This Thesis explores the role of structural defects and disorder and their relationship to experimental data, with a particular emphasis on molecular framework materials. The question of how we can build atomistic models of amorphous materials from experimental data without needing to make system-specific assumptions is addressed. The role of 'structural invariance', <i>i.e.</i> the limited range of distinct local atomic environments within a material, as a restraint within reverse Monte Carlo refinement (RMC) is investigated. The operation of these invariance restraints operate is shown to be system-dependent and the challenges associated with effective refinement, <i>e.g.</i> configurational 'jamming', are also investigated. A generalisation to the 'structural simplicity', <i>i.e.</i> the simplest model, holding all else constant, is most likely to be correct. Three new metrics of structural simplicity are proposed: two intrinsically three-dimensional measures of local geometric invariance and one measure of local symmetry. These metrics are shown to robustly quantify the configurational quality. The ability of these metrics to act as effective restraints for the RMC refinement of amorphous materials is demonstrated by the construction of the first data-driven tetrahedral models of amorphous silicon. The role of defects and disorder within metal–organic frameworks (MOFs) is investigated through the canonical MOF UiO-66(Hf). Through a combination of techniques, including X-ray diffuse scattering, anomalous diffraction, total scattering and electron diffraction measurements, the existence of correlated metal-cluster absences in UiO-66(Hf) is demonstrated. Furthermore the ability to synthetically tune both the interactions and concentration of defects is shown. The thermomechanical properties of defective UiO-66(Hf) are also examined. UiO-66(Hf) is shown to rapidly densify by up to 5% (ΔV/V ) on ligand elimination. The resultant densified phase exhibits colossal (≥100MK^-1) volumetric negative thermal expansion (NTE); the largest reported value for any MOF. Finally, the capability to tune the physical properties of MOFs through defect incorporation is demonstrated through the defect-dependence of both the densification and the NTE.

The Type Three Secretion Systems (T3SSs) of Gram-negative bacteria, including Shigella, Salmonella, and Enteropathogenic/Enterohaemorrhagic Escherichia coli (EPEC/EHEC), pass virulence factors directly into the host to mediate invasion. Prior to secretion down the narrow T3SS channel, effector proteins associate with chaperone proteins. The binding enables the T3SS to keep effectors soluble and partially unfolded for secretion. In the first part of this thesis, the association of one promiscuous chaperone, Spa15 of Shigella flexneri, with three of its cognate effectors has been studied. In addition to the role this plays in secretion, the binding of one particular substrate leads to Spa15 being involved in the regulation of the T3SS. The oligomerisation and impact of substrate binding upon Spa15 has been determined by crystallography and EPR. Once secreted, T3SS effectors subvert the host cytoskeleton for the benefit of the bacteria. Soluble homologues of Spa15 effectors from EHEC and Salmonella have been purified, and their interactions with host GTPases which lead to stress fibre phenotypes observed. The Twin Arginine Translocation (Tat) pathway provides a contrasting view of bacterial secretion. Instead of preventing folding in the cytoplasm, it is a criterion of transport that the protein be folded. One of the reasons for internal folding is the necessity to insert cofactors which could not be incorporated externally. In the second part of this thesis, a protein which exemplifies this necessity is studied. This is PhoD, the model protein for Tat export from Bacillus subtilis. PhoD is an alkaline phosphodiesterase expressed to scavenge phosphate in times of phosphate deficiency. The structure of PhoD has been solved, and the protein is shown to be able to cleave a component of its own cell wall. It uses an unusual catalytic site more reminiscent of the eukaryotic purple acid phosphatases than of other currently known alkaline phosphatases. Furthermore this site appears to require metal binding before export from the bacterial cytoplasm.

This Thesis describes the synthesis and characterisation of new alkaline earth- and rare earth-transition metal complexes. Experimental and computational studies were performed to investigate the structure and bonding in these complexes. Their reactivity was also studied. Chapter 1 introduces metal-metal bonded complexes and current alkaline earth- and rare earth-transition metal bonded complexes. Chapter 2 describes experimental and computational studies of new alkaline earth- and lanthanide-Fe complexes possessing the [CpFe(CO)2]- anion. Chapter 3 presents experimental studies of the reduction of Fe3(CO)12 with Ca. Chapter 4 describes experimental and computational studies of new alkaline earth- and lanthanide-Co complexes containing the [Co(CO)3(PR3)]- anion. Chapter 5 presents full experimental procedures and characterising data for the new complexes reported. Appendix describes the attempted synthesis of [Ca{CpRu(CO)2}2(THF)x]y and study by DFT of [CaRp2(THF)3]2 CD Appendix contains .cif files for all new crystallographically characterised complexes described.

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 &Aring; resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

Fibronectin is an extracellular multidomain glycoprotein that directs and regulates a variety of cell processes such as proliferation, development, haemostasis, embryogenesis, and wound healing. As a major component of blood, fibronectin exists as a soluble disulphide linked dimer, but it can also be incorporated into an insoluble cross-linked fibrillar network to form a major component of the extracellular matrix. Fibronectin is composed of an extended chain of module repeats termed Fn1, Fn2, and Fn3 that bind to a wide range of transmembrane receptors and extracellular matrix components, including collagen. The gelatin binding domain of fibronectin was first isolated as a 45kDa proteolytic fragment and has since been found to be composed of six modules: 6Fn1-1Fn2-2Fn2-7Fn1-8Fn1-9Fn1 (in this notation nFX represents the nth type X module in the native protein). This domain has been reported to bind to both collagen and denatured collagen (gelatin), but with 10-100 times higher affinity to the latter; it can be purified to homogeneity on a gelatin affinity column. In the work presented here, fragments of the gelatin binding domain are expressed in P. pastoris, purified to homogeneity, and investigated at the molecular level. Through a dissection approach, surface plasmon resonance (SPR) is used to characterise the recombinantly produced protein, to accumulate more information about the function of the full domain. NMR is used to assess the folding of the protein fragments at atomic resolution. In particular, the secondary structure of 8Fn1-9Fn1 is mapped using inter-strand NOEs, which suggests that the construct takes the fold of a pair of typical Fn1 modules. Gelatin affinity chromatography is used to confirm that both Fn1 and Fn2 modules contribute to gelatin binding, possibly in two clusters (1Fn2-2Fn2 and 8Fn1-9Fn1). The 7Fn1 module may perform a structural role in linking together these two interaction sites, in the same way as suggested for 6Fn1, which is thought to act in a structural manner to enhance the binding of 1Fn2-2Fn2 to gelatin. Three carbohydrate moieties are found on this domain, one on 2Fn2 and two on 8Fn1. Here, by means of expressing different protein length fragments, and by site directed mutagenesis, the role of each sugar chain is investigated independently. The sugar chain on 2Fn2 does not appear to promote binding to collagen, nor does the first sugar chain on 8Fn1 (N-linked to N497), implying another role for these sugars such as protection from proteolysis. However, the presence of at least a single GlcNAc sugar residue on the second sugar chain site on 8Fn1 (N- linked to N511) is essential for full affinity binding to collagen. Direct binding of the 8Fn1-9Fn1 module pair to collagen is assessed with a short collagen peptide and the binding is monitored by NMR. The peptide appears to bind, predominantly to the final strand of 8Fn1, the first β- strand of 9Fn1, and the linker between the two modules, with μM affinity. A model for bound peptide is proposed. The highly conserved amino acid motif Ile-Gly-Asp (IGD) is found on four of the nine N-terminal Fn1 modules of fibronectin. Tetrapeptides containing the IGD were demonstrated to promote the migration of fibroblast cells into a native collagen matrix. Two of these “bioactive” IGD motifs are found within the gelatin binding domain, one on 7Fn1 and one on 9Fn1. In this study, the motif in the 8Fn1-9Fn1 module pair is shown to be located in a tightly constrained loop within 9Fn1. By site directed mutagenesis, the IGD motifs of 7Fn1 and 9Fn1 are subjected to single amino acid substitutions, and their ability to stimulate cell migration assessed in our assay. By NMR, the fold of the IGD mutant proteins is found to be unaffected by the mutation with respect to the wild type, with the exception of small perturbations around the substitution site. While the wild type module is able to stimulate fibroblast migration, the mutant proteins show reduced or negligible bioactivity. The larger fragments show far more potency in stimulating fibroblast migration, with 8Fn1-9Fn1 (one IGD motif) 104 times more potent than the IGD peptide, and the full gelatin binding domain (two IGD motifs) 106 times more potent than the 8Fn1-9Fn1. Potential mechanisms for this enormous enhancement of the IGD potency in different contexts are discussed.

This thesis explores the capacity of sterically and electronically unsaturated boranes to bind substrates of biological and environmental interest, and transduce such binding events into a photo-physical and/or electrochemical response, hence reporting the presence of these substrates. Chapter three details the synthesis of a range of novel ferrocenyl boranes featuring either a proximal hydrogen-bond donor or a second Lewis acidic centre. These novel boranes were shown to be competent at binding both cyanide and fluoride anions, with the role played by a proximal hydrogen-bond donor or a second Lewis acidic centre in anion binding investigated by both NMR and crystallographic studies. Chapter four reports the synthesis of novel pyridinyl and related boronic esters, as well as unexpected mixed alkenyl/aryl boranes. The capacity of both types of system to bind fluoride or cyanide anions in solution was investigated by UV-Vis and NMR studies. The photo-physical responses to these anions were also probed, leading to the establishment of both switch-on and switch-off fluorescent responses. Chapter five extends the knowledge derived from selective anion receptor design and combines this with recent developments in the field of frustrated Lewis pairs (FLPs) to activate, bind and report the presence of nitrous oxide (N₂O) molecule. Thus, the syntheses of novel, highly Lewis acidic ferrocenyl boranes that incorporate a high degree of steric loading around the boron centre are reported. The electrochemical and photo-physical response of an FLP system to the presence of N₂O was investigated leading to the development of a novel N₂O reporting system.

This Thesis describes the synthesis and characterisation of sulfonamide supported titanium, zirconium and aluminium complexes and their use as ring opening polymerisation catalysts for ε-caprolactone and rac-lactide. Chapter 1 introduces polyester use, development and characterisation in general. Metal catalysed ring opening polymerisation of cyclic esters is considered in a literature review of the field. Titanium, zirconium and aluminium complexes supported by polydentate sulfonamide ligands are also discussed. Chapter 2 describes the synthesis and characterisation of new sulfonamide supported titanium amide, isopropoxide and zirconium isopropoxide complexes. Their application as catalysts for the ring opening polymerisation of ε-caprolactone and rac-lactide is discussed and compared with known zirconium isopropoxide complexes supported by bis(phenolate) amine ligands. Chapter 3 describes the synthesis and characterisation of Cs symmetric titanium amide and alkoxide complexes supported by dianionic, tri- and tetradentate sulfonamide ligands. Zirconium alkyl and amide complexes supported by C3- symmetric trianionic ‘tren’ type ligands bearing three different sulfonamide groups are also presented. The application of these complexes for the ring opening polymerisation of ε-caprolactone and rac-lactide is described and compared with the complexes presented in Chapter 2. Chapter 4 provides an overview of the synthesis and characterisation of aluminium alkoxide and alkyl complexes supported by dianionic, tri- and tetradentate sulfonamide ligands. Solution state behaviour and solid state structures are presented and discussed. An assessment of these complexes for the ring opening polymerisation of rac-lactide is presented. Chapter 5 presents full experimental procedures and characterisation data for the new complexes reported. CD Appendix contains .cif files for all new crystallographically characterised complexes described, and additional polymerisation graphs.