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Abstract: Marine picocyanobacteria are essential drivers of global biogeochemical cycles, playing a crucial role in ecosystem functioning. Their significant phylogenetic and functional diversity allow them to thrive across various marine regions. However, the mechanisms underpinning their adaptation to diverse oceanic niches remain poorly understood, especially with changing nutrient landscapes driven by climate change and anthropogenic activities. A critical factor in their success are nutrient uptake systems, particularly ATP-binding cassette (ABC) transporters, which account for over half of their transport capabilities. This thesis explores the substrate specificity, and ecological roles of substrate-binding proteins (SBPs) associated with these transporters, highlighting how these systems aid niche adaptation in marine picocyanobacteria. SBPs are key to ABC transporter function, binding substrates with high specificity and affinity, facilitating their cellular uptake. This study integrates structural biology, genomics, biophysical studies, and environmental genomics to elucidate the functions and ecological roles of these proteins, providing insights into the adaptive strategies of marine picocyanobacteria. Functional characterisation of three phosphate binding protein homologs (PstS), three urea binding protein homologs (UrtA) and two glycine-betaine binding protein homologs (ProX) from Synechococcus strains inhabiting distinct oceanic niches was conducted. The study of PstS homologs in WH8102 revealed that PstS1b, unique to Synechococcus clade III strains, is the highest affinity binding protein, providing a competitive advantage in ultraoligotrophic environments. Analysis of UrtA homologs in CC9311 and WH8102 revealed UrtA from CC9311 had higher binding affinity to urea compared to both UrtA proteins from WH8102. This is intriguing as CC9311 originates from a high-nutrient mesotrophic environment, contrasting with WH8102’s habitat in a nutrient-poor oligotrophic region. Investigation of ProX homologs from MITS9220 and WH8102 revealed a functional distinction between the two proteins, with MITS9220_ProX likely behaving as a generalist solute-binding protein, while WH8102_ProX acts as a specialist binding protein for higher salinity environments. The findings of this thesis highlight the versatility and adaptability of SBP-dependent ABC transporters in marine picocyanobacteria. The study demonstrates that these transport systems modulate their substrate affinities and specificities in response to environmental conditions, facilitating niche adaptation. This research is part of a larger project aimed at characterising the full repertoire of SBPs in marine picocyanobacteria, enhancing our understanding of nutrient acquisition mechanisms in these important microorganisms. The integration of various scientific approaches in this study provides a comprehensive framework for investigating the physiological and ecological significance of ABC uptake systems, offering valuable insights into the adaptive strategies of marine microbes in the face of changing oceanic conditions.

Open Access

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Abstract: Enzymes from salt-in halophiles are stable in conditions of low water activity with applications in chiral synthesis requiring organic solvents, yet the origins of such stability remains poorly understood. Here we describe the molecular basis of the reaction mechanism and dual NADH/NADPH-specificity of D2HDH, a 2-hydroxyacid dehydrogenase from the extreme halophile Haloferax mediterranei, an organism whose proteins have to remain active in high intracellular concentrations of KCl. Halophilic adaptations of D2HDH include the expected acidic surface and a reduction in hydrophobic surface resulting from a lower lysine content. Structure determination of crystals of D2HDH grown with KCl showed that bound K+ ions were coordinated predominantly by clusters of main chain protein carbonyl ligands, with no involvement of the numerous exposed surface carboxyls. Structural comparisons identified similar sites in other halophilic proteins suggesting that the generic use of carbonyl clusters to coordinate K+ ions may also contribute in a carboxylate-independent way to the stabilisation of the folded state of the protein in its high salt environment.

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Abstract: Perlecan is an essential multi-domain, disulfide bond rich basement membrane protein. Mutations in perlecan cause Schwartz-Jampel syndrome and dyssegmental dysplasia. While there has been a large body of experimental work reported on perlecan, there is only minimal structural information available to date. There is no prior structural data for region 3 of perlecan in which some Schwartz-Jampel syndrome causing point mutations have been reported. Here, we produce constructs of the disulfide rich region 3 of perlecan along with five mutations previously reported to cause Schwatz-Jampel syndrome. Four of the mutations resulted in decreased yields and thermal stability compared to the wild-type protein. In contrast, the P1019L mutation was produced in good yields and showed higher thermal stability than the wild-type protein. The crystal structures for both the wild-type and P1019L mutation were solved. As expected, both showed laminin IV-like and laminin-type EGF-like domains, with the P1019L mutation resulting in only a minor conformational change in a loop region and no significant changes in regular secondary or tertiary structure.

Open Access

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Abstract: Voltage-dependent anion channel 1 (VDAC1) is a key protein in cellular metabolism and apoptosis. Here, we present a protocol to express and purify milligram amounts of recombinant VDAC1 in Escherichia coli. We detail steps for a fluorescence polarization-based high-throughput screening assay using NADH displacement, along with procedures for thermostability, fluorescence polarization, and X-ray crystallography. In this context, we demonstrate how 2-methyl-2,4-pentanediol (MPD), a crystallization reagent, interferes with VDAC1 small-molecule binding, hindering the detection of these ligands in the crystal.

Open Access

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Abstract: Analytical absorption corrections are employed in scaling diffraction data for highly absorbing samples, such as those used in long-wavelength crystallography, where empirical corrections pose a challenge. AnACor2.0 is an accelerated software package developed to calculate analytical absorption corrections. It accomplishes this by ray-tracing the paths of diffracted X-rays through a voxelized 3D model of the sample. Due to the computationally intensive nature of ray-tracing, the calculation of analytical absorption corrections for a given sample can be time consuming. Three experimental datasets (insulin at λ = 3.10 Å, thermolysin at λ = 3.53 Å and thaumatin at λ = 4.13 Å) were processed to investigate the effectiveness of the accelerated methods in AnACor2.0. These methods demonstrated a maximum reduction in execution time of up to 175× compared with previous methods. As a result, the absorption factor calculation for the insulin dataset can now be completed in less than 10 s. These acceleration methods combine sampling, which evaluates subsets of crystal voxels, with modifications to standard ray-tracing. The bisection method is used to find path lengths, reducing the complexity from O(n) to O(log2 n). The gridding method involves calculating a regular grid of diffraction paths and using interpolation to find an absorption correction for a specific reflection. Additionally, optimized and specifically designed CUDA implementations for NVIDIA GPUs are utilized to enhance performance. Evaluation of these methods using simulated and real datasets demonstrates that systematic sampling of the 3D model provides consistently accurate results with minimal variance across different sampling ratios. The mean difference of absorption factors from the full calculation (without sampling) is at most 2%. Additionally, the anomalous peak heights of sulfur atoms in the Fourier map show a mean difference of only 1% compared with the full calculation. This research refines and accelerates the process of analytical absorption corrections, introducing innovative sampling and computational techniques that significantly enhance efficiency while maintaining accurate results.