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Abstract: We have investigated the role of oxygen stoichiometry and structural properties in the modulation of Co valence and spin state in single-layer La2–xAxCoO4±δ (A = Sr, Ca; 0 ≤ x ≤ 1) perovskites as well as the interplay between their local structural properties and the magnetic and charge-ordering phenomena. We show the results of high angular resolution powder X-ray diffraction and Co K-edge X-ray absorption and emission spectroscopy experiments on polycrystalline and single-crystal samples. The different doping-induced changes in the Co valence and spin state by Ca (or Sr) substitution can be understood in terms of the evolving oxygen stoichiometry. For Ca doping, the interstitial oxygen excess around the La/Ca atoms in underdoped samples is rapidly lost upon increasing the Ca content. The creation of oxygen vacancies leads to the stabilization of a mixed-valence Co2.5+ independently of the Ca content. In contrast, Sr substitution leads to almost stoichiometric samples and a lower oxygen vacancy concentration, which allows higher mixed-valence states for Co up to Co2.9+. The Co mixed-valence state along the two series is fluctuating between two valence states, Co2.4+ as in La2CoO4.2 and Co2.9+ as in LaSrCoO3.91, that become periodically ordered for the charge-ordered phases around the half-doping. The X-ray emission derived spin states agree well with the Co fluctuating mixed-valence state derived from X-ray absorption spectroscopy on consideration of a distribution of high-spin Co2+ and low-spin Co3+. Furthermore, there is no quenching of the orbital contribution for the high-spin Co2+, as concluded from a comparison with macroscopic magnetization measurements. Doping holes are mainly located in the ab plane and have a strong oxygen 2p character. The major lattice distortions, which are different for Sr and Ca doping, occur along the c axis, where changes in the oxygen stoichiometry take place. Moreover, charge-order transitions are clearly shown from the anomalous increase of the c lattice parameter with an increase in the temperature above 500 K but there is no signature for a temperature-dependent spin-state transition.

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Abstract: In this study we show how non-trivial equilibrium and kinetic adsorption behaviour in a flexible zeolite can be understood through a combination of experimental characterisation and modelling. Flexible zeolites, such as those in the RHO-family, can exhibit unusual stepped isotherms in the presence of CO2, but their structural complexity makes it hard to attribute a clear mechanism. Here we present a structural and kinetic study on (Na,TEA)-ZSM-25, an extended member of the RHO-family, and show that by combining diffraction data, lattice fluid modelling and dynamic column experiments, we obtain a plausible mechanism for CO2 adsorption and transport in this material. It is evident that by using any single technique, the behaviour is too complex to be readily understood. This is to our knowledge the first study to measure and model the changing kinetics due to adsorption induced framework flexibility.

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Abstract: The β‐propeller fold is adopted by a sequentially diverse family of repeat proteins with apparent rotational symmetry. While the structure is mostly stabilised by hydrophobic interactions, an additional stabilisation is provided by hydrogen bonds between the N‐and C‐termini, which are almost invariably part of the same β‐sheet. This feature is often referred to as the “Velcro” closure. The positioning of the termini within a blade is variable and depends on the protein family. In order to investigate the influence of this location on protein structure, folding and stability, we created different circular permutants, and a circularised version, of the designer propeller protein named Pizza. This protein is perfectly symmetrical, possessing six identical repeats. While all mutants adopt the same structure, the proteins lacking the “Velcro” closure were found to be significantly less resistant to thermal and chemical denaturation. This could explain why such proteins are rarely observed in nature. Interestingly the most common “Velcro” configuration for this protein family was not the most stable among the Pizza variants tested. The circularised version shows dramatically improved stability, which could have implications for future applications.

Open Access

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Abstract: Notum inhibits Wnt signalling via enzymatic delipidation of Wnt ligands. Restoration of Wnt signalling by small molecule inhibition of Notum may be of therapeutic benefit in a number of pathologies including Alzheimer’s disease. Here we report Notum activity can be inhibited by caffeine (IC50 19 µM), but not by demethylated caffeine metabolites: paraxanthine, theobromine and theophylline. Cellular luciferase assays show Notum-suppressed Wnt3a function can be restored by caffeine with an EC50 of 46 µM. The dissociation constant (Kd) between Notum and caffeine is 85 µM as measured by surface plasmon resonance. High-resolution crystal structures of Notum complexes with caffeine and its minor metabolite theophylline show both compounds bind at the centre of the enzymatic pocket, overlapping the position of the natural substrate palmitoleic lipid, but using different binding modes. The structural information reported here may be of relevance for the design of more potent brain-accessible Notum inhibitors.

Open Access

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Abstract: A protein’s amino acid sequence determines its structural, chemical and physical properties, yet how sequence variation influences protein function is still incompletely understood. Protein fitness landscapes powerfully describe the sequence-function relationship by dividing sequence space into functional hills and valleys. This representation is often invoked yet lacks experimental evidence; the immense vastness of possible sequence space makes comprehensive high-quality datasets difficult to obtain. Laboratory directed evolution has focused on optimal utilisation of substitution libraries, however examination of functional innovation in Nature shows that short insertions and deletions (InDels) also play a key role. Beyond rare targeted studies of specific InDels, high-throughput data on fitness landscape for mutations other than substitutions are lacking entirely. In my PhD, I worked towards experimentally describing the fitness landscapes of InDels and substitutions in three systems: GFP, phosphotriesterase (PTE) and the kinase MKK1 docking domain. Towards this goal, I established two experimental assays (GFP, PTE) for deep mutational scanning and a new software toolkit, InDelScanner, for interpreting resulting data that contain InDels. With GFP, I sorted the deletions and substitution libraries into three activity fractions using FACS, then deep sequenced them with Illumina MiSeq to obtain a pilot dataset. The comparison of deletion effects between different lengths of deletions (-3, -6 and -9 bp) indicates that deletions are partially tolerated in eGFP, with tolerance improved for short deletions and in the stabilised starting point GFP8. Further interpretation of data was complicated by limited resolution in the sequencing dataset stemming from poor FACS separation, so I optimised the conditions for better sorting resolution using the mKate2 fluorescent protein as an expression reporter. In the second iteration of the activity sorting I additionally included UMIs in the plasmid design to improve the utilisation of NGS capacity. In the case of PTE, I performed proof-of-concept experiments for microfluidic droplet sorting in an integrated device with an in-line incubation line and a fluorescent sorting design. In parallel, testing of solubility and activity of random InDel variants showed that functional InDels do not necessarily suffer from a stability handicap, making InDel mutagenesis a viable strategy for gene randomisation in directed evolution. One challenge of InDel library data analysis is that InDels are not compatible with existing, substitution-focused software. Using the GFP deletions dataset, I developed the InDelScanner scripts which accurately detect, aggregate and filter insertions, deletions and substitutions. Using the scripts for composition analysis of TRIAD libraries in PTE showed these libraries are well balanced and highly diverse. Finally, I used the InDelScanner scripts to interpret a deep mutational scanning dataset that recorded the sequence preferences in the MKK1 docking domain, acting to activate ERK2. This experiment showed that the fitness landscape in this kinase pair is shaped by the activating effect of hydrophobic residues in the docking groove, as well as widespread positive epistasis. Together, the projects in this thesis demonstrate that deep mutational scanning experiments are a powerful method for exploring the sequence-function relationship in proteins, which can extend into comparison of different types of mutations as well as probing their (epistatic) interactions.