The mitochondrial tyrosyl tRNA synthetases (mtTyrRS) from certain fungii are found to be bifunctional enzymes that aid in group I intron splicing in addition to charging tRNA[superscript Tyr]. This splicing activity is conferred by several insertions that are unique to these mtTyrRS. Initial biochemical evidence suggested the similar tertiary structures of the tRNA and the intron enable binding of the protein to both. However, a recently solved co-crystal structure showed that the tRNA and intron were bound on opposite faces of the protein. The intron was bound almost exclusively by a novel surface formed by several insertions in the protein. This work presents the structure of the C-terminal domain of the A. nidulans mtTyrRS (PDB ID -- 2ktl). NMR results show that the C-terminal domain contains an S4 fold with a mixed [beta]-sheet and two anti-parallel [alpha]-helices that pack against these strands. The strands [beta]1 and [beta]5 are parallel, and [beta]2 to [beta]5 are arranged anti-parallel to each other. The C-terminal domain from A. nidulans mtTyrRS has three insertions in its sequence that make it almost twice the size of bacterial TyrRS. NMR results show that insertion 3 at the N-terminus of the domain is flexible. Insertion 4 is contained in the loop connecting [beta]2-[beta]3 and does not have a well defined structure. Insertion 5 and the C-terminal extension form two helices, [alpha]5 and [alpha]6 that fold away from the core of the protein. An extended helix ([alpha]4) between strands [beta]3 and [beta]4 was identified by NMR. Based on structural alignments with bacterial TyrRS, this helix was classified as a novel insertion 4b in the C-terminal domain. Conserved positively charged residues used to bind the tRNA are found in the turn between the anti-parallel [alpha]-helices and the turn connecting strands [beta]4-[beta]5. Based on a comparison with other TyrRS structures, the three insertions are positioned away from the tRNA binding site. The insertions form a novel RNA binding surface that could interact with the intron. Since these insertions are found in loop and termini regions, they could be a structural adaptation acquired by these splicing mtTyrRS. NMR spectra of the full length TyrRS from B. stearothermophilus and mtTyrRS from A. nidulans indicate that the motion of the C-terminal domain is coupled to that of the full length protein. This provides new information regarding the organization of the full length TyrRS. / text

Fibronectin (Fn) is an extracellular matrix (ECM) protein involved in embryonic development, wound healing and tumorigenesis. Structurally Fn is mainly composed of three repeated modules: FI, FII and FIII, together with an alternatively spliced type III connecting segment (IIICS). The IIICS has no sequence homology to these repeated modules and contains integrin, proteoglycan and zinc binding sites. These sites facilitate adherence and spreading of leukocytes, peripheral neurons and melanoma cells, which can lead to disease states such as inflammation, autoimmunity and cancer metastasis. Therefore, there is the potential to develop therapeutic agents based on the IIICS structure. In this study, nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the structure and dynamics of both the IIICS and its adjacent FIII15 module, two of the few Fn regions for which a structure has not been elucidated. An ensemble of solution state NMR structures calculated for the isolated FIII15 module showed that FIII15 forms a rare six-stranded FIII fold, homologous to a typical seven-stranded FIII fold, with a disordered N-terminal linker sequence. NMR relaxation data and chemical shift analysis showed that the IIICS is an intrinsically disordered region with no areas of well-defined secondary structure. A structure was also calculated for FIII15 within a construct containing the IIICS, which showed that contrary to a previous hypothesis, the IIICS does not contribute to the FIII15 structure. In addition, structural comparisons between IIICS splice variants suggested that alternative splicing confers no stable structural features to the IIICS. Furthermore, ligand binding studies showed that, under conditions tested, neither zinc nor the proteoglycan heparin, induced the formation of any secondary structure to this region. Zinc binding did, however, induce oligomerisation of a IIICS containing construct and appeared to enhance the binding of heparin to the IIICS. Data was obtained to suggest that FIII15 forms a transient interaction with an adjacent module, which is likely to be FIII14. It is hoped that the work presented will contribute to further studies into this important area of Fn and may aid in the future development of novel therapeutics.

Protein-protein interactions are involved in various biological processes and detailed characterization of their structural basis by the means of structural biology is often instrumental for rigorous understanding of underlying molecular mechanisms. This information is important not only for fundamental biology but also plays an important role in search for sites amenable for therapeutic intervention. Nuclear magnetic resonance spectroscopy is alongside X-ray crystallography and single-particle cryo-electron microscopy one of the key high-resolution techniques in structural biology. Although its applicability to larger systems has a well-known physical limit, it offers unique capabilities in addressing highly dynamic or inherently heterogeneous systems. In this doctoral thesis, the solution-based NMR approach was used for detailed structural characterization of selected biologically important proteins and their complexes that provided important insights into their biological roles. In three distinct projects, I (i) studied the relationship between the structural effects of particular modifications in the insulin-like growth factor II (IGF-II) and their selectivity to the insulin axis receptors; (ii) the specific binding mechanism of the SH3 domain from the Crk-associated substrate (CAS); (iii) and...

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This thesis describes the isolation and structure elucidation of secondary metabolites formed in static culture from a number of endophytic Xylariaceous fungi. Four Xylaria endophytes isolated from a palm tree in Thailand were surface cultured on an aqueous malt extract-glucose medium. They all produced cytochalasin D, coriloxin, (S)-mellein and (3R,4R)-4-hydroxymellein as the main secondary metabolites suggesting that the four endophytes could be the same species. The endophytic fungus A116 produced cytochalasin D as the main secondary metabolite. Another non-endophytic fungus B315, produced cytochalasin D, (R)-mellein, a mixture of two isomers of 4-hydroxymellein and phloroglucinol. X.62, an endophytic fungus, produced 19,20-epoxycytochalasin C from the mycelium as the main secondary metabolite. The fungus Engleromyces sinensis produced engleromycin acetate as the main secondary metabolite. Fungus X. polymorpha produced (3E)-4-(3'-acetyl-2',6'-dihydroxy-5'-methylphenyl)-2-methoxybut-3-enoic acid.

The conserved secondary structural RNA motifs of EncephaloMyoCarditis Virus (EMCV) have been well characterised biochemically and shown to play an important role in translation initiation by a novel cap-independent mechanism called Internal Ribosomal Entry Site (IRES). However, the three dimensional structure and interactions of these conserved motifs are not known, and hence the mechanism is not fully understood. The NMR results described in this thesis have provided, for the first time, new structural knowledge on the conformation of these motifs, their affinity for Mg2+ and their intermolecular interactions. RNA motifs selected from two separate domains (I and J) of the IRES structure were investigated using a range of 2D and 3D NMR techniques. The apical ‘hammerhead’ region of the I domain contains a highly conserved 16mer RNA which hosts a stable and mutationally sensitive G547CGA550 tetraloop. Sequence specific assignments were carried out on this motif, along with its Mg2+ complex, and a large number of NMR experimental constraints were generated for the RNA structure determination. Similarly, high resolution NMR structures of a distal 17mer RNA, which has been predicted to be a potential receptor for the GCGA tetraloop, and its Mg2+ complex were also produced. Thus, we were able to demonstrate that Mg2+ stabilises the RNA tertiary structure via non-specific interactions. Since the largest changes were induced at the tetraloop motif, we propose that Mg2+ stabilises the 16mer into an optimum conformation which is essential for IRES function. The determination of the structures of the above motifs led us to investigate the 16mer-17mer binary (1:1) complex at 1 GHz, in the presence of Mg2+. Significant changes were observed in the 1H and 31P chemical shift, NOE intensity and line width, clearly demonstrating RNA-RNA interactions taking place between the two components. The most interesting result to emerge was the distinct absence of NOEs from G547{NH} of the stable tetraloop, thus highlighting an important structural role for this functionally critical residue. Since no previous work has shown a clear interaction between the two RNAs, the results obtained in this project provide the first direct experimental evidence for intramolecular interactions in the I domain of EMCV IRES.Finally, we show how isotopically labelled RNAs can be successfully used as an aid in NMR assignment, analysis and structure determination. The J domain of EMCV IRES binds to eIF4GII protein and is essential for translation initiation. A suite of 3D NMR techniques were carried out on a highly enriched and uniformly 13C, 15N-labelled 39mer RNA. Several key features of the RNA, which may be involved in protein recognition, were identified. Further, a selectively 19F-labelled 16mer RNA from the I domain, was also studied to show how fluorine NMR can be used to probe RNA structure, dynamics and interactions. The RNA motifs of the EMCV IRES were shown to exhibit high stabilities, which are brought about by the complex folding of the various secondary structural elements involving RNA- Mg2+, RNA-RNA and RNA-protein tertiary interactions. It is these vital interactions that enable the IRES to recruit the ribosome in the translation initiation step of protein synthesis, and have laid a strong foundation for further NMR investigation of the whole IRES.

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