer and other diseases marked by rapidly dividing cells. To aid this effort, past research on TS has developed two models to explain how TS works. A detailed, sequential chemical mechanism explains the methylene and hydride transfers from one cofactor to the substrate. And, a two state, dynamical model explains the conformational change that TS undergoes during its catalytic cycle. Combining these two models will lead to a fuller understanding of protein structure, function, and dynamics interrelationships. Two of the new structures contain cofactor in a heretofore unseen state, bound in the active site with its imidazolidine ring intact. Finding that this is an allowed enzyme-cofactor state indicates that ring opening and formation of the highly reactive iminium cation may occur relatively late in the methylene transfer, after preparation of the substrate; and, the reaction may perhaps be concerted. Further, details of these two structures show that protonation of the correct imidazolidine ring nitrogen (N10) may be selected by the geometry and environment imposed on the bent cofactor by TS. N5, the "wrong" ring nitrogen, is blocked and in a hydrophobic environment, while N10 is rehybridized to sp3 and its lone pair (nascent hydrogen) is pointed into an aqueous cavity trapped within the enzyme. A proposal coming from this dissertation is for a combination of the two models describing TS catalysis. The chemical mechanism model and the conformational change model both describe the same phenomena and these models should be connected and combined into one larger model to further increase our knowledge of the connections between structure, dynamics and function. The four structures reported here begin that connection process.

Small heat shock proteins (sHsps) are oligomeric proteins expressed by cells in response to high temperatures. It is believed that sHsps are produced as a defensive mechanism against temperature stress and act as molecular chaperones by binding and protecting heat-labile proteins from irreversible aggregation. Binding results in the formation of sHsp/substrate complexes from which substrate can later be refolded by ATP-dependent chaperones. Despite past investigations, many aspects of this model remain poorly defined. Results presented here provide new insight into the mechanism of sHsp action. sHsp chaperone activity and sHsp oligomerization are closely linked. Therefore, an understanding of the oligomeric structure, subunit number, and subunit dynamics is essential to understanding sHsp action. Three sHsps were analyzed for these properties: PsHsp18.1 from pea, TaHsp16.9 from wheat, and SynHsp16.6, from the cyanobacterium Synechocystis. In solution, SynHsp16.6 is a duodecamer, while TaHsp16.9 and PsHsp18.1 are dodecamers. An equilibrium between an oligomeric and suboligomeric state was observed for PsHsp18.1 and SynHsp16.6. Increasing temperatures resulted in the reversible dissociation of the TaHsp16.9 oligomer into a suboligomeric species. These results indicate that subunit dynamics are important for sHsp function. Interactions between sHsp and substrate in sHsp/substrate complexes and the mechanism by which substrate is transferred to refolding chaperones are poorly defined. C-terminal affinity-tagged sHsps were used to investigate these issues. This analysis revealed that while some sHsp subunits within sHsp/substrate complexes remain dynamic, complex size remains unchanged and association of substrate with sHsp is not similarly dynamic. These data suggest a model in which ATP-dependent chaperones associate directly with sHsp-bound substrate to initiate refolding. The homologous TaHsp16.9 and PsHsp18.1 are structurally similar. However, TaHsp16.9 interacts differently with substrate and is less effective at protecting substrate than PsHsp18.1. Studies with chimeric sHsps made between PsHsp18.1 and TaHsp16.9 revealed that the N-terminal arm is involved in subunit affinity, substrate protection, and substrate refolding, but interactions between the N-terminal arm and C-terminal domain are also critical for these aspects of chaperone activity. Additionally, the first ten residues of the N-terminal arm play a role in sHsp subunit affinity and substrate protection, but are unimportant for substrate protection.

Thymidylate synthase (TS) catalyzes the conversion of dUMP into dTMP via a methyl transfer from the cofactor, 5,10-methylenetetrahydrofolate (CH2THF), which is converted to dihydrofolate (DHF) during the reaction. Because this reaction is a step in the only de novo pathway leading to thymidine nucleotides, there has been considerable interest in TS inhibition for the treatment of proliferative disease. This had led to a large body of biochemical and structural data and a proposed reaction mechanism. Despite this extensive study, there are a number of unanswered questions regarding TS function. The role of the large ligand-induced conformational change is poorly understood, and many steps in the proposed mechanism of catalysis are unconfirmed. The roles of many evolutionarily conserved residues are also poorly understood, notably those located away from the active site. In this work, 23 structures of E. coli TS are presented. These structures are of eight site-specific mutants bound to several ligand combinations. The results of this work cast doubt on a number of aspects of previously published models of the reaction mechanism, and lead to a new proposed model. TS appears to use strain, electrostatic interactions, and conformational change to influence the stability, and thus reactivity, of the catalytic enzyme-substrate covalent bond. This work adds to a growing body of data suggesting that the stability of this bond is variable. Instability of this bond is central to the new proposed reaction mechanism. The mechanism proposed here accounts for the reaction without requiring a large isomerization of the ligand complex that was previously thought to be necessary. Also presented is evidence that CH2THF binds to TS without opening of the 5-membered ring and in a conformation similar to that of other TS-bound folates but different from the conformation observed in solution. The roles played by many conserved residues appears to be subtle, as evidenced by the small structural changes of mutants when compared to wild-type. The conservation of these residues suggests that TS is a highly optimized enzyme under strong selective pressure to maintain maximum catalytic activity.

Thioredoxins are a family of small redox proteins that mediate a number of cytosolic processes in the cells of all organisms. Human thioredoxin has a number of additional functions, including the apparent stimulation of NF-κB and AP-1 transcriptional activation. It can also be exported out of the cell, where it apparently has additional functions including the ability to stimulate cell growth. The crystal structure of human thioredoxin revealed that the protein can form a homodimer. The dimer contains a disulfide bond between the Cys 73 residues of each monomer, and a novel hydrogen bond between the Asp 60 residue of each monomer that is responsible for the pH dependent behaviour of dimerization. This work was undertaken with two goals. First, three mutant protein with changes in the dimer interface, Asp58 → Ser (D58S), Ala66 → Arg (A66R) and Trp31 → Ala (W31A) were isolated and characterized. The ability of the mutant proteins to form dimers, their activities with respect to the reduction of insulin disulfides and their interaction with thioredoxin reductase were examined. The three mutations affected thioredoxins ability to form dimers: the W31A and A66R mutant proteins formed dimers less well, and the D58S mutant protein lost the pH dependence for dimer formation. Thioredoxin with a D58S mutation behaved very similarly to wild type in the activity assays, while A66R showed a 6-fold increase in K(m) and W31A showed large changes to both K(m) and V(max). From these results I suggest that the dimer interface plays a role in defining the binding site for thioredoxin reductase. The second goal was to examine the role that oxidation of the active site plays in dimerization. Oxidation, which results in structural changes to the dimer interface, also caused thioredoxin to form dimers much less readily. The reduction in dimer formation offers a possible mechanism through which thioredoxin could play a role in signaling oxidative stress. The findings detailed here begin to describe the role that the dimeric form of thioredoxin could play in physiological situations. The structural bases for the changes in dimerization are not yet fully characterized, as the monomeric form of thioredoxin has not yet been crystallized.

Thymidylate synthase (TS, EC 2.1.1.45) is the enzyme responsible for the synthesis of 2'-deoxythymidine 5'-monophosphate (dTMP), using 2'-deoxyuridine 5'-monophosphate (dUMP) as the substrate and 5,10-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄ folate) as both carbon donor and reductant. Inhibition of TS stops growth of rapidly dividing cells, and for decades TS inhibitors such as 5-fluorouracil, and more recently folyl-analogs, have been used as anticancer drugs. However, prior to my studies, there were no structures available of any ligand-bound mammalian TS. In this dissertation I present the crystal structure of rat TS bound to the substrate dUMP and the anticancer drug Tomudex. Unexpectedly, the enzyme has an open conformation, with ligands bound but no covalent adduct between the catalytic cysteine (Cys 189) and the nucleotide, unlike that reported for the same complex with the E. coli enzyme. Three changes in amino acid sequence between the E. coli and rat TS proteins, namely ecT78 → R101, ecW83 → rN106 and ecV262 → rM305, result in loss of van der Waals contacts with Tomudex. These changes coupled with the loss of a hydrogen bond between the Tomudex 2-quinazoline position, which has been changed from the amino group of the cofactor to a methyl group, suggest that Tomudex may inhibit mammalian TS by stabilizing the open conformation. In a second project, I have studied the role of two conserved residues, K48 and R166 in catalysis of E. coli TS. Mutation of each of these residues to glutamine produces nearly inactive proteins. Crystallographic analyses of K48Q and R166Q suggest that the loss of these charged groups reduces binding of the ternary covalent intermediate. Superposition of the mutated structures with a previously determined wild type structure containing a close analog of this intermediate (TS-FdUMP-CH₂H₄ folate), reveals that the mutants are either more open or distorted, and therefore unable to contact the ligands properly. Both K(m) and k(cat) are altered for the two enzymes, with K(m) increasing about 10-fold, and kcat reduced 400-fold (K48Q) and 3,400-fold (R166Q). Taken together, these data suggest that K48Q and R166Q bind weakly one or more of the reaction intermediates, leading to near inactivity.