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The unfolding and refolding of a number of oligomeric enzymes have been studied. These were: fumarase from pig heart, the NAD+ -dependent isocitrate dehydrogenase from yeast, the citrate synthases from pig hean, Acinetobacter anitratum and Thermoplasma acidophilum and the chaperone protein GroEL from Escherichia coli. In each case the unfolding by guanidinium chloride (GdnHCI) was monitored by enzyme activity (to detect possible perturbations at the active site), protein fluorescence (to detect changes in tertiary structure) and far U.v. circular dichroism (to detect changes in protein secondary structure). In general the losses in secondary and tertiary structure were found to run broadly in parallel, whereas the enzyme activity was lost at much lower concentrations of GdnHCl. This sensitivity to mild, denaturing conditions may reflect the greater flexibility of the active site compared with the molecule as a whole. Interestingly) the bacterial citrate synthases were activated in the presence of low concentrations of GdnHCl. Following denaturation) refolding was initiated by lowering the concentration of GdnHCI by dilution or dialysis. Only the dimeric citrate synthases (from pig heart and Thermoplasma acidophilum) could be reactivated to a moderate extent using the dilution procedure; less than 5% reactivation was observed for the other enzymes. In the cases of fumarase, NAD+ -dependent isocitrate dehydrogenase and the dimeric citrate synthases the degrees of reactivation following dialysis were significantly greater (approximately 50-75% of the native enzymes) than those obtained following the dilution procedure. Factors such as protein concentration and the inclusion of dithiothreitol in the dialysis or dilution buffer were found to influence significantly the extent of reactivation. The greater yield of reactivation of unfolded protein using the dialysis procedure probably reflects the ability of the enzyme to make the correct structural adjustments between intermediates when the concentration of GdnHCI is lowered gradually. In the case of Thermoplasma acidophilum the recovery of citrate synthase activity was much greater at 20 ·C than at 55 ·C (the optimal temperature for growth of this organism). This has implications for the folding process in vivo under the extreme growth conditions of thermophiles and possibly other extremophiles. The hexameric citrate synthase from Acinetobacter anitratum and the tetradecameric chaperonin, GroEL could not be reactivated following denaturation. Far u.v. circular dichroism measurements on GroEL indicated that the native secondary structure of this protein was regained to a large extent. In vivo a number of the proteins studied (fumarase and citrate synthase from pig hean and yeast NAD+ -dependent isocitrate dehydrogenase)are translocated into mitochondria as precursors in a non-native state prior to processing, folding and assembly. The lack of complete refolding of the proteins studied in this work points to the existence of specialised mechanisms in vivo to promote efficient folding. Chaperone proteins have been implicated in the assistance of protein folding in vivo. Intriguingly. the studies on the inefficient refolding of the chaperonin GroEL support the proposal that this protein may fold in vivo by way of a "self chaperoning" mechanism.

The bacterial toxin pyolysin (PLO) is a member of the family of Cholesterol-Dependent Cytolysins, which form large, oligomeric pores in cholesterol-containing membranes. The general CDC structure has an elongated shape and consists of four domains rich in β-sheet structure. Upon binding to a membrane, molecules diffuse laterally on the surface and oligomerize to form a pre-pore complex, then insert into the membrane yielding pores of unusually large size, approximately 30 nm in diameter. In this work, the oligomerization properties of PLO were investigated. In particular, the role of the C-terminal domain in the oligomerization process, the effects of a disulphide-tethered mutant on the activity of the wild type toxin, and the pore-forming ability of oligomers pre-formed in solution were characterized. Chapter 2 characterizes the functional properties of a recombinant fragment that corresponds to the C-terminal domain 4 of PLO. It is shown that this fragment can form hybrid oligomers with intact PLO toxin molecules, and is also capable of self-oligomerization. The fragment has no haemolytic activity of its own; nevertheless, it can to some degree increase the haemolytic activity of the wild type toxin. In addition, in a mixture domain 4 and wild type interact in such a way as to form unusual shapes on cholesterol crystals that have not been previously observed. Chapter 3 describes the effects of a disulphide bond linking domain 2 to a membrane-inserting region of domain 3 on the oligomerization process. The disulphide mutant was not able to oligomerize on its own, and when combined with active PLO toxin, the haemolytic activity of wild type was significantly inhibited. Also, the combination of the disulphide-tethered mutant with intact toxin resulted in the formation of hybrid oligomers. This, in turn, caused an increase in incomplete ring formations on cholesterol surfaces which correlate to a reduction in functional pore size, suggesting that insertion of subunits is partially cooperative. The results of the investigation of the pore-forming ability of solution-derived oligomers (SDO) are described in Chapter 4. Here, the fluorescence emission of an environmentally-sensitive probe on the SDO after membrane insertion was a fraction of that observed with the monomeric control, which was supported by hydrophobic quenching analyses. This suggests that the formation of SDO may block necessary conformational changes in the intact toxin to allow membrane insertion.

Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally attached fatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolarization. It has long been assumed that the mode of action of daptomycin involves the formation of oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not been experimentally demonstrated. In the work described in this thesis, I have used fluorescence energy transfer (FRET) between native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the antibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly and is stable for a length of time longer than required for the bactericidal effect. Through variation of the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have determined that the oligomer consists of approximately 6–7 molecules, or, depending on the structure of the oligomer, possibly up to twice that number. Oligomer formation on liposomes and on bacterial membranes was confirmed using excimer fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was also used to demonstrate a stoichiometric interaction between daptomycin and PG. It has previously been shown that the bactericidal activity of daptomycin requires calcium and correlates with the concentration of PG in the bacterial cell membrane; these requirements mirror those observed here for oligomer formation. Furthermore, membrane permeabilization is selective, and electron microscopy of bacterial membranes exposed to daptomycin has revealed no discontinuities or accretions of electron density. Both of these findings suggest formation of a small membrane lesion, which is compatible with the small size of the oligomer that was determined here. In conjunction with these previous findings, the experiments contained in my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin.

Thermolysis of Cp*W(NO)(CH ₂CMe₃) ₂,CpW(NO)(CH ₂CMe₃)₂, Cp*W(NO)(CH₂SiMe₃)(η²-CPhCH₂), or Cp*W(NO)[CH(Ph)CH₂CH(nPr)CH₂]in cyclic olefins results in the formation of ring-retaining oligomers having lengths up to dodecamers. The main cyclohexene dimer is 3-cyclohexylcyclohexene. A small percentage of oligomers contain neopentyl or CH=CHPh end groups. Turnover frequencies for the Cp*-tungsten precatalysts range from 5.5 to 6.5 mol/h at 100 °C. In room temperature solutions, Cp*Mo(NO)(CH₂CMe₃)₂ generates the alkylidene intermediate [Cp*Mo(NO)(=CHCMe₃)], which couples with cyclic olefins to form cismetallacycles. The isolable cyclopentene-derived cis-metallacycle, Cp*Mo(NO)[cis-η² CH(CH₂)₃CHCHCMe₃], converts in the solid state to the allyl-hydride complex Cp*Mo(NO)(H)(η³-CH(CH₂)3CCHCMe₃). With larger cyclic olefins (cyclohexene through cyclooctene) the initial cis-metallacycles isomerize to trans-metallacycles of the form Cp*Mo(NO)[trans-η²-CH(CH₂)nCHCHCMe₃] (n = 4, 5, 6), and these subsequently convert with loss of dihydrogen to η⁴-diene complexes, Cp*Mo(NO)[η⁴-CHCH(CH₂)n-₁CCHCMe₃]. Thermolysis of the η⁴-diene complexes in cyclohexene results in decomposition of the organometallic complex with small amounts of oligomer formation. Thermolysis of Cp*W(NO)CH₂CMe₃)₂ in cyclic-olefm substrates generates the alkylidene intermediate [Cp*W(N0)(=CHCMe₃)], which couples with cyclic olefins in a manner analogous to the Cp*Mo-system. Tungsten trans-metallacycles are observed by ¹H NMR spectroscopy, but the organometallic subsequently reacts further with loss of the coupled neopentyl-cyclic olefin and coordination of two substrate molecules to form the putative Cp*W(NO)(cyclic olefm)₂ complex. Two additional cyclooctene products are isolated, the 1,4- diene Cp*W(N0)[η⁴-CHCH(CH₂)₅CHCCH(CH₂)₆] and the allyl hydride Cp*W(NO)(H)[η³- CH(CH₂)₆CCCHCH(CH₂)₅], both containing two coupled cyclooctene molecules. A tungsten cis-metallacycle forms with 2,5-dihydrofiiran, but a ring-opened alkoxy-allyl complex forms with 3,4-dihydro-2H-pyran, and 1,2,3,6-tetrahydro-pyridine undergoes N -H bond activation to afford an amido product. CpW(NO)(CH₂CMe₃)₂ produces some oligomers of cyclohexene, but in all other reactions bimetallic decomposition pathways predominate. / Science, Faculty of / Chemistry, Department of / Graduate

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Ph.D. (Chemistry) / Please refer to full text to view abstract

Hepatitis C Virus (HCV) is a positive-strand RNA virus that has infected more than 3% of the world population. Chronic infections by the virus lead to cirrhosis and hepatocellular carcinoma. HCV is currently the leading cause for liver transplantation in the US. The nonstructural protein NS5B of HCV is the RNA-dependent RNA polymerase (RdRp) that replicates the viral RNA on host derived membranous structures. Structurally NS5B has the characteristic fingers, thumb and palm domains seen in all polymerase proteins. However, extensive interactions between the fingers and thumb domains completely encircle the active site of NS5B as seen in solved X-ray diffraction crystal structures. These interactions are primarily mediated by a short (35 residues) flexible loop called the Delta 1 loop. NS5B produced from heterologous systems can initiate RNA synthesis by a de novo initiation mechanism from 3?ends of RNA templates or can also extend from 3'ends of primers that are annealed stably to a template RNA in biochemical assays. The closed conformation of NS5B as per X-ray crystal structures can only accommodate a ssRNA but not a dsRNA, hence necessitating a conformational change between de novo initiation and elongation. The details of these conformational changes are not known and will prove to be important to design potent polymerase inhibitors. The study performed for this dissertation focused on the conformational requirements of NS5B during de novo initiation and primer extension (or elongation). Biochemical assays utilizing template RNAs that can lead to both de novo initiation and primer extension products were utilized, and a systematic mutational analysis of the template channel of the RdRp was performed. Mutants W397A and H428A were identified that showed only primer extension but no de novo initiation. Structural analysis of NS5B suggested that these residues were important contact points in the Delta 1 loop and thumb domain interactions. A deletion mutant, m26-30 with a five amino acid deletion at the apex of the Delta 1 loop also failed in de novo initiation but not primer extension reactions. Biophysical and gel shift assays showed that m26-30 was in a more open conformation than the WT enzyme. Furthermore, oligomerization of NS5B was demonstrated and its role in RNA synthesis was examined. It was found that the de novo initiation competent conformation of NS5B is maintained by oligomeric contacts between individual subunits, likely by stabilizing the Delta 1 loop and thumb domain interactions. Mutations disrupting the Delta 1 loop and thumb domain interactions as well as those in the allosteric GTP binding site induced conformational changes in the protein partially explaining the defect in de novo initiation activity in enzymes carrying those mutations. These results not only contribute to the overall mechanism of RNA synthesis in viral RdRps but also open new avenues for developing HCV polymerase inhibitors.

One of the most intriguing questions in evolutionary biology is how biochemical and structural complexity arise through small and incremental changes; however answering this question requires an explicit set of candidate residues and an experimental system in which to test them. This dissertation aims to understand how biochemical complexity evolves and assesses the structure-function relationship in the green fluorescent protein (GFP) protein family using an ancestral reconstruction approach. In the second chapter, I studied the evolution of biochemical complexity in Kaede-type red fluorescent proteins (FPs) from Faviina corals. An increase in biochemical complexity is represented by the emergence of red fluorescence because it necessitates the synthesis of a tri-cyclic chromophore from a precursor bi-cyclic chromophore through an additional autocatalytic reaction step. The autocatalytic reaction is fully enabled by as many as twelve historical mutations. Here, I showed that the red fluorescent chromophore evolved from an ancestral green chromophore by perturbing the ancestral protein stability at multiple levels of protein structure. Moreover, only three historical mutations are sufficient to initiate the selection-accessible evolutionary trajectory leading to emergence of red fluorescence. The third chapter investigates six mutations proximate to the chromophore in the Kaede-type FP that could have facilitated autocatalytic synthesis of the red chromophore by enlarging the chromophore-containing cavity and modifying its microenvironment. Two of these six mutations were found to strongly affect the protein’s stability and oligomeric tendency. Additionally, I showed that the dimeric least divergent Kaede-type FP, R1-2, evolved from the tetrameric green ancestor. Taken together the results of these studies indicate that the step-up in biochemical complexity in the Kaede-type FPs was achieved via disruption of the existing stable interactions at tertiary and quaternary protein structure levels. In the fourth chapter, I resurrected the common ancestor of all FPs cloned from the order Leptothecata (class Hydrozoa), which are characterized by the highest known homo-oligomeric diversity. I showed that the ancestor was a green monomeric FP with a large Stokes shift. The ancestral FP together with the extant Leptothecata FPs could server as a model system to study the evolution of function and homo-oligomerization, and the desirable photophysical characteristics would make this ancestral FP a useful bio-marker in bio-medical research. / text