Dissertations / Theses on the topic 'Crystallographic investigations'

Salt formation provides a means of altering the physicochemical and resultant biological characteristics of a drug entity without modifying its molecular structure. Many published reviews have indicated the importance of the selection of the most appropriate salt form. This work is an investigation into the salt formation of two heterocyclic drugs. This is done by the physicochemical and the crystallographic studies of 19 high resolution single crystal diffraction studies. The particular targets of the work are the selection of the most appropriate salt forms, investigations into the tautomerism and polymorphism (or pseudopolymorphism) and an understanding of the interactions most likely between these heterocyclic drugs and their specific receptor sites. Section 1 describes the effect of protonation on the absorption of drugs, the rationale for using various salt forms and the resultant effect this has on a number of physicochemical properties of the parent compound. Section 2 is a description of the experimental techniques used in the physicochemical investigations and in crystal structure determination. In Sections 3 and 7, the preparation and characterisation of the salts and modifications of the two heterocyclic drugs, GU and IM is described. In Sections 4 and 8, the physicochemical investigations into the hygroscopicity and solid-state stabilities of the salts of GU and IM is described. Van't Hoff solubility studies are used to determine the enthalpies of solution and where appropriate the relative thermodynamic stabilities of the various phases produced. The structures of 19 of the salts or modifications of GU and IM, together with their packing and hydrogen bonding interactions is described in Sections 5 and 9. Sections 6 and 10 describe the ionisation properties of these molecules. Both the guanidine and imidazole moieties of GU and IM, respectively, are tautomeric, the particular form(s) found in these investigations and the effect of protonation is discussed. The conformations of these structures are discussed and the effect of protonation, especially on the puckering of the piperazine ring, is described.

In this work, the phase behavior of binary systems comprising nitrogen and a noble element (nitrogen + argon and nitrogen + krypton) was studied at high density in the condensed state. Following the work of Lotz et al.[2001], the main goal of this work was to further investigate the pressure-concentration phase diagram as well as to look for the possible formation of van der Waals compounds at elevated pressures and room temperature and study their physical properties, using both vibrational spectroscopy and X-ray diffraction. The observed phases, formed by single atoms and/or simple molecules in the binary systems were solved and modeled for their corresponding crystalline structures. From experimental results, lattice parameters for all crystalline structures and phase transitions, if detected, have been observed to shift with respect to that of the pure substances. The analyses and characterization of these binary systems are discussed in detail.

In the first article, the seismic properties for a suite of rocks along the West Cycladic Detachment System (Greece) are calculated, using Electron backscatter diffraction (EBSD) measurements and the minerals’ elastic stiffness tensors. Muscovite and glaucophane well defined crystallographic preferred orientation increases the seismic anisotropy. Maximum Pwave velocities have the same orientation as the Miocene extension and maximum S-wave anisotropy is subhorizontal, parallel with mineral alignment, suggesting strong radial anisotropy with a slow subvertical axis of symmetry. In the second article, teleseismic receiver functions are calculated for an array of stations in the Cyclades and decomposed into back-azimuth harmonics to visualise the variations in structure and anisotropy across the array. Synthetic receiver functions are modeled using the first order structural observations of seismic discontinuities and EBSD data. They indicate 5% of anisotropy with slow symmetry axis in the upper crust, and demonstrate the importance of rock textural constraints in seismic velocity profile interpretation.

Ultramarine is a blue pigment, formula ideally Na7.5A16 S1 6024S4.5 manufactured by Reckitt's Colours Ltd. at a rate of >10,000 tonnes per annum. The structure is based on a sodalite cage and the pigment is manufactured from kaolin, sulphur, sodium carbonate and minor ingredients, in furnaces with small surface area to volume ratios. To simulate industrial furnacing while examining a sample by PXRD (Powder X-Ray Diffraction) a novel 'Birkbeck Cell' was developed for use in a Guinier-Lenng camera. This cell is based on a stainless steel former with mica windows, and together with a gas rinsing system this arrangement produced the first time-resolved PXRD study of ultramarine formation: kaolinite is first separately calcined to the amorphous phase metakaolinite, to which the sulphur and sodium carbonate are added; in the second part of the furnace cycle sulphur and sodium carbonate react to form a sodium polysulphide, with metakaolinite later reacting with sodium salts to form a carnegieite, which then combines with the sodium polysulphides to form the initial ultramarine lattice ('primary ultramarine') ; in the latter part of the furnacing an 'oxidation' stage forms blue ultramarine and sodium sulphate is produced as a by-product. These results are supported by differential thermal analysis and thermogravimetric analysis; model furnacing; room temperature PXRD studies of various components and stages in the process; visible/uv spectrophotometry and electron microscopy. The structure of ultramarine was refined by Rietveld analysis of the neutron powder diffraction data and showed that the aluminium and silicon are not .OW0 ordered, the space group is 143m , the sodium ions lie just off the six-fold faces and the S 3 - group is in the centre of the cage, in a disordered arrangement. This analysis is supported by magic-angle spinning nuclear magnetic resonance, PXRD and the current literature on other spectroscopic methods. Finally the role of minor ingredients is examined and initial experiments to indicate possible alternative manufacturing methods are described.

RNA maturation involves several steps prior to export of the mRNA out of the nucleus and translation in the cytoplasm. PremRNA 3’end processing is one of such steps, and comprises the endonucleolytic cleavage and polyadenylation of the 3’end of the premRNA. These two steps involve more than 14 processing factors that coordinate multiple proteinprotein and proteinRNA interactions necessary to coordinate efficient cleavage and polyadenylation. To date, many of these interactions have been investigated biochemically and require additional structural characterization both to confirm and highlight key residues involved in substrate contacts. Further structural characterization will also open investigation into the mechanism of 3’end processing by providing structural insight into the coordination of multiple binding components. The cleavage factor Im, CF Im, is a component of the 3’end processing machinery and plays an important role early, during endonucleolytic cleavage, and additionally to increase polyadenylation efficiency and regulate poly(A) site recognition. CF Im is composed of a small 25 kDa subunit, CF Im25, and a large, either 58 kDa, 68 kDa, or 72 kDa subunit. The 25 kDa subunit of CF Im interacts with both the RNA and other processing factors such as the poly(A) polymerase, Clp1, and the larger subunit of CF Im. It is our goal to crystallize CF Im25 alone and in complex with one of its interacting partners to better understand CF Im25 contributions to premRNA 3’end processing. The structural investigation of CF Im25 and its binding partners has accomplished four major objectives: 1) Characterized the crystal structure of CF Im25 alone and bound to diadenosine tetraphosphate, 2) Provided insight into the oligomeric state of the CF Im complex, 3) Determined the binding properties of the Nudix domain of CF Im25 and its function in 3’end processing, 4) Further characterize the interactions between CF Im25 and PAP, CF Im68, and Clp1. These results demonstrate CF Im25 is a dimer both in solution and in the crystal suggesting that it is likely to be a dimer in the CF Im complex. The nucleotide binging capability of CF Im25 has no apparent role in 3’end processing in vitro but may provide a function outside of 3’end processing or may directly be involved in RNA recognition. The additional investigation of complex interactions with the 25 kDa subunit of CF Im25 suggests that although these factors interact during the 3’end processing event additional mechanisms may play a role in stabilizing those interactions.

The most common pathology in neonatal period is transient renal, which under adverse conditions can lead to the development of acute renal failure. Diagnosis of renal neonatal asphyxia is difficult because of the lack of specific clinical symptoms and lack of informativeness of traditional survey methods.

Kidneys are very sensitive to the deficit of oxygen. Renal dysfunction can occur within 24 hours after an episode of ischemia and may provoke the development of cortical necrosis. Relevance of the study determined the lack of highly sensitive and at the same time, the available non-invasive diagnostic methods for early detection of kidney damage in newborns.

The halogen bond has recently risen in prominence as a non-covalent interaction for use in supramolecular chemistry, allowing for the rational design of materials, pharmaceuticals, and functional molecules. The occurrence of the σ-hole opposite to the C-X covalent bond (X = F, Cl, Br, I) renders the halogen bond a highly directional and tuneable interaction, offering desirable features to crystal engineers. The halogen bond can be divided into its two components: the halogen bond donor bearing the halogen atom, and the electron-rich halogen bond acceptor. In this thesis, we investigate the nature of the halogen bond, its role in supramolecular assembly and impact on the local dynamics, along with developing synthetic methods to prepare this class of materials. We begin by fully characterizing the halogen bond donor by using 35Cl ultra-wideline solid-state nuclear magnetic resonance (NMR) spectroscopy on a series of single-component chloronitriles exhibiting the C-Cl···N halogen bond. We then perform the first modern nuclear quadrupole resonance (NQR) investigations of the halogen bond, observing the 79/81Br and 127I nuclei in a series of cocrystals exhibiting the C-Br···N and C-I···N halogen bond, respectively. Computational results attribute the observed increases in the quadrupolar coupling constants (CQ) to a reduction in the carbon-halogen σ-bonding contribution to V33 and an increase in the lone-pair and core orbital contributions, providing the first model of the electronic changes occurring on the halogen bond donor upon the formation of the halogen bond. Attention is then turned on characterizing the halogen bond acceptor and its surrounding environment, beginning by investigating a solid-state NMR approach relying on the 19F nucleus to characterize perfluorinated cocrystals. This strategy has reduced analysis times from hours to minutes while providing higher sensitivity and resolution, with the resulting chemical shifts permitting the unambiguous identification of the halogen bond and allowing for the refinement of X-ray crystal structures. The halogen bond acceptor is then investigated in a series of isomorphous dimers exhibiting both the halogen bond and hydrogen bond in the C≡C-I···X-···H-N+ motif, revealing the halogen bond’s relative contribution to the electric field gradient increasing in the order of Cl- > Br- > I-, contrasting the contributions of the hydrogen bond. We then explore the impact of the halogen bond on the surrounding environment, using the rotating methyl groups of 2,3,5,6-tetramethylpyrazine as a model. Upon the introduction of a halogen bond, we observe a reduction in the rotational energy barrier of 56% on average, overshadowing the 36% reduction observed in the hydrogen bonded cocrystals. This is the first instance of the halogen bond directly catalyzing the local dynamics, coining the term “dynamics catalyst”. These results provide an effective strategy of enhancing the dynamics in molecular systems, such as molecular machines, supramolecular catalyst, as well as correcting the faulty dynamics encountered in diseased proteins. The role of halogen bonding in crystal engineering is then explored, reporting the first supramolecular triangle, a series of discrete charged dimers, and supramolecular architectures built from 1,3,5-tri(iodoethynyl)-2,4,6-trifluorobenzene, with the potential of creating fully organic porous structures for gas absorption. Mechanochemistry is then investigated as a synthetic method, allowing for the preparation of cocrystals featuring 3-iodoethynylbenzoic acid as the donor, with the resulting structures exhibiting concurrent halogen and hydrogen bonding. Mechanochemical ball milling is shown to reduce preparation times of powdered cocrystals from days to a single hour, while using a fraction of the organic solvent. Lastly, we pioneer cosublimation as a solvent-free synthetic technique for rapidly preparing halogen bonded cocrystals, yielding quality single crystals within a few hours, and a microcrystalline product within 15 minutes. Among its advantages, cosublimation offers a significant acceleration of discovery, while eliminating the environmental footprint associated with conventional synthetic methods.

This thesis work addresses the characterisation of structural and magnetic properties of Ni2MnGa based Heusler alloys. The alloy series Mn1+xNi2-xGa has been investigated experimentally by magnetization measurements, X-ray and neutron scattering. The systematics is established as a function of Mn content of the magnetic and structural properties. Of particular interest is the development of the martensitic phase as a function of Mn content, its hysteresis behaviour and the influence of chemical order as established by a suitable heat treatment. A comprehensive experimental characterisation is given of the alloys series Mn1+xNi2-xGa for 0≤x≤1. The experimental investigation is complemented by modelling of domains in pure Ni2MnGa. Based on a detailed experimental determination of transformation matrices for the austenite martensite transformation a characterisation is given of domain walls and their crystallographic orientation. The descriptions and models which exist in the literature are extended to enable the modelling of individual domain walls within a single crystal of Ni2MnGa and its extension to several domain walls. The framework for this modelling is extended to enable the description of domain wall networks. Particular domain wall network configurations are identified and described. Within the topic of shape memory materials in general, and of the ferromagnetic shape memory Ni2MnGa based alloys in particular, this thesis work offers insight into some important aspects of their physical properties. A range of approaches is used to address some of the characterisation issues for these compounds.

Voltage-gated calcium channels (Cay) have functions ranging from regulating release of hormones and neurotransmitters, generating cardiac action potentials, and excitation-contraction coupling. At nerve terminals, N- and P/Q- type Cavs convert the action potential into aC²⁺ signal that in turn triggers neurotransmitter release. Neurotransmitter release requires several components, such as SNARE proteins. SNAREs, as well as many other presynaptic proteins, can interact with Cavs and inhibit them by increasing their inactivation. The interaction is localized in the intracellular loop between domains II and III of the CL 1 subunit, in a domain termed ‘synprint’ (synaptic protein interaction site). In this study, we tried to solve the structure of the synprint site by crystallography. To date, long needle-shape crystals were obtained; however, the quality of these crystals was not good enough for X-ray diffraction. in addition, isothermal titration calorimetry (ITC) was used to determine the interaction between SNARE protein syntaxinlA and the synprint site. It turned out that not any binding was detected, suggesting that the interaction between SNARE proteins and the presynaptic Cas, if at all present, is weak.

Title of the thesis On the investigation of nucleation, growth, structure and morphology of functional materials through advanced crystallographic techniques Description of the research work done I-Use of gel technique for the crystallization of coordination polymers A gel is a system where a liquid phase is dispersed within a solid phase. A network of micrometric channels and pores filled by solution characterizes the obtained material. Any mass transport mechanism but slow diffusion is avoided. This creates an interesting media to perform crystal nucleation and growth under purely diffusive conditions. Recently gel technique for crystallization has been extensively adopted, leading to intriguing results about crystals quality, dimension, mechanical and chemical stability. For certain organic molecule were reported the gel ability to induce the formation of new phases and polymorphs. Inside gel media, slightly different crystallization conditions can be simultaneously created. For a class of compounds known as coordination polymers or MOF, little difference in the crystallization environment can lead the synthesis towards the formation of network completely different from the point of view of dimensionality, topology, coordination geometry of metal centres and ligand behaviours. The aim of the following projects is to map the relevance of gel technique if applied for the synthesis of coordination polymers, considering parameters as solvent composition, gel nature, reagents concentrations ([L]+[M]) and ratio([L]/[M]). I-A system Cd(II) bpp We considered water/EtOH solutions of CdCl2 (M) and bis-Pyridyl-propane (bpp) and agarose gel at different concentration. Three different crystalline phases can be obtained: 1D [Cd(bpp)3Cl2]∙H2O (1D-Cd) (L/M=3) 3D [Cd(bpp)2Cl2]·H2O (3D-dia) (L/M=2) 3D [Cd2(bpp)3Cl2]·H2O (3D-sra) (L/M=1,5) The phases show different compositions in terms of L/M ratio, and also different dimensionality and topology. Their morphologies also are peculiar enough to let they be straightforwardly identified by optical microscopy. We made a strict comparison between system behaviour inside solution and jellified solution, to point out the most important gel effects on crystallization process. For solution studies, we applied batch technique. For gel studies, we used counterdiffusion methods. We used two different experimental set up: (a) Layering of the solutions of a reagent over the gelified solution of the other reagents inside a test tube; (b) Layering in the opposite branches of a U-shaped tube the solutions of the reagents over a gelified solvent. I-A.1 solution behaviour We determined the precipitated phases considering different reagent concentration and ratio at fixed EtOH amount (50%) and different amounts of EtOH and reagents ratio at a fixed concentration (20mM). To confirm the results obtained, we also study powders precipitated from solutions at higher concentrations with the same experimental conditions. 3D-sra phase forms at low L/M value (<2) for any solvent compositions. Increasing [L]/[M] value bring to two different behaviours. For EtOH quantity below than 30% 1D-Cd precipitates as powder, above 30% we observe precipitation of 3D-dia. From solutions, we never observe co-precipitation of all the three phases. But 3D-sra and 3D-dia coexist inside alcoholic solutions. I-A.2 Gel technique modifies the phase diagram When we laid ligand alcoholic solutions over water-gelified solutions of the metal salt, the crystals form more in the solutions than in gel. The sra phase appears for a narrower range of L/M values, while dia phase becomes predominant. Instead, when we layer metal salt solution over the ligand solution, the two phases coexist for all the considered value of L/M. They are segregated: sra phase inside the solution, the 3D-dia inside the gel. We suggest that the gel slows selectively the diffusion of bpp, segregating the phases. The gel strongly affects crystals morphologies. Dia phase forms largely elongated and distorted structures inside the gel, while 3D-sra inside gel media forms little spherulites. I-A.3 The phase diagram can be mapped inside a U-shaped tube We perform counterdiffusion experiment inside a U-shaped tube. In the first place we consider tubes whit the same gelled solutions (1% agarose, 40% ethanol), varying only reagents concentrations and ratio. We observed the formation, starting from the ligand charged branch, of phase dia and then phase sra. Inside tube containing the excess of metal, dia phase forms only inside ligand solution, while using ligand excess we observed a transition from a tube containing both the phases to the disappearance of the sra phase. This behaviour confirms that the gel phase probably slows the diffusion of the ligand solution. After that, we studied the effect of the solvent composition. Inside gel, we consider %EtOH from 15% to 40%. We observe coprecipitation of all the three phases for %EtOH<20%. They form starting from the ligand branch and their order is 1D-Cd, 3D-dia, 3D-sra. I-A.4- The use of different gel influences the phase diagram and crystals morphologies Agarose dispersed in water o ethanol-water solvents is the gel media that we choose. Agarose dispersion forms gel at different concentrations, expressed as %w/v, ranging from 0.1% to 3%. Increasing agarose % creates a stiffer material. We didn’t observe relevant effects on the phase diagram, while crystal morphology shows increasing habit distortion increasing gel strength. Then we considered different gel media to fill the U-shaped tube. As gelator, we considered silica, Polyacrylamide (PAA), polyvinylpyrrolidone (PVP). All gels considered kept the same reciprocal disposition of the phases. But 1D-Cd isn't formed inside PVP, PAA gels strongly inhibit 3D-dia nucleation, also shifting the crystal position nearer to the metal solution branch, while silica gels move phases position in opposite directions. Crystals morphology is strongly affected by gel strength; in fact, we observe no modification only inside the very light PVP gel. Instead, inside silica gels, 3D-dia crystal modifies its habit. In fact, We observed the prismatic form {100} becoming predominant. I-B System Cu(II)/isonicotinic acid Isonicotinic acid (HIso) is a widely used ligand. It is a linear, rigid connector. Its coordinative sites are also donor or acceptor of hydrogen bonds useful for the assembly of supramolecular frameworks. It’s an anionic ligand, so its MOF structures are neutral, avoiding any templating effect of the counter anion. In CSD databased is reported a large number of structures with different topologies and dimensionality. In particular, Copper(II) atom containing structure show peculiar ligand behaviour and coordination geometry. In this project, we mapped the species obtained from the mixing of Copper chloride salts and isonicotinic acid at room temperature, the parameters considered are the same as the previous project, total concentration ([Cu(II)]+[HIso]), the molar ratio([HIso]/ [Cu(II)]). As the solvents, we considered water, THF, and DMSO. We considered the previously described crystallization method. Five phases were obtained: 1 - 0D complex [Cu(Iso)2(H2O)4] formed by single distorted copper octahedral complexes. 2 - 2D MOF [Cu2(Iso)3Cl(H2O)2] ∙ THF formed by a 2D network staked along [001] directions 3 – 2D MOF [Cu(Iso)2(H2O)]∙THF formed by a 2D network staked along [001] directions 4 – 2D MOF [Cu4(Iso)6Cl2(H2O)2(DMSO)] formed by a 2D network staked along [110] directions 5 - 2D MOF [Cu2(Iso)2(H20)] ∙ DMSO formed by a 2D network staked along [001] Working inside water/THF solutions, phase 1 and 2 have been isolated. Phase 2 is strongly favoured both by low values of L/M ratio and high %THF in the precipitating solvent. Phase 1 precipitates from water solutions or solutions with high L/M value. To observed gel effect we used a limited amount of experimental conditions. We consider an equal amount of the reagents (L/M=1) or metal excess (L/M=0,5), and concentration 50 or 75 mM. About solvent compositions, working inside test tubes, we consider all the possible combination of water and water/THF solutions. The use of only water or water gel bring to the formation exclusively of phase 1, instead, water/THF gels and THF rich environment are characterized by the formation of phases 2 (L/M=0,5) and 3 (L/M=1). This last phase is highly unstable out of the gel and has never been detected in the solution. Counterdiffusion inside U-shaped tube, using water/THF solutions of equal concentration, lets to precipitate phase 1 (near ligand charged branch) and phase 3 (right behind it). This tells us an higher mobility of the metal salt respect to the ligand molecule inside the gel media, and the existence of a range of L/M value was phase 3 is favoured respect to phase 1, where from the solution we can only precipitate phase 1. Has to be determined if phase 3 is stabilized due to peculiar interaction interplayed with the gel media or it’s a metastable phase that can be observed due to the slowing of the degradation kinetic inside the gel media. Using as solvent DMSO/THF mixtures we obtain interesting results. Also in this case from solution can precipitate two different phases, both of them are 2D MOF. For this mixture, the presence of THF doesn’t change the nature of the obtained phase. For L/M=0,7 we obtained phase 4 while phase 5 was obtained for a higher concentration of ligand (L/M=2,7). The two phases can coexist for L/M=1,3, but phase 5 is unstable outside solution. Considering crystallization conditions, stability outside solution, topology, dimensionality, coordination geometry, and ligand behaviour phase 2 and 4 show strong similarity, as phases 3 and 5. We proposed that a simple parameter as L/M can have deep effects on the solution behaviours of the reagents and then determine the topology and dimensionality of the precipitated phase. II-Morphology prediction Inside gel media, the only viable mass transport mechanism is the diffusion troughs the small pores of the media. If reagent diffusion is enough slowed, it is the rate-determining step of crystal growth. That means that the observed morphology is determined only by the stability of the faces, that is proportional to the surface tensions. This happens because the energy request to bind a molecule on a certain face (attachment energy) that is the most important kinetic parameter, is less determining. The former situation leads to the formation of the equilibrium morphology, while the latter to growth morphologies. Being gel media pure diffusive, in principles crystals grew inside gel media should show a morphology nearer to the equilibrium one, not considering peculiar interactions between faces and gel media itself. The simulation of crystal habit and morphology is not trivial for coordination polymer due to two issues: the selection of a proper building unit that is actually added to the growing crystal and the choice of an appropriate potential to model the interactions. Our purpose is to study from an experimental point of view the effect on crystal habit of gel media, and then, to verify the feasibility of PIXEL package for simulation of the morphology. The 0D Cu-HISO (phase 1, project B) system shows important morphological modification if growth inside gel or inside solutions with different concentrations. We identify four different crystal habits. All of them can be described by some the following six forms: {101 ̅}, {010}, {011 ̅ }, {11 ̅0},{111 ̅},{001}. Inside the solutions, the crystals are platelets and the most important form is the {101 ̅ }. We observed an evolution of the habit increasing the reagents concentration, from rhombic platelet (forms {101 ̅}, {010}, {011 ̅}) to hexagonal platelets with increasing importance of form {001}. Inside gel, we observe only prisms, where forms {101 ̅} and {010} are predominant and they have the same importance. We embarked on the simulation of both equilibrium and growth morphology, in the framework of Hartman-Perdok (HP) theory, using PIXEL method for energetic calculations, to verify if habit simulation can shed some light on these behaviours. HP approach considers a crystal a fabric of chain of molecules bounded by strong interactions (polymeric bond chain, PBC). Every chain represents a crystallographic direction of fast growth. If a face is parallel to at least two of these chains, the energy request to attach a molecule over it is presumably higher than the energy requested to attach a molecule to its border. As a result, this faces will grow in extension and is classified as F (flat). The equilibrium morphology contains only flat faces. Attachment energy calculations, considering as the starting point a reasonable bunch of faces (the flat ones), let us know the growth morphology. Then, towards the broken bonds model, from this, we can calculate the surface tension of the single face and then the equilibrium morphology. All energetic evaluations were performed using PIXEL formalism. Originally developed for closed-shell, neutral organic compounds, it has been subsequently extended to deal with ionic compounds, salts, and zwitterions. The PIXEL formalism and parameterization have been recently extended systematically to compounds including first-row transition metals in combination with organic moieties. This extension has been carried out using an extensive set of thermochemical data, successfully reproducing the sublimation enthalpies of a wide range of crystalline materials. PIXEL treat pecking energies as a summation of single dimer interactions, bringing straightforwardly to the identification of strong bonds and then of PBC. The identified PBC let us classify as flat all the observed crystal forms apart from the plane (001), that is classified K (kinked) the unstable kind. Then, we calculate the attachment energies and surface tensions for all the identified flat faces. The calculated equilibrium morphology is very similar whit the bars morphology. This morphology is typical of low concentration environment and gel media. This is the experimental equilibrium habit. This confirms the goodness of PIXEL simulation. This also confirms that crystals grew inside gel media shows their equilibrium morphology, suggesting that inside gel mass transport is only diffusive and it’s the rate determining step of the growth. The balance between kinetic and thermodynamic factors for the determination of the morphology is well described by PIXEL calculated values. Form {111 ̅} from both calculation and experiments reveal to be relevant in the equilibrium morphology but almost invisible during the growth process, while form {011 ̅} shows exactly the opposite behavior. The importance of form {101 ̅} is not explained by energetic values. However, some considerations about its structure suggest that this is the only face without a net charge over itself, creating an easily poisoned surface. Form {001} is completely different, and here we can suggest an opposite behaviour, where the large negative charge on its surface can probably create a kinetic barrier that became more effective at high concentration fast growth process. However, its K nature is significant considering that its appearance is strongly affected by experimental conditions. III-Cholesterol crystallization from model lipid bilayers Atherosclerotic diseases are one of the major death cause in the western world. A crucial step in the development of the disease is the rupture of the atherosclerotic plaques end the resulting inflammatory response. It's well known that plaques are formed by different lipids, in particular, cholesterol, and the presence of cholesterol crystals is indicative of the pathologic ones. The presence of cholesterol crystals can be related also with the triggering of the inflammatory response itself, but a clear correlation was not yet established. There is not much information about the process of cholesterol crystal formation and deposition inside plaque, a lipid-rich environment. Different studies where performed inside bile, because cholesterol crystal triggers the formation of the gallbladders. From these studies was observed the ability of cholesterol to form different crystalline materials, from the typical platelet of triclinic monohydrate cholesterol to more unusual material as curved fibres and helices. In recent years some observations were performed on the behaviours of lipid mono and bilayer. A result from this study was the identification of 2D lipid structures called ‘’rafts’’, usually formed by saturated lipids as dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM), that move inside a liquid 2D phase of unsaturated lipids as palmitoyl oleoyl phosphatidylcholine (POPC). Cholesterol molecules also form ordered lipid domains, sometimes by mixing with other saturated lipids. For all the cited lipids was identified a concentration threshold above which Cholesterol phase separates and forms such rafts. Cholesterol rafts are characterized by a rectangular 2D symmetry, not compatible with the already known triclinic structure. Recently was discovered a correlation between the presence of such ordered cholesterol domains and the appearance of cholesterol crystal over lipid bilayer. The purpose of the project was to study the crystallization process. In the first place identifying the cholesterol phases that can be obtained, then, adopting super-resolved fluorescence microscopy (STORM method), obtaining more information about nucleation and growth processes. Lipid bilayers were formed by vesicles fusion and were cholesterol-enriched using a well know procedure adopting a cholesterol: cyclodextrin complex. We considered the mixture of cholesterol with unsaturated (POPC) and saturated (DPPC) lipids. For fluorescence microscopy, we used an IgM antibody developed in Lia Addadi group, able to recognize ordered cholesterol patterns as crystals or domains. TEM microscopy and electron diffraction were performed to identify the crystalline phases. Over POPC/Chol bilayer we observed exclusively the formation of square platelet crystals, while over POPC/DPPC/Chol bilayer we identified also a large number of curved fibres, needle, and hexagonal platelets. TEM diffraction demonstrated that the square platelets consist of triclinic cholesterol monohydrate phase. While all the other morphology belongs to crystals of a monoclinic cholesterol monohydrate phase, that was never be identified before. The morphology of such phase is intriguing because atherosclerotic plaques often contain needle crystal, that cannot be formed by the triclinic phase, suggesting a relevant role for the monoclinic phase for pathological environments. Crystals grown from murine cells fed by HDL particles show the same characteristics. Further analysis performed with STORM technique revealed very different behaviours of cholesterol domains inside the two considered bilayers (POPC/Chol, POPC/DPPC/Chol). Also, the crystallization environment of the two phases shows interesting differences. POPC bilayer is characterized by the presence of small Cholesterol domains, accordingly with the low solubility of Cholesterol in unsaturated lipids. They can aggregate forming larger domains that already show shapes similar to the triclinic platelet, suggesting a sudden aggregation and formation of triclinic phase crystals. DPPC bilayers show the presence of slightly bigger domains, homogenously diffused over the bilayer. The triclinic phase crystal is often surrounded by large not labelled areas, that can be related with large cholesterol ordered domains, suggesting a slower process of aggregation and crystal formation over the bilayer. Instead, monoclinic crystals are often found grown inside the bilayer itself. They reveal to be very flexible, bending and assuming curved morphologies from their very first appearance.

The importance of crystals to the pharmaceutical industry is evident - over 90% of pharmaceutical products contain a drug in crystalline form. However, the crystallization phenomena of drug compounds are poorly understood. An increased understanding of these processes may allow a greater degree of control over the crystallization outcomes, such as morphology, purity, or stability. In these studies, we have applied Atomic Force Microscopy (AFM) to the in situ investigations of drug crystal growth. We utilized AFM to assess the growth on the (001) face of aspirin crystals at two supersaturations, elucidating both the growth mechanisms and kinetics at each supersaturation. We also investigated the nucleation of aspirin crystals, using microcontact printing to arrange aspirin-binding and non-binding self-assembled monolayers (SAMs) onto surfaces. This facilitated the visualization, using AFM, of the growth of aspirin crystals adhered to the surface. Additionally, secondary nucleation was observed on the growing crystals. The effect of the additives acetanilide and metacetamol on the morphology and growth on the (001) face of paracetamol was investigated. The presence of metacetamol significantly reduced the growth rate on the face, with respect to pure paracetamol solutions. The growing steps exhibited a pinned appearance, consistent with the Cabrera and Vermilyea model. Conversely, acetanilide caused dissolution to occur. Finally, we assessed the capabilities of AFM in following the structural transformations of crystals, which can occur in unstable pharmaceutical compounds. We employed AFM to determine the process by which anion exchange, and the subsequent structural transformations, of the co-ordination polymers {[Ag(4,4'-bipy)]BF4} and {[Ag(4,4'-bipy)]NO3} occur. AFM data verified that the anion exchange process is solvent-mediated. The mechanisms underlying this process are discussed herein. These results reiterate the capability of AFM to monitor dynamic events on crystal surfaces. Analogous studies could be applied to numerous pharmaceutical compounds, thus facilitating the optimization of their crystallization parameters. In essence, future experiments using AFM may afford greater control over crystallization, and prevent the production of unwanted or unstable pharmaceutical compounds.

A new approach to the investigation of the optical properties of polytypic minerals that combines spindle stage techniques, X-ray diffraction methods, electron microprobe analysis, and dielectric tensor calculations has been developed and applied to zinc sulfides and chloritoids. For the first time, X—ray diffraction studies of natural anisotropic zinc sulfides have documented the simultaneous occurrence of twinning and stacking disorder along more than one of the four symmetry equivalent <111> directions of sphalerite. Precession photographs of optically anisotropic zinc sulfides are characterized by twin—equivalent diffraction maxima and diffuse diffraction streaking along lattice rows with (h-k) ≠ 3n (equivalent hexagonal indices) in one or two <111> directions. A system of linear equations has been used to calculate the approximate volume fractions of each twin domain and the sphalerite host domain. Dielectric tensor calculations have been performed to illustrate that mixtures of cubic and hexagonal zinc sulfide may be optically biaxial if the intergrowth occurs along more than one of the symmetry equivalent <111> directions of sphalerite (cubic). The dependence of the optical properties upon the chemical variation and polytypic intergrowth in the Hg-Fe chloritoids has been investigated. The effects of the variation in chemical composition of specific polytypic compositions were analyzed first. The refractive indices of 10 approximately pure 2M₂ Hg-Fe chloritoids show strong correlations (R² ≥ 0.094) to the proportion of Hg cations in the H(1B) site, Hg/(Hg + Fe + Hn) - HGN. Correlations between the optical orientation angles and HGN were weaker (R² ≤ 0.87). The optical orientation is very sensitive to small variations in the polytypic composition, especially orientation angles that have fixed values in 2M₂ chloritoid. The parameter showing the most sensitivity is ∠ X ⋀ b, which is 0° in an ideal 2M₂ chloritoid, but increases to about 6° for a chloritoid containing 10% by volume 1Tc polytype. The sum of ∠ Y ⋀ c* and ∠ Z ⋀ c* for an ideal 2M₂ chloritoid has a value of 90°, whereas a chloritoid with 10% 1Tc has a sum of 92°. Although not as sensitive as ∠ X ⋀ b, this parameter can be determined with only a spindle stage or universal stage. The observed dependence of the optical properties on polytypic intergrowth and chemical variation has been modeled using dielectric tensor calculations based on the properties of a 1Tc layer and assuming that the 2M₂ polytype is derived by twinning the 1Tc polytype about [010] with an (001) composition plane.
Ph. D.

The phosphothreonine lyase class of enzymes represents a recently discovered set of enzymes that catalyze a dephosphorylation reaction. The catalysis is carried out using a unique elimination mechanism without any involvement of cofactors. Crystallographic studies of SpvC, a phosphothreonine lyase, and its mutant show that the mutation of the general catalytic acid does not result in any significant perturbations to the tertiary and the secondary structure of the protein. Using results from the structural studies and a deuterium isotope exchange experiment, we conclude that the reaction catalyzed by SpvC may not involve formation of a carbanion at the active site.

Macromolecular X-ray crystallography (MX) is currently the dominant technique for the structural eluci- dation of macromolecules at near atomic resolution. However, the progression and deleterious effects of radiation damage remains a major limiting factor in the success of diffraction data collection and subsequent structural solution at modern third generation synchrotron facilities. For experiments conducted at 100 K, protein specific damage to particular amino acids has been widely reported at doses of just several MGy, before any observable decay in average diffraction intensities. When undetected, such artefacts of X-ray irradiation can lead to significant modelling errors in protein structures, and ultimately the failure to derive the correct biological function from a model. It is thus vital to develop tools to help MX experimenters to detect and correct for such damage events. This thesis presents the development of an automated program, RIDL, which is designed to objectively quantify radiation-induced changes to electron density at individual atoms, based on F_obs,n − F_obs,1 Fourier difference maps between different dose states for a single crystal. The high-throughput RIDL program developed in this work provides the ability to systematically investigate a wide range of macromolecular systems. To date, damage to the broad class of nucleic acids and nucleoprotein complexes has remained largely uncharacterised, and it is unclear how radiation damage will disrupt the validity of such models derived from MX experiments. This thesis presents the first systematic investigations on a range of nucleic acid, protein-RNA and protein-DNA complex case studies. In general, it is concluded that nucleic acids are highly robust to radiation damage effects at 100K, relative to control protein counterparts across the tested systems. For protein crystals at 100K, cleavage of the phenolic C-O bond in tyrosine has disseminated through the MX radiation damage literature as a dominant specific damage event at 100K, despite the absence of any energetically favourable cleavage mechanism. To clarify the radiation susceptibility of tyrosine, this thesis presents a systematic investigation on radiation damage to tyrosine in a wide range of MX protein radiation damage series retrieved from the Protein Data Bank. It is concluded that the tyrosine C-O bond remains intact following X-ray irradiation, however the aromatic side-group can undergo radiation-induced displacement. This thesis also presents further applications of the RIDL program. A protocol is introduced to calculate explicit half-dose values for the electron density at individual atoms to decay to half of their initial value at zero absorbed dose. In addition, a methodology is developed to detect radiation-induced changes to electron density occurring over the course of the collection of a single MX dataset of diffraction images, all of which are required for structural solution. These protocols aim to advise experimenters of when previously-undetected site-specific damage effects may have corrupted the quality of their macromolecular model. Overall, the work in this thesis is highly applicable to both the future understanding of radiation damage in macromolecular structures, as well as of interest to the wider crystallographic community.

The opportunistic pathogen Clostridium difficile is the most common cause of antibiotic-associated diarrhoea, with severity of disease ranging from mild diarrhoea to fulminant pseudomembranous colitis and death. It poses a major burden on healthcare providers, costing millions of pounds each year due to ward closures, isolation measures and prolonged illness. The current antibiotic therapy for C. difficile infection is effective, but high rates of relapse lead to ongoing misery for patients and spiralling costs for healthcare providers. Novel therapeutics for C. difficile are therefore desperately sought, and the antibiotic-induced nature of the disease has led to interest in development of non-antibiotic therapies. Sortase enzymes are responsible for covalent anchoring of specific proteins to the peptidoglycan of the cell wall of gram-positive bacteria. Following the discovery that the Sortase A enzyme of Staphylococcus aureus is essential for pathogenesis, sortase inhibitors are under investigation as novel therapeutics. Being ubiquitous in gram-positive bacteria, it is likely that other gram-positive pathogens require sortase enzymes for their pathogenesis and may be targets for development of sortase inhibitors. This work describes a characterisation of the sortase enzyme of C. difficile. To provide evidence for a role of the sortase in the cell wall biogenesis, a C. difficile sortase knockout strain was constructed by intron mutagenesis. Characterisation of this mutant led to the discovery that the putative adhesin CD0386 is anchored to the peptidoglycan of C. difficile by the sortase SrtB. To provide structural insight into the catalytic mechanism of the C. difficile sortase, an active site mutant was crystallised and its structure solved to 2.55Å by X-ray diffraction. The wall-linked protein CD0386 was also crystallised and subject to successful test diffraction. Analyses of SrtB reaction products by chromatography and mass spectroscopy indicate that the enzyme cleaves an SPKTG peptide motif and catalyses a transpeptidation reaction with a component of the C. difficile peptidoglycan.

The relationship between enzyme dynamics and enzymatic catalysis has become a central topic in modern enzymology, and studies in this area promise to enrich our current understanding of catalysis in biological systems. Escherichia coli dihydrofolate reductase (EcDHFR) has been a frequent subject of study in the context of protein dynamics, due to its small size, biological ubiquity, and the fact that its structural, kinetic and mechanistic characteristics are well established. Intrinsic kinetic isotope effects (KIEs) have proven to be highly sensitive probes of the role of dynamics in EcDHFR catalyzed reaction, as they circumvent the kinetic complexity of the enzyme-catalyzed reactions, and extract information directly pertaining to the chemical step. Previously, studies of their temperature-dependence were used to probe the effect of mutations at residues distant from the active site upon the hydride-transfer reaction catalyzed by EcDHFR. The results of these experiments supported the presence of a network of residues that were dynamically linked to the hydride-transfer step, and were in excellent agreement with computational studies predicting the presence of such a network. This thesis aims to extend upon these results to study the nature and extent of the dynamic network in EcDHFR, both by using an established experimental methods and by developing new biophysical probes of protein dynamics in this system. The major experimental methodology utilized in the following chapters is the determination and analysis of KIEs in a variety of EcDHFR mutants. To facilitate these measurements, new synthetic routes to a range of isotopically labeled nicotinamide cofactors have been developed. Some of the labeled materials have been used to establish a sensitive, triple-isotope technique to competitively measure deuterium isotope effects in enzyme-catalyzed reactions in EcDHFR. Synthesized materials were usd to measure the temperature dependence of intrinsic KIEs in selected dynamically altered mutants of EcDHFR, viz. W133F and F125M DHFR. Crystal structures have been obtained for both these mutants as well as for the previously studied G121V isozyme, and the combination of kinetic and structural information discussed in the context of catalytically important dynamic fluctuations in EcDHFR. Pressure-dependence of deuterium KIEs is also developed as a tool to probe the role of dynamics and tunneling in the EcDHFR reaction, with the ultimate aim of establishing high-pressure KIE measurements as a complementary method to variable temperature measurements. Finally, molecular recognition force spectroscopy (MRFS) measurements of an EcDHFR self-assembled monolayer (SAM) on gold are described. The surprisingly active enzymatic SAM has been shown to be a promising platform for future MRFS experiments to measure the forces involved in EcDHFR dynamics. All together, these studies advanced our ability to study the role of enzyme dynamics and quantum tunneling in enhancing their chemistry.

Spectroscopy is increasingly used to investigate and monitor the solid state forms of pharmaceutical materials and products. Spectroscopy�s speed, nondestructive sampling, compatibility with fibre optics and safety also make it attractive for in-line monitoring. In this thesis, the spectroscopic techniques Fourier transform Raman spectroscopy, terahertz pulsed spectroscopy and second harmonic generation were used to characterise and quantify polymorphism and crystallinity of pharmaceutical compounds. Where possible, the multivariate analysis technique partial least squares was used for quantitative analysis. Fourier transform Raman spectroscopy detects polarisability changes mainly associated with molecular vibrations. Terahertz pulsed spectroscopy is a new spectroscopic technique that operates between the infrared and microwave regions of the electromagnetic spectrum and detects dipole moment changes mainly associated with crystalline phonon vibrations in the solid state. Second harmonic generation is a nonlinear optical phenomenon that depends on the dipole moment in crystals and crystal symmetry. Several materials capable of existing in different solid state forms were used. FT-Raman spectroscopy was able to differentiate carbamazepine forms I and III, enalapril maleate forms I and II and γ-crystalline and amorphous indomethacin. Combined with partial least squares the technique could quantify binary mixtures of CBZ forms I and III with a limit of detection as low as 1%, and mixtures of enalapril maleate with a limit of detection of as low as 2%. Terahertz pulsed spectroscopy obtained very different spectra for carbamazepine forms I and III, enalapril maleate forms I and II, γ-crystalline and amorphous indomethacin, crystalline and supercooled thermotropic liquid crystalline fenoprofen calcium, three forms of lactose, and five forms of sulphathiazole. At present the modes in the spectra cannot be attributed to specific phonon modes. Quantitation of binary mixtures of different forms of a compound using partial least squares analysis usually resulted in a limit of detection of about 1%. Second harmonic generation was used to quantify binary mixtures of different forms of enalapril maleate and lactose, as well as binary mixtures of enalapril maleate form II and polyvinylpyrrolidone. A quantitative relationship was present for each of the mixtures, however the limits of detection were usually above 10%. The high value is probably due to the machine being a prototype and univariate analysis associated with a single output variable. Future improvements to the apparatus and measurement parameters are likely to reduce the limits of detection. Ranitidine hydrochloride polymorphs could also be differentiated using second harmonic generation, however γ-crystalline and amorphous indomethacin and forms I and III of carbamazepine could not. The methods used in this thesis were successfully used for qualitative and quantitative analysis of polymorphism and crystallinity of pharmaceutical compounds. TPS and SHG are useful additions to the range of experimental techniques that can be used to investigate and monitor properties of pharmaceutical solids.

A major challenge in cancer treatment is acquired or intrinsic multidrug resistance (MDR) to chemotherapeutics. A notorious mediator of MDR is P-glycoprotein (P-gp, ABCB1), product of the human MDR1 gene, which actively effluxes cytotoxic drugs from cancer cells, resulting in sub-therapeutic intracellular concentrations. Understanding how P-gp interacts with drugs has been severely limited by the lack of high-resolution structures of P-gp. Although numerous efforts to obtain an X-ray crystal structure of P-gp have been attempted, human P-gp has never been crystallized. However, mouse P-gp (87% homologous to human P-gp) has been crystallized, and several structures of mouse P-gp have been recently reported. Despite a high degree of homology, it is currently unknown why mouse P-gp can be crystallized while human P-gp cannot. The studies presented in this thesis describe the creation of novel chimeras of mouse and human P-gp as an approach to investigate whether specific protein domains are responsible for differences in the ability to form crystals between mouse and human P-gp. A range of chimeras, created by protein domain swapping, were expressed in mammalian cells and all were found to retain MDR transport function demonstrating that P-gp can tolerate major structural changes. High-level expression of all chimeras was achieved by baculovirus-mediated heterologous protein expression. Chimeric proteins were purified by a multi-step process including immobilized metal affinity chromatography and size exclusion chromatography. Crystallization screening obtained protein crystals for two of the chimeras, indicating the approach adopted is a successful strategy, and an advance along the path towards a high-resolution structure of human P-gp.

The catalytic mechanism of the Pdx1 subunit of the pyridoxal 5’-phosphate (PLP) syn-thase enzyme complex has been the subject of intense study since its discovery in 1999. Pdx1 uses two active sites (P1 and P2) to perform a complex reaction combining ribose 5-phosphate, ammonia and glyceraldehyde 3-phosphate to form PLP. While some aspects of the Pdx1 mechanism are now understood, several questions re-main, in particular how the enzyme transfers the reaction from one active site to the next and how the reactions in the two sites are coordinated. In this investigation, X-ray crystallography and UV-Vis spectrophotometry have been used to determine the struc-ture of the protein in various intermediate states and elucidate the catalytic mechanism. The role of specific active site residues in catalysis of PLP biosynthesis by the Arabidop-sis thaliana Pdx1 protein is investigated using site-directed mutagenesis. The crystal structures presented in this thesis demonstrate that the Pdx1 enzyme uses a novel relay mechanism to covalently transfer intermediates between active sites and to coordinate substrate binding, intermediate shuttling and catalysis. Chapter 1 provides an introduction to the biological role of pyridoxal 5’-phosphate and its biosynthesis by the PLP synthase enzyme complex. Chapter 1 also describes the use of X-ray crystallography to understand how proteins function and how complementary methods such as UV-Vis spectrophotometry can be used to track the effects of site-specific radiation damage during collection of X-ray diffraction data. The methods used to produce, purify and investigate the Pdx1 enzyme are described in Chapter 2. Chapter 3 provides an analysis of the activity of wild type AtPdx1.3 using UV-Vis spectropho-tometry and X-ray crystallography to monitor the accumulation of chromophoric inter-mediates and the product, PLP. Chapter 4 describes the use of site-directed mutagenesis to trap the Pdx1 protein in additional intermediate states and the characterisation of these intermediate states using UV-Vis spectroscopy and crystallography. UV-Vis spectra of Pdx1 crystals were collected to ensure that the Pdx1 enzyme was in the desired intermediate state before collection of X-ray diffraction data. It became clear that the UV-Vis spectra of the Pdx1 crystals in some intermediate states were changing during X-ray data collection. Chapter 5 describes the experiments that were performed to check that the Pdx1 structures obtained were not affected by site-specific radiation damage. The use of multi-crystal data collection strategies and UV-Vis spectroscopy showed that changes in the spectra were instead caused by radiolysis of the solvent surrounding the crystals.

The Streptomyces avidinii protein streptavidin binds the small molecule biotin (vitamin H / B₇) with extraordinary stability, resulting in the streptavidin-biotin interaction being one of the strongest non-covalent interactions known in nature (K_d ~ 10^-14 M). The stable and rapid biotin-binding, together with high resistance to heat, pH and proteolysis, has given streptavidin huge utility, both in vivo and in vitro. Accordingly, streptavidin has become a widely used tool in many different biotechnological applications. Streptavidin has also been the subject of extensive research efforts to glean insights into this paradigm for a high-affinity interaction, with over 200 mutants of the protein reported to date. Despite the high stability of the streptavidin-biotin interaction, it can and does fail under certain experimental conditions. For example, streptavidin-biotin dissociation is accelerated by an increased temperature or lower pH (conditions often encountered in cellular imaging experiments), and by mechanical stress, such as the shear force arising from fluid flow (encountered when streptavidin is used as a molecular anchor in biosensor chips and arrays). This study details efforts made at increasing further the utility of streptavidin, by increasing the stability of biotin and biotin-conjugate binding. A rational site-directed mutagenesis approach was used to create 27 mutants, with eight of these mutants possessing higher-stability biotin-binding. The most stable biotin-binding mutant was named traptavidin and was extensively characterised. Kinetic characterisation revealed traptavidin had a decreased dissociation rate from biotin and biotin-conjugates when compared to wildtype streptavidin, at both neutral pH and pH 5. Atomic force microscopy and molecular motor displacement assays revealed the traptavidin-biotin interaction possessed higher mechanical stability than the streptavidin-biotin interaction. Cellular imaging experiments revealed the non-specific cell binding properties of streptavidin were unchanged in traptavidin. X-ray crystallography was also used to generate structures of both apo- and biotinbound traptavidin at 1.5 Å resolution. The structures were analysed in detail and compared to the published structures of streptavidin, revealing the characteristics of traptavidin arose from the mutations stabilising a flexible loop over the biotin-binding pocket, as well as reducing the conformational change on biotin-binding to traptavidin. Traptavidin has the potential to replace streptavidin in many of its diverse applications, as well as providing an insight into the nature of ultra-stable noncovalent interactions.

Biogenesis of the multivesicular body (MVB) organelle is an important process for regulation of signalling in the cell. Signal receptors embedded within the outer MVB membrane can be sorted into intralumenal vesicles which bud away from the cytosol to within the MVB preventing further signalling. Sorting of receptors, invagination of the membrane and release of vesicles into the MVB lumen are mediated by the endosomal sorting complexes required for transport (ESCRT) along with a range of accessory proteins including histidine domain protein tyrosine phosphatase (HD-PTP). HD-PTP is a multidomain protein which makes several interactions with ESCRT partners, including ESCRT-0, ESCRT-I and ESCRT-III. This thesis focusses specifically on the interaction between HD-PTP CC domain and Ubap1 (ESCRT-I), and the two interactions of HD-PTP Bro and PRR domains with STAM2 (ESCRT-0) SH3 and Core domains. To address the structure of HD-PTP, multiple techniques were used: X-ray crystallography, which gives high resolution structural information; small angle X-ray scattering (SAXS), which gives low resolution data for large non-crystallisable units in their solution state; and double electron-electron resonance (DEER) spectroscopy, which gives high resolution nanometre-range distance constraints between cysteines labelled with methanethiosulfonate spin label (MTSL). It was shown by X-ray crystallography that HD-PTP has an elongated CC domain, in stark contrast to its homologues ALIX and Bro1 which both have V-shaped CC domains. The CC domain showed limited flexibility both by SAXS and DEER. Further investigation showed that there was no significant conformational change upon binding its ESCRT-I partner Ubap1. The multidomain structure of HD-PTP Bro1-CC-PRR was described by SAXS, showing that these domains form an extended arrangement in solution. In addition, SAXS was also used to analyse the structure of these domains in complex with STAM2 (ESCRT-0), which showed that STAM2 is simultaneously tethered by the Bro1 domain and PRR. The Bro-CC-PRR portion of HD-PTP, has 9 cysteines, so with the aim of measuring local structural information in the CC domain alone, alternative spin labelling methods were investigated. Use of a bromoacrylaldehyde spin label (BASL), instead of MTSL, allowed more selective labelling of surface exposed cysteines, and avoided labelling most of the cysteines in the Bro1 domain. This novel method allowed the shape of the CC domain to be monitored during STAM2 binding and showed that there is no induced conformational change.

The Morita-Baylis-Hillman (MBH) reaction is a carbon-carbon (C-C) bond forming reaction between an activated alkene and an aldehyde. It is a synthetically useful reaction due to the high atom economy and retention of multiple functional groups. Unfortunately, harsh reaction conditions are required during the MBH reaction and unpredictable product stereospecificity have hampered the widespread application of this reaction. Catalysis of the MBH reaction by enzymes has the potential to allow the reaction to occur at ambient conditions, while offering scope for improving the stereospecificity. This thesis focussed on the enzyme design of a MBH enzyme using thermostable fructose-1,6- bisphosphate (FBP) aldolases as scaffolds. These enzymes were chosen because there are common features between the aldol and MBH reactions, both making use of an enol intermediate to attack the aldehyde. In addition, aldolases typically accept a wide variety of substrates. Thermostable aldolases were selected for increased temperature tolerance creating a more desirable catalyst for industrial purposes. Thermoproteus tenax FBP aldolase (TtFBPA; WT and W144L, W144Y, K177A variants) and Staphylococcus carnosus FBP aldolase (ScFruA) were expressed and purified from E. coli. While the retro-aldol reaction catalysed by these enzymes could be easily monitored, the reverse reaction (aldol synthesis) is more difficult to quantify. Multiple methodologies for high throughput spectrophotometric detection of aldol activity were developed as a method of monitoring constructs made during directed evolution of the FBP aldolases. However, none of these proved successful in robustly determining aldol activity. The dihydroxyacetone phosphate (DHAP) mimic 1-hydroxy-3-buten-2-one phosphate (HBOP) was used to assay for MBH catalysis. While crystallographic studies with TtFBPA suggest that HBOP is bound to W144L TtFBPA in a manner compatible with the MBH reaction. NMR studies could not detect any corresponding activity. This suggests further protein engineering will be required to evolve this FBP aldolase to an MBH catalyst. In addition, our crystallographic and NMR studies with TtFBPA reveals this enzyme is capable of catalysing the formation of both FBP and tagatose-1,6-bisphosphate (TBP).Additionally, we determined the first structure of ScFruA. Interestingly, NMR experiments suggested ScFruA lacks significant control of the stereospecificity of the aldol condensation reaction and appears to catalyse the formation of FBP, TBP, xyluose-1,6- bisphosphate and psicose-1,6-bisphosphate. We conclude that while FPB aldolases could indeed provide useful scaffolds for the development of an MBH catalyst, the enzymes lack any inherent activity, necessitating the need for future creation of variants. The success of this approach will depend on the ability to screen mutant libraries for MBH product formation.

This thesis combines solid-state nuclear magnetic resonance (NMR) spectroscopy, X-ray diffraction (XRD), chemical synthesis, isotopic enrichment and density-functional theory (DFT) calculations to provide insight into a number of microporous materials. The first class of materials studied is metal-organic frameworks (MOFs), where the presence of paramagnetic ions has a range of effects on the ¹³C NMR spectra, depending on the nature of the ligand-metal interactions. For the Cu²⁺-based MOFs, HKUST-1 and STAM-1, the assignment of the NMR spectra is non-intuitive, and unambiguous assignment requires specific ¹³C labelling of the organic linker species. It is shown that ¹³C NMR spectra of these two MOFs could act as a sensitive probe of the nature of “guest” molecules bound to the Cu²⁺. The second class of materials is aluminophosphates (AlPOs). It is shown that, using a series of relatively simple linear relationships with the crystal structure, the NMR parameters calculated by DFT (with calculation times of several hours) can be predicted, often with experimentally-useful accuracy, in a matter of seconds using the DIStortion analysis COde (DISCO), which is introduced here. The ambient hydration of the AlPO, JDF-2, to AlPO-53(A) is shown to occur slowly, with incomplete hydration after ~3 months. The resulting AlPO-53(A) is disordered and some possible models for this disorder are investigated by DFT. The final class of materials is gallophosphates (GaPOs), particularly GaPO-34 and related materials. The two as-prepared forms of GaPO-34 are characterised by solid-state NMR, and their calcination investigated by TGA and in-situ powder XRD. An unusual dehydrofluorinated intermediate phase is isolated and characterised for the first time by solid-state NMR. The fully calcined material is shown to be stable under anhydrous conditions, but hydrates rapidly in air. The hydrated material is stable under ambient conditions, but collapses upon heating. Partial dehydration without collapse is achieved by gentle heating or room-temperature evacuation. The impurity phases, GaPO₄ berlinite and GaPO-X are investigated by solid-state NMR and, while the structure of GaPO-X remains unknown, much structural information is obtained.