Abstract The hydratase/isomerase superfamily consists of enzymes having a common evolutionary origin but acting in a wide variety of metabolic pathways. Many of the superfamily members take part in β-oxidation, one of the processes of fatty acid degradation. One of these β-oxidation enzymes is the Δ3-Δ 2-enoyl-CoA isomerase, which is required for the metabolism of unsaturated fatty acids. It catalyzes the shift of a double bond from the position C3 of the substrate to the C2 position. In this study, the Δ 3-Δ 2-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae was identified, overexpressed as a recombinant protein and characterized. Subsequently, its structure and function were studied by X-ray crystallography. The yeast Δ 3-Δ 2-enoyl-CoA isomerase polypeptide contains 280 amino acid residues, which corresponds to a subunit size of 32 kDa. Six enoyl-CoA isomerase subunits assemble to form a homohexamer. According to structural studies, the hexameric assembly can be described as a dimer of trimers. The yeast Δ 3-Δ 2-enoyl-CoA isomerase is located in peroxisomes, the site of fungal β-oxidation, and is a necessary prerequisite for the β-oxidation of unsaturated fatty acids; the enoyl-CoA isomerase knock-out was unable to grow on such carbon sources. In the crystal structure of the yeast Δ 3-Δ 2-enoyl-CoA isomerase, two domains can be recognized, the N-terminal spiral core domain for catalysis and the C-terminal α-helical trimerization domain. This overall fold resembles the other known structures in the hydratase/isomerase superfamily. Site-directed mutagenesis suggested that Glu158 could be involved in the enzymatic reaction. Structural studies confirmed this, as Glu158 is optimally positioned at the active site for interaction with the substrate molecule. The oxyanion hole stabilizing the transition state of the enzymatic reaction is formed by the main chain NH groups of Ala70 and Leu126. The yeast Δ 3-Δ 2-enoyl-CoA isomerase hexamer forms by dimerization of two trimers, as in the other superfamily members. An extensive comparison of the five known structures of this family showed that the mode of assembly into hexamers is not a conserved feature of this superfamily, since the distance between the trimers and the orientation of the trimers with respect to each other varied. Marked differences were also detected between the two yeast enoyl-CoA isomerase crystal forms used in this study, one being crystallized at low pH and the other at neutral pH. The results suggest that the yeast Δ 3-Δ 2-enoyl-CoA isomerase could occur as a trimer at low pH.

No description available.

No description available.

A series of 31 sulfur-nitrogen donor ligands and 64 metal(II) complexes have been investigated. The thiomethylated aniline ligands 2–(methylthiomethyl)aniline 2MT and 2–(methylthio)aniline 2MA were synthesized with their substituted derivatives (-Me, -MeO, -Cl, -Br, -NO2) to serve as chelating agents. These ligands behave as bidentate ligands with SN donor group with Co(II), Ni(II) and Cu(II). The Co(II) and Ni(II) complexes have the ML2Cl2 molecular formula while the Cu(II) complexes formed with MLCl2 stoichiometry where L is the bidentate ligand. The ligands and their metal(II) complexes have been characterized by elemental analysis and with spectroscopic techniques. The trend observed in the NMR spectra and IR frequencies of the thiomethylated compounds shows there is a significant difference between the 2MT and 2MA series as a result of sulfur lone pairs extending the conjugation of the aromatic ring in the case of the latter. The effect of the position and electronic nature of ring substituent on the NMR shifts of the amine protons is discussed. The 6- and 5-membered chelate complexes formed by the 2MT and 2MA ligands respectively do not show significant diversity in their spectroscopic properties. From the elemental analysis for the Co(II) and Ni(II) complexes, their compositions reveal 1:2 M:L stoichiometry with 2 chlorine atoms from the respective metal salts. In addition, the spectroscopic data are largely indicative of tetragonally distorted structures for these solid complexes. The X-ray crystallography data reveal the Cu(II) complexes exist as square pyramidal dimers and with long Cu–Cl equitorial bonds fit into the tetragonally distorted octahedral structure. The electrolytic nature of Co(II) and Cu(II) complexes in DMF were found to be similar, they behave as non electrolytes in contrast to Ni(II) complexes which are 1:1 electrolytes. The electronic spectra of these metal(II) complexes were found to be different for both their solid forms and in solutions of DMF and DMSO and this has been discussed. The thiomethylated aniline ligands possess the amine and thioether groups which are present in many known biologically active compounds, hence the biological activity of the ligands and their metal complexes were tested against three strains of bacteria and one fungus. The methoxy-substituted derivatives were found to possess better inhibitory activity and this was similarly reflected in the metal(II) complexes. The activity of the complexes can be said to be in the order, Cu(II) > Co(II) > Ni(II). The Schiff-base derivatives were prepared from the ligands and para-methoxysalicylaldehyde and their Cu(II) complexes were synthesized in order to determine their biological activity. The Schiff-base ligands were found to be less active than their parent ligands. The Cu(II) complexes are not soluble in water, DMSO or DMF, as a result and could not be evaluated for their biological activity. Based on the good results from the antimicrobial evaluation, the antiplasmodial activity of some of the Co(II), Ni(II) and Cu(II) complexes of the thiomethylated ligands against Plasmodium falciparum (FCR-3) was determined. At 50 μM concentration level, the Cu(II) complexes show activity equal or better than the prophylactic chloroquine. The Cu(II) complexes with the methoxy-substituted demonstrated exceptional activity but their Co(II) and Ni(II) analogues did not show any activity. The cytotoxicity of the active Cu(II) complexes at 50 μM concentration was determined against the breast cancer cell line (MDA-MB-231). The compounds destroyed the cancer cell in the range of 28–40%, thus showing their preferred activity against the parasitic cell instead of the cancer cell. The selectivity demonstrated by these compounds have shown them to be potential antimalarial agents and this could be further investigated.

Cytoplasmic dynein is a molecular motor that moves cargos along microtubules. Dynein, together with its large co-factor dynactin, is responsible for the vast majority of traffic towards the centre of the cell. The largest subunit of the dynein complex is called the dynein heavy chain (DHC). The DHC includes a C-terminal motor domain, which converts ATP hydrolysis into mechanical force, an N-terminal tail domain, and a flexible linker domain to join the two together. An intermediate chain (DIC) and light intermediate chain (DLIC) bind directly to the DHC tail, while light chains (DLCs) bind to the DIC. This tail complex is important for both cargo binding as well as homodimerisation of the DHC, which is necessary for processive movement. Previous studies suggest that the DLCs play an important role in homodimerisation, but it remains unclear how else the DHCs are held together. Using S. cerevisiae as a model system, I co-expressed all four dynein subunits and purified functional dynein motors. In this background, I found that truncating the DHC to include only the first 1004 residues (out of the total 4092) eliminates the motor domain as well as the flexible linker domain, while preserving binding to the DIC, DLIC and DLC. However, truncating just another 50 residues off of the C-terminus led to a loss of all accessory subunits. I developed a protocol for expressing and purifying large quantities of the 1004 residue construct, thus I provide the first description of a recombinant dynein tail domain. Using negative stain electron microscopy (EM), I also present the first 3D structural information for the tail region of the cytoplasmic dynein motor. I then describe a construct including only the first 557 residues of the DHC, which dimerises despite not being able to bind any of the other subunits. I present a crystal structure of this smaller DHC fragment, which shows that the N-terminal 180 residues of the DHC constitute an intricate dimerisation domain made up of a β-sheet sandwiched between α-helices. Not only is this the first crystal structure of any part of the DHC N-terminus, but it reveals a previously undocumented dimerisation domain within the DHC itself. Furthermore, information garnered from this crystal structure allowed for interpretation of a recent cryo-EM structure of a triple complex containing the dynein tail, dynactin and the cargo adaptor BICD2 (TDB) that was solved by my colleagues in the Carter group. Only by docking the DHC N-terminus crystal structure within the TDB EM density did it become clear that the N-terminus of the DHC is responsible for the majority of the contacts the dynein tail makes with both dynactin and BICD2. Therefore the work that I present here sheds new light on the unexpected importance of the DHC N-terminus and allows two important conclusions to be made. First, the N-terminal 180 residues of the DHC constitute a dimerisation domain of its own. Second, the next ~400 residues of the DHC form a domain that plays a key role in the complex interface between dynein, dynactin and BICD2.

No description available.

No description available.

The mixed lineage leukemia (MLL) proteins regulate an array of developmental and differentiation processes. Similar to other members of the SET1 family, association of MLL1-4 with Ash2L, RbBP5 and WDR5, collectively termed the MLL core complex, is required for MLL mediated histone H3 Lys-4 di/tri-methylation. Each member of the core complex has a unique role in modulating the activity of MLL1. WDR5 is key in nucleating the formation of the core complex by acting as a structural scaffold, whereas Ash2L and RbBP5 are responsible for stimulating MLL methyltransferase activity. Currently, the structural and biochemical mechanisms utilized by the core complex to regulate MLL1 activity are unknown. Through structural and biochemical dissection of the core complex we have assigned specific functions to core complex subunits and have identified the minimal structural requirements for methyltransferase activity. Furthermore, through structure based drug design, we have identified a peptidomimetic inhibitor of MLL1 methyltransferase activity.

Ligand-protected metal nanoclusters (NCs), which are ultra-small nanoparticles marked by their atomic precision, are distinctly importance for contemporary nanomaterials. NCs have attracted significant research attention for utilizing their novel optical and physicochemical properties in various applications, including fluorescence sensing, catalysis, and biomedical applications. This dissertation deals with ligand-protected atomically precise silver NCs and is divided into two main parts. The first part is focused on the exploration and design of well-defined silver NCs through surface co-ligand engineering. The second part is related to the development of silver NC-based frameworks (NCFs). In the first part, we designed a synthetic strategy based on engineering the structure of the phosphine co-ligands with thiols to generate the large box-shaped [Ag67(SPhMe2)32(PPh3)8]3+ (referred to as Ag67) NC. The strategy demonstrates that the combined use of judiciously chosen thiol and phosphine co-ligands can result in stable highly anisotropic box-like shapes. The optical absorption spectrum of the Ag67 NC displays highly structured multiple sharp peaks. The crystal structure shows a Ag23 core formed of a centered cuboctahedron (an unprecedented core geometry in silver clusters), which is encased by a layer with a composition of Ag44S32P8 arranged in the shape of a box. The electronic structure of this box-shaped cluster resembles a jellium box model with 32 free electrons. In the second part, a novel approach is developed for the assembly and linkage of atomically precise Ag NCs into one-dimensional (1D) and two-dimensional (2D) NC-based frameworks (NCFs) with atomic-level control over cluster size and dimensionality. With this approach three novel, but related, crystal structures (one silver NC and two NCFs) were synthesized. These structures have the same protecting ligands, and also the same organic linker. The three structures exhibit a similar square gyrobicupola geometry of the building NC unit with only a single Ag atom difference. The critical role of using a chloride template in controlling the NC’s nuclearity was demonstrated, as well as the effect of a single Ag atom difference in the NC’s size on the NCF’s dimensionality, optical properties, and thermal stability.

Glutamate carboxypeptidase II (GCPII) is a zinc-dependent carboxypeptidase with high expression levels in prostate carcinoma. As the enzyme represents a validated target for cancer therapy and imaging, the development of new GCPII-specific ligands is still a focus of an active academic and industrial research. However, existing assays to screen inhibitor libraries and determine inhibitor efficacy are suboptimal at best. This thesis is aimed at the development of small internally quenched probes that could be used for continuous measurement of the GCPII enzymatic activity. These probes are derived from natural GCPII substrates and consist of a fluorophore/quencher pair connected by a GCPII-hydrolysable linker. I first characterized biophysical properties of the probes and then determined kinetic parameters of their hydrolysis by GCPII. The optimized activity assay was then used to determine inhibition constants of several GCPII-specific inhibitors. Finally, complexes between the inactive enzyme and several probes were co-crystallized and one of the complexes refined and analyzed. Our data show that the probes are involved in non-covalent interactions with the same amino acid residues of the enzyme's active site as natural substrates. The developed assay could be optimized for high-throughput...