Doctoral Theses / Doktorsavhandlingar Institutionen för kemi och molekylärbiologi

Permanent URI for this collectionhttps://gupea-staging.ub.gu.se/handle/2077/29054

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Allostery at Work: Mapping, Modulating, and Monitoring PKL Function with Chemotype Diversity
(2025-08-19) Nilsson, Oscar
Understanding the processes of protein regulation and our ability to control enzyme activity in the human body lie at the core of drug development. The liver isoform of pyruvate kinase (PKL) is a metabolic enzyme crucial for energy production. Because of its metabolic role, PKL and other pyruvate kinase isoforms are interesting drug targets for metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). In this thesis, the allosteric regulation of PKL was explored using multiple approaches to expand our knowledge about this extensively regulated enzyme. Known allosteric ligands were derivatized into fluorescent reporter probes to monitor ligand engagement with the allosteric pocket of PKL. This approach centred around the direct incorporation of fluorescent dye molecules into the scaffolds of the allosteric ligands, exploiting structurally overlapping features. A set of probes containing the environment-sensitive dye 4-sulfamoyl-7-aminobenzoxadiazole (SBD) was developed and used to measure the occupancy of unlabelled ligands in indicator displacement assays. The concept was further developed by applying this design philosophy to a more potent ligand class, generating a cell-permeable fluorescent tracer. In combination with NanoBRET technology, this tracer enabled the development of a target-engagement assay capable of detecting allosteric ligand binding to PKL in living cells. This thesis also proposes a model for how the allosteric regulation of PKL works. By modulating the enzyme with compounds derived from the known drug mitapivat, minor modifications were identified that had significant effects on functional outcomes. Biophysical techniques and molecular dynamics simulations were employed to investigate a structurally similar activator/inhibitor pair, demonstrating that they differentially affect the stability of the protein-protein interfaces. Furthermore, a fragment-based drug design effort was undertaken to map the allosteric pocket and identify novel ligand scaffolds capable of allosterically inhibiting PKL. An iterative design process starting from simple fragments yielded moderate inhibitors with large structural diversity, providing valuable insights into key binding interactions and serving as a foundation for future optimization toward more potent modulators. These findings collectively deepen our understanding of PKL allosteric regulation and provide useful chemical tools for further study. They may also inform future efforts aimed at developing ligands to modulate PKL activity in a physiological context.
Mechanisms of male germ cell development: Centriole regulation, sperm tail formation, and membrane fluidity
(2025-05-16) Stojanovic, Nena
Male infertility affects ~7 % of men and arises from a complex interplay of genetic, physiological, and environmental factors. This thesis explores novel gene functions crucial for spermatogenesis, offering insights into the molecular basis of male fertility. The number and position of centrioles are strictly regulated in mitotic cells, however, centriole dynamics during male meiosis remain largely unexplored. Beyond their role in genetic segregation, centrioles in male germ cells transform into basal bodies forming sperm flagellum. The first study identifies MEICEN as a novel testis-specific regulator of centriole dynamics and sperm tail formation. MEICEN is a centriolar satellite protein, that controls CETN1 and 2 (Centrin1 and 2) distribution between centrioles and centriolar satellites, ensuring centriole stability and integrity during remodeling. Meicen knockout (KO) mice exhibit supernumerary centrioles, leading to disorganized sperm tails, and ultimately male infertility. These findings establish MEICEN as a meiosis-specific regulator of centriole width and number in spermatogenesis, shedding light on the intricate mechanisms governing male germ cell development. Impaired sperm motility presents a substantial challenge in reproductive medicine contributing to male infertility cases. The second study identifies coiled-coil domain containing protein 28 A (CCDC28A), a germ cell upregulated protein, as essential for sperm tail movement. Knockout models reveal that CCDC28A deficiency disrupts the head-tail coupling apparatus (HTCA), causing sperm tail defects and reduced motility. Transmission electron microscopy reveals disruptions at the capitulum-basal plate junction of the HTCA in CCDC28A mutants, resulting in head bending within the neck region, often accompanied by midpiece thickening. These findings establish CCDC28A as a critical factor in male fertility, contributing to sperm tail morphogenesis through HTCA formation. Membrane composition is vital for male germ cell development. The third study identifies AdipoR2 as a key regulator of the meiosis-specific lipidome. AdipoR2 upregulates the expression of the fatty acid elongase ELOVL2, both transcriptionally and post-transcriptionally, facilitating synthesis of very long chain polyunsaturated fatty acids (VLC-PUFA). AdipoR2 knockout testes show VLC-PUFA depletion, palmitic acid accumulation, resulting in cellular membrane stiffening and nuclear envelope invagination. This condition disrupts the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Additionally, the stiffened membrane impairs intercellular bridge formation and the germ cell syncytium, disrupting the orderly arrangement of cell types within the seminiferous tubules. These findings highlight the AdipoR2-ELOVL2 pathway’s crucial role in maintaining membrane fluidity for proper chromosome dynamics during meiosis.
The PAQR-2 and HIF-1 pathways are physiologically essential for unsaturated fatty acid homeostasis in C. elegans
(2025-05-05) Kaper, Delaney
Since the 1930s, scientists have known that certain unsaturated fatty acids are essential in the human diet and act as precursors for further fatty acid synthesis. Unlike humans, the nematode C. elegans can de novo synthesize omega-3 and omega-6 polyunsaturated fatty acids, making it a useful species for studying fatty acid function. Fatty acids form the tails of phospholipids, the main component of cellular membranes, and modulating the identity of these tail fatty acids has important implications for membrane homeostasis. Saturated fatty acids have membrane rigidifying effects, while unsaturated fatty acids fluidize membranes; thus, a proper balance between the two is crucial for several membrane properties. One way by which cells achieve membrane homeostasis is through the PAQR-2 membrane fluidity regulator that responds to membrane rigidification by increasing fatty acid desaturation and incorporation of unsaturated fatty acids into phospholipids. In the first part of this thesis, the effect of excessively rigid and excessively fluid membranes on several cellular and physiological traits were studied in C. elegans and revealed that deviation from optimal membrane composition in either direction is deleterious. Next, we further characterized the molecular basis of PAQR-2 activity, revealing that PAQR-2 recruits a complex containing enzymes important for fatty acid elongation and for channeling of unsaturated fatty acids into phospholipids. In the final part of this thesis, a forward genetics screen led to the discovery that the HIF-1 pathway can potentiate desaturase activity in a C. elegans mutant that is almost wholly devoid of polyunsaturated fatty acids. We conclude that the PAQR-2 and HIF-1 pathways are regulators of unsaturated fatty acid homeostasis essential for the physiological health of C. elegans.
Unconventional Protein Functions through Liquid-Liquid Phase Separation in Stress Responses and Aging
(2025-04-30) Gao, Yuan
Just as human society relies on individuals specializing in distinct roles to ensure its proper functioning, a cell depends on organelles to coordinate essential biological processes. Traditionally, organelles have been defined as membrane-bound structures that establish distinct biochemical microenvironments through physical compartmentalization. However, in recent decades, the discovery and growing recognition of membraneless organelles, such as stress granules (SGs) and the pre-autophagosomal structure (PAS), have reshaped our understanding of intracellular organization and biological processes. These dynamic, non-membranous biomolecular condensates maintain their functional integrity through liquid-liquid phase separation (LLPS), a specialized phase transition in which a homogeneous solution spontaneously demixes into two immiscible liquid phases: a dense phase and a dilute phase. This biophysical process is driven by weak, multivalent interactions among macromolecules, such as proteins and nucleic acids. LLPS enables the reversible formation of spatially and functionally distinct compartments, allowing cells to dynamically regulate essential processes such as stress responses and aging, all without the need for lipid membranes. LLPS has thus opened a new dimension for scientific inquiry, enabling researchers to both observe and influence life’s fundamental processes. This thesis provides new perspectives on traditionally well-studied proteins through the lens of LLPS, focusing on stress responses and aging. Specifically, it uncovers new roles for Lsm7 and thioredoxin reductase 1 (Trr1) in stress responses and aging, respectively, mediated through LLPS. For Lsm7, the mechanism of SG initiation via its phase separation, coupled with a conserved signaling pathway identified in this study, provides new insights into SG formation and their involvement in SG-associated human diseases. For Trr1, our findings reveal an unexpected connection between the autophagy process and the antioxidant system, with Trr1’s phase separation playing a key role in initiating ER-phagy during aging. These discoveries offer fresh perspectives on aging, age-related diseases, and the regulation of autophagy. Altogether, these findings offer new insights into fundamental biological processes and lay the groundwork for future research aimed at leveraging LLPS to better understand and potentially manipulate life itself.
Glycans at the Core: Computational-Experimental Investigations of Complex Carbohydrates
(2025-04-28) Lundstrøm, Jon
Next to nucleic acids and proteins, glycans represent a third class of biological sequence, composed of monosaccharides assembled into complex and often branched structures. Glycans modify various biological molecules, most commonly proteins and lipids, and engage in a diverse range of functions, primarily through interactions with specific glycan-binding proteins known as lectins. Throughout this thesis, multiple aspects of glycans, lectins, and glycosylation mechanisms are explored. The remarkable diversity of both glycans and lectins imposes experimental challenges for characterizing the binding specificity of newly discovered lectins. To address this, we introduce LectinOracle, a deep learning model that combines transformer-based protein representations with graph convolutional neural networks for glycans, enabling accurate prediction of lectin-glycan interactions. In parallel, we employ an extensive array of experimental techniques to thoroughly characterize a newly identified plant lectin from Cucumis melo, investigating its glycan-binding specificity, binding kinetics, and solving the structure of the N-terminal domain in complex with glycan ligands. Finally, we challenge a long-standing paradigm in the field of glycobiology: that O-GalNAc glycosylation is restricted to proteins destined for secretion. In this study, we conclusively demonstrate that nuclear proteins can be modified with extended O-GalNAc-type glycans through a mechanism that depends on Golgi-resident biosynthetic enzymes. Our findings suggest the existence of a novel pathway in which nuclear proteins are actively shuttled to and from the secretory pathway. Altogether, the work presented in this thesis contributes significantly to the advancement of key areas in glycobiology, spanning computational modeling, structural biology, and fundamental insights into glycosylation mechanisms.
AI-based Spectra Processing and Analysis for NMR
(2025-04-11) Jahangiri, Amir; Jahangiri, Amir
Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful analytical technique for obtaining atomic-level information across various scientific fields. However, direct interpretation of raw NMR data is impractical due to its complexity. While traditional signal processing methods are widely used, they face limitations in handling advanced tasks. Consequently, advanced computational approaches are required to optimize spectral reconstruction and improve analytical precision. Artificial Intelligence (AI), particularly deep learning, has demonstrated significant potential in addressing these challenges. This thesis explores AI-based signal processing in NMR spectroscopy, focusing on spectral reconstruction, resolution enhancement, and quality assessment. The first study introduces Low-Rank Decoupling (LRD), a method leveraging prior knowledge of J-coupling for homonuclear virtual decoupling. LRD enhances spectral resolution while maintaining sensitivity and minimizing artifacts, outperforming conventional decoupling approaches. The second study presents Magnetic Resonance processing with Artificial intelligence (MR-Ai) as an alternative to conventional nonlinear NMR processing. A 1D WaveNet-based NMR Network (1D WNN) is developed to address non-uniformly sampled (NUS) reconstruction as a pattern recognition problem, surpassing traditional methods in stability and accuracy. MR-Ai is also adapted for virtual decoupling, demonstrating robustness against variations in J-coupling values. The third study extends MR-Ai beyond traditional NMR processing. A 2D WNN architecture is designed to reconstruct Echo (or Anti-Echo) spectra, correcting phase-twist distortions in incomplete quadrature detection. Additionally, MR-Ai introduces a reference-free evaluation metric, estimating uncertainty in spectral reconstructions for direct quality assessment without external references. The fourth study introduces Peak Probability Presentation (P3), a novel AI-driven spectral visualization technique. To achieve this, an nD WNN architecture is developed for pattern recognition in nD NMR spectra. Unlike traditional intensity-based representations, P3 assigns a probability score to each spectral point, providing artifact-free, ultimate-resolution spectral interpretation. The results demonstrate P3’s superior performance in peak detection, spectral clarity, and noise differentiation. Additionally, P3 is integrated into Targeted Acquisition (TA) to develop a quantitative spectrum quality score for real-time spectral quality assessment and optimized data acquisition. Overall, this thesis demonstrates that AI-driven NMR processing not only enhances existing methods but also introduces fundamentally new approaches to spectral reconstruction, resolution enhancement, and quality assessment. As AI evolves, its integration into NMR workflows is expected to revolutionize the field, making high-quality spectral data more accessible, interpretable, and efficient.
Development of Carbonyl Functionalization Methods Towards Sustainable Synthetic Strategies
(2025-04-10) Bacaicoa García, Sara
Carbonyl compounds represent a fundamental and versatile class of organic mole-cules that play a central role in both synthesis and biology. In synthetic chemistry, traditional methods for carbonyl functionalization generally involve the use of haz-ardous reagents or the production of large amounts of waste all of which contribute to environmental problems. Consequently, the development of sustainable strate-gies for carbonyl functionalization is of utmost importance. This thesis explores two strategies for carbonyl functionalization: aerobic oxidative N-heterocyclic carbene (NHC) catalysis and the visible-light induced Zimmerman-O’Connell-Griffin (ZOG) rearrangement. The objective is to develop efficient carbon-yl functionalization methods that contribute to the advancement of sustainable chemistry through the investigation of these strategies. Aerobic oxidative NHC catalysis is a synthetic strategy for the direct conversion of aldehydes to activated carbonyl species known as acyl azolium intermediates. These intermediates are pivotal for the synthesis of various substances, including highly substituted benzene derivatives. Within this approach, aerial oxygen is the terminal oxidant, a desirable choice due to its cost-effectiveness and the production of water as the sole byproduct. However, the low reactivity of oxygen necessitates the development of a specialized catalytic system. By incorporating electron transfer mediators to the reactions (ETMs), mild aerobic oxidation conditions can be achieved. These ETM-assisted, aerobic NHC-catalyzed transformations demonstrate high selectivity and reduced waste generation. The ZOG rearrangement represents a strategy for the generation ketenes another form of an activated carbonyl species. In this reaction we use visible light to trigger a rearrangement of a photosensitive substrate leading to in situ formation of the ketene. Ketenes are highly reactive intermediates capable of engaging with a diverse array of substrates, thereby enabling broad synthetic utility. Remarkably, no addi-tional reagents or catalysts are needed to trigger this photochemical reaction. With only the reactants and visible light, a broad range of products can be obtained under mild conditions and generating a minimal amount of waste.
Advancing the use of serial crystallography in drug discovery
(2025-04-02) Dunge, Andreas
This thesis explores the application of serial synchrotron crystallography (SSX) in drug discovery, focusing on the structural studies of soluble epoxide hydrolase (sEH) and cytochrome P450 3A4 (CYP3A4). Utilizing SSX, a relatively novel technique that collects data from a large number of small crystals at room temperature (RT), this research aims to investigate the structural dynamics and ligand interactions of these enzymatic targets. A workflow is developed that enables the transition from macrocrystals to microcrystals, and this study incorporates ligand soaking to explore protein-ligand interactions in their near-native states. We successfully established a method for obtaining microcrystals and soaking compounds, which facilitated a comprehensive fragment study. This study led to the identification of 40 active site-binding fragments out of 384 tested for sEH, showcasing the method's effectiveness. Additionally, the RT structures revealed conformational nuances of F497 in sEH, with inward movement observed in response to potent inhibitors. For CYP3A4, an RT structure provided valuable comparisons to cryogenic analyses, emphasizing structural differences. The thesis highlights SSX's capability to capture structural information that may be obscured under traditional cryogenic conditions, demonstrating its utility alongside conventional cryogenic macromolecular crystallography to enhance understanding of protein-ligand complexes. Additionally, the work addresses the logistical and technical challenges inherent in SSX and proposes strategies to optimize experimental conditions effectively. By focusing on these targeted studies, this research highlights the capability of SSX to advance drug discovery efforts. SSX offers a novel approach for detailed structural analysis of proteins, enabling the identification of interactions which could be hidden at cryogenic temperature which can be useful for designing effective therapeutic agents.
Structural and functional insights into ba3-type cytochrome c oxidase using X-ray crystallography and In Crystallo spectroscopy
(2025-03-17) Kabbinale, Arpitha
Cellular respiration extracts the energy stored in glucose through a series of electron and proton transfer reactions so as to produce ATP, the primary energy currency of living cells. A key step in this process is oxidative phosphorylation, whereby electrons pass through an electron transport chain while generating a proton concentration gradient across an energy-transducing biological membrane. Cytochrome c oxidase (CcO) is the terminal enzyme of this electron transport chain and facilitates the final electron transfer to molecular oxygen, reducing it to water while simultaneously driving proton translocation across the cellular membrane. Understanding CcO structural and functional properties, including electron and proton transfer and oxygen binding dynamics, is crucial for unraveling the molecular mechanisms of energy conversion. This knowledge provides insights into bioenergetics, metabolic regulation, and potentially has biomedical applications since several diseases are linked to the dysfunction of CcO. This PhD thesis focuses upon the ba3-type CcO from Thermus thermophilus. To gain functional insights into CcO we used X-ray crystallography, a structural biology technique that provides high-resolution three-dimensional snapshots of the enzyme. Since static X-ray structures do not fully capture the enzyme’s dynamic nature, we employed time-resolved serial X-ray crystallography to visualize structural changes in the ba3-type CcO after dioxygen is reduced to water. Additionally, UV-Vis spectroscopy was used to monitor the redox state of the enzyme, allowing greater control over diverse experiments, including during X-ray-induced photoreduction studies. As this work proceeded, numerous experimental limitations had to be addressed, including difficulties in working with photocaged oxygen as a tool for reaction initiation, maintaining an aerobic conditions to ensure a fully reduced enzyme, and the reducing effects of photoelectron. Nevertheless, when taken together our structural results build a consistent picture of conformational changes associated with the ba3-type CcO, as the enzyme is reduced by photoelectron, as the enzyme slowly turns over in the presence of residual dioxygen, as the enzyme rapidly turns over after UV laser pulse releases dioxygen from photocaged oxygen, and as the pH changes. These structural results enhance our understanding of the mechanism by which the enzyme performs its catalysis and translocates protons. These findings are discussed in detail within this thesis.
Boron Directed RegioselectivAromatic Ortho Functionalizations
(2025-03-14) Shinde, Ganesh
Selectivity is crucial in modern organic synthesis, as it allows for precise molecular modifications while minimizing undesired side reactions. Controlling selectivity is essential for improving reaction yields, reducing purification steps, and enhancing overall synthetic efficiency. This is particularly important in synthetic methodologies, where precise control over regioselectivity is essential for constructing complex molecules efficiently and reliably. In this work, we have developed a series of selective deborylative transformations that leverage the unique reactivity of dibromoboracycles to achieve regioselective C–halogen, C– aryl, and C–benzyl bond formations in 2–aryl–N–heteroarenes, aldehydes, N–aryl amides and ureas. By integrating nitrogen and carbonyl-directed borylation with subsequent functionalization, we have introduced efficient, metal-free, and scalable methodologies that address long-standing challenges in site-selective C–H functionalization. Our oxidative halodeboronation strategy provides a direct and regioselective approach to ortho-halogenation, eliminating the need for transition-metal catalysts while ensuring high functional group tolerance. This protocol expands the synthetic utility of boron, enabling the precise installation of halogens in 2–aryl–N–heteroarenes and N–aryl amides under mild conditions. Furthermore, we have demonstrated that dibromoboracycles can be directly employed in ortho-arylation and ortho-benzylation, bypassing the conventional BBr₂-to-BPin conversion. This unique approach facilitates the efficient synthesis of biaryl amides, diarylmethane amides, and dibenzoazepines, unlocking new avenues for selective C(sp²)–C(sp²) and C(sp²)– C(sp³) bond formation. Additionally, our method enables one-pot diagonal diarylation, allowing streamlined access to complex molecular architectures such as tetraarylbenzenediamines and N–doped fulminenes. Finally, we introduce a scalable and chromatography-free synthesis of aryl-difluoroborane (Ar–BF₂) compounds, which exhibit enhanced stability and reactivity. These Ar–BF₂ species serve as highly versatile intermediates for late-stage functionalization, enabling diverse transformations, including radioiodination, halogenation, hydroxylation, azidation, and Suzuki-Miyaura cross-coupling. Their broad applicability highlights their potential as powerful tools in pharmaceutical synthesis and beyond. Overall, this work represents a significant advancement in boron-mediated functionalization, establishing a unified platform for regioselective C–H activation and cross-coupling reactions. By harnessing the intrinsic reactivity of dibromoboracycles, we provide highly selective, operationally simple, and scalable strategies that eliminate unnecessary synthetic steps, paving the way for future developments in boron-directed transformations and late-stage functionalization.
PCET-Mediated Photocatalytic Radical Generation from Alcohols: Mechanistic Insights and Synthetic Applications
(2025-03-03) Patehebieke, Yeersen
Visible-light photoredox catalysis has emerged as a powerful tool for forging carbon–carbon bonds under mild, sustainable conditions. This thesis presents new methodologies for the direct generation and synthetic exploitation of alkyl radicals from unfunctionalized aliphatic alcohols—traditionally challenging precursors due to their high O–H bond dissociation energy. By harnessing both metal-free and synergistic dual catalytic approaches, this work demonstrates that readily accessible alcohols can be transformed into versatile C-centered radicals, enabling efficient C(sp³)–C(sp³) and C(sp³)–C(sp²) bond formations. In the first part of this thesis, a metal-free photocatalytic protocol is developed for the alkylation of electron-deficient alkenes. Mild reaction conditions, relying on aliphatic secondary or tertiary alcohols as redox auxiliaries, deliver structurally diverse alkylated products with broad substrate scope. Mechanistic investigations, supported by density functional theory (DFT) calculations and steady-state as well as nanosecond transient absorption spectroscopy, reveal a complex manifold of competing pathways—most notably a base-independent, non-proton-coupled electron transfer (non-PCET) fragmentation and a concerted PCET pathway. These findings challenge the previously accepted view that stepwise PCET exclusively drives alkoxy radical formation, instead showing that multiple routes can operate in parallel. The second part of the thesis expands this reactivity to aryl halides via a dual photoredox–nickel catalytic platform. Here, alkyl radicals are generated through concerted PCET-mediated β-scission of aliphatic alcohols, facilitating the formation of C(sp³)–C(sp²) bonds under exceptionally mild conditions. Femtosecond transient absorption experiments confirm that PCET outcompetes direct fragmentation in this system, delivering high chemoselectivity and functional-group tolerance across a wide range of substrates, including challenging tertiary alcohols. Late-stage functionalization of bioactive molecules further underscores the synthetic utility of these methods. Finally, in the third part, the developed PCET-mediated generation of C-centred radicals are extended to monosaccharides, which are structurally more complex compounds. In this work, we also broadened the chemistry to allow for fragmentation-initiated hydrogenation, which presents a novel synthetic entry to this compound class. Collectively, the results presented in this thesis establish aliphatic alcohols as robust, readily available radical precursors for visible-light-driven bond formations. Beyond providing valuable synthetic transformations, the mechanistic insights gleaned—particularly regarding the interplay of non-PCET and PCET pathways—offer a deeper understanding of radical generation from alcohols. These discoveries pave the way for more efficient, sustainable, and versatile photoredox methodologies that harness readily accessible feedstocks in complex molecule construction.
Structural studies of the photosynthetic reaction center of Blastochloris viridis using XFEL and Synchrotron sources
(2025-01-28) Banacore, Analia
Life on Earth as we know it has been possible because of photosynthesis, a process by which hydrocarbons and chemical energy are generated using the energy extracted from sunlight. As with any other biological process, there is a protein responsible and it is called the reaction center. It is found in various photosynthetic organisms, including bacteria, algae, and plants. A photosynthetic reaction center has a conserved structure and function, where light absorption triggers an electron transfer process leading to energy production. This thesis is focused on the bacterial reaction center from Blastochloris viridis, analogous to photosystem II in plants. This study is focused on characterization of protein structural changes upon light absorption. For decades, X-ray crystallography has been a key method for determining protein atomic structures. X-ray free-electron lasers have facilitated new approaches, called serial femtosecond X-ray crystallography (SFX), which enable new experimental possibilities with smaller crystals. This approach requires new crystal preparation methods for specialized delivery systems. In this thesis, a technique for growing membrane protein micro-crystals using lipidic cubic phase crystallization and adding crystal seeds is presented. With this approach, a 2.3 Å resolution SFX structure of the reaction center was obtained. Detergent grown micro-crystals were used to validate a fixed target device, yielding a 3.3 Å structure from data collected using serial synchrotron X-ray crystallography. LCP grown micro-crystals were used in time resolved SFX studies, where structural movements of the co-factors in the protein on a sub-picosecond timescale after photon absorption were assessed. Further, time resolved SFX results show multi-photon absorption induces a thermally driven expansion of the protein structure surrounding the co-factors. These findings contribute to a sequence of studies that highlight the intricate interplay between light absorption and structural changes within the protein.
Time-Resolved X-ray Crystallography and Quantum Chemical Calculations of the Proton Pumping Mechanism in Cytochrome c Oxidase
(2024-12-13) Johannesson, Jonatan
Aerobic respiration and photosynthesis are arguably the two most essential processes for life on earth as known to us. These two processes occur on an immense scale every day and the electron transport chain of aerobic organisms effectively utilizes the energy available from the exergonic reduction of the high potential electron acceptor oxygen. In order to couple the exergonic reaction to energy available for the cell, an electrochemical gradient is generated across the cell membrane which can be used for energy demanding process. The last step of the electron transport chain's generation of the gradient is performed by the enzyme cytochrome c oxidase which catalyses the formation of water and makes use of the free energy by the translocation of protons across the membrane against this gradient. How the enzyme couples this proton translocation to the exergonic redox events of the catalytic mechanism is currently not known in detail, although many of the requirements of the event have been elucidated. Insights of the mechanistic details can help not only to understand this vital process, but also essential aspects of energy transduction efficiency with potential future applicability. Time-Resolved Serial Femtosecond X-ray crystallography enables the 3D visualization of protein structures by their illumination in the crystalline form. By collection of diffraction images at certain time delays in relation to the reaction initiation, structural information of the catalytic mechanism can be retrieved. There are many technical barriers to this however, and those are the topic of the first half of this thesis where overcoming them is the main hurdle. Novel structural information is successfully retrieved in three different papers while one paper investigates and optimizes the chemical barriers to enabling the experiment to as great extent as possible. In the last part of the thesis additional insights of the mechanistic details of the catalytic cycle are gained by applying computational chemistry to study processes within the active site of the enzyme. These methods can give even further detailed information for where temporal and spatial resolution limit the experimental techniques and the insights gained from these are presented in the last two papers.
Design and development of bioresponsive pseudoglucosinolates, non-cytotoxic coumarins with antibiofilm properties and studies towards the synthesis of [13]cytochalasans
(2024-12-10) Chakrabarti, Aishi
The main aim of this thesis was the development of multifunctional pseudoglucosinolates (psGSLs) and artificial multivalent glucosinolates (mv-GSLs) as ITC releasing prodrugs. They were utilized as imaging, enzyme labelling and controlled drug delivery tools. Beyond that it also dealt with the development of 6,7-dihydroxycoumarins as non-cytotoxic antibiofilm agents. Lastly, studies towards the total synthesis of [13]cytochalasans (chaetoglobosin B and D) were conducted. The first chapter focused on the exploration and expansion of the utility of glucosinolate (GSL) inspired compounds for diverse biochemical and therapeutic applications. The first project within this chapter involved the development of enzyme-responsive pseudoglucosinolates (psGSLs) as prodrugs for the controlled release of isothiocyanates (ITCs). Incorporating nitroreductase (NTR) and azoreductase (AzoR)-responsive masking groups and a polyethylene glycol (PEG) chain enhanced solubility and functionality of the probes. Further attachment of fluorophores enabled imaging of enzymatic activation. Synthesized via multistep routes, psGSLs demonstrated successful ITC release upon enzymatic cleavage, confirmed by LC-MS and covalent binding of the ITCs were confirmed by SDS-PAGE analysis. In vivo validation in C. elegans further highlighted the covalent binding of ITCs. These results showcase the potential of psGSLs as flexible tools for imaging, and enzyme dependent labelling of biomolecules. The second project within this chapter investigated the design and synthesis of psGSL-based antimicrobial prodrugs for controlled drug release upon enzymatic activation. Antibiotics were attached to the psGSL thiohydroximate unit via carbamates, replacing the O-sulfonate group. LC-MS analysis showed release of antibiotics upon activation of the antimicrobial prodrugs by nitroreductase (NTR). Although the conversions were slow and incomplete, proof-of-principle for the activity of these probes was provided. Photo-cleavable psGSLs were also developed to bypass enzymatic incompatibility by incorporating o-nitrobenzyl moiety for UV-activated drug release at 365 nm. Additionally, azide groups in the psGSL structure allowed for fluorophore attachment or immobilization on surfaces, enabling potential applications as imaging agents or antimicrobial coatings. The third project within this chapter is introducing the first artificial multivalent glucosinolates (mv-GSLs). Compounds which bear more than one glycosidic thiohydroximate-O-sulfonate moiety, potentially amplifying the bioactivities observed for natural monovalent antetypes. Multistep synthesis and biochemical evaluation of mv-GSLs, featuring aliphatic and aromatic cores, were conducted. Myrosinase from Sinapis alba efficiently released multivalent isothiocyanates (mv-ITCs) for selected substrates, while specifier protein AtNSP3 facilitated only partial conversion to nitriles. Further optimization of the structures and developing a bigger panel of compounds could facilitate better conversion efficiency with myrosinase. The second chapter focused on the synthesis and bio evaluation of novel 6,7-dihydroxycoumarin-5-carboxylates (DHCou and 4-MeDHCou) as non-cytotoxic antibiofilm agents. These compounds were synthesized through multistep route in 11% and 8% overall yield respectively. Biological evaluation revealed significant retention of biofilm inhibition activity, with 4-MeDHCou demonstrating efficacy against Staphylococcus aureus and Candida albicans, while DHCou selectively inhibited C. albicans biofilms. Importantly, both derivatives exhibited no cytotoxicity against mammalian cell lines, unlike their parent compounds, esculetin (II-2) and 4-methylesculetin (II-29). Therefore, incorporation of a carboxylate moiety at the C5 position successfully produced compounds with reduced cytotoxicity and retained biofilm activity. These results establish a basis for further exploration of dihydroxycoumarin derivatives as antibiofilm agents, with potential applications in developing artificial siderophores or antimicrobial drug conjugates. The third chapter focused on the studies towards the total synthesis of [13]cytochalasans chaetoglobosin B and D. Efforts toward the total synthesis of chaetoglobosin D were centered on the construction of its isoindolone core (III-148) and macrocyclic fragment (III-149). Initial strategies faced significant challenges, including unsuccessful attempts to form the Wittig salt (III-145) during the construction of the macrocyclic fragment as well as decomposition of intermediates (III-122, III-128a, III-128b) during Diels-Alder reactions for construction of the isoindolone core. These drawbacks necessitated a shift to the synthesis of chaetoglobosin B, employing a revised strategy. Key precursors, diene III-163 and dienophile III-171, were synthesized via multistep routes, though the processes were hindered by low yields and issues with product selectivity, such as inseparable E/Z-isomer mixtures and material loss during intermediate preparation. While the assembly of the isoindolone core (III-173) and macrocyclic fragment (III-180) was not completed, a new synthetic pathway was proposed, incorporating intramolecular Diels-Alder reactions and optimized strategies for coupling and protecting intermediates. Future efforts could refine these routes, improve yields, and enable the total synthesis of chaetoglobosin B and related compounds. This work lays a foundation for advancing synthetic methodologies toward complex natural products.
Computational tools for the analysis of time-resolved serial X-ray crystallography data.
(2024-11-22) Vallejos, Adams
Many transmembrane proteins play an essential role by facilitating energy transduction in various biological systems. Whether powered by light, as in bacteriorhodopsin or photosynthetic reaction centres, or by oxygen reduction, as in cytochrome c oxidase, these proteins enable directional proton transport across the membrane. For bacteriorhodopsin and cytochrome c oxidase, this is achieved through structural changes that alternate the accessibility of their active sites to either side of the membrane, and for photosynthetic reaction centres through redox reactions coupled to electron movements across the membrane. These mechanisms allow these proteins to establish electrochemical gradients crucial for cellular functions including ATP synthesis, photosynthesis, and cellular respiration. In this research, we use time-resolved serial crystallography to resolve the three-dimensional structures of these three molecular systems and investigate the time-dependent conformational changes they undergo after light excitation. We developed and employed a variety of computational methods to characterize protein dynamics using statistics. We investigate the role of occupancy in popular structural reﬁnement strategies, namely partial-occupancy reﬁnement or reﬁnement against extrapolated data to assess the structural shifts associated with light activation events. In addition, we integrate these structural reﬁnement strategies with resampling methods to estimate coordinate uncertainties, ensuring robust and reliable interpretation of these transient states. To further analyze structural dynamics, we use difference Fourier maps, which minimize phase bias and enhance our ability to resolve subtle but functionally signiﬁcant changes, and employ singular value decomposition to analyze them in two ways: First, as a tool for enhancing the signal-to-noise of these maps, and second, in combination with resampled maps to improve our conﬁdence while assigning time-dependent difference density features. We also examine residual densities for transient water molecules by comparing difference Fourier and Polder omit maps, allowing us to characterize transient hydration states involved in the energy transduction mechanisms. Together, these methods provide a comprehensive framework for elucidating the precise structural dynamics in these proteins, advancing our understanding of how energy transduction is coordinated at the molecular level. These approaches offer valuable insights into the mechanisms underlying key biological processes, from photosynthetic energy capture to cellular respiration.
A tale of two songs: oligomeric and monomeric functions of the molecular chaperone CCT
(2024-11-07) Córdoba Beldad, Carmen Maria; Córdoba Beldad, Carmen Maria
The chaperonin-containing tailless complex polypeptide 1 (CCT) is a 1 MDa barrel shaped molecular chaperone present in the cytoplasm of all eukaryotes. Eight different subunits, located in a fixed position, form CCT, which is mostly studied as the folding machinery of the cytoskeletal proteins actin and tubulin. Roles beyond folding have been reported for the CCT oligomer and together with the increasing evidence of monomeric CCT subunit functions, have elevated the importance of studying the chaperone CCT. Here, we show that the transcription factor STAT3, a previously reported oligomeric binding partner of CCT, does not behave as an obligate folding substate but rather is sequestered by CCT. To expand our knowledge of the monomeric roles of the CCT subunits, we have studied the interaction between CCTδ and p150Glued, a component of the dynactin complex involved in crucial biological processes such as mitosis. We show that monomeric CCTδ is required for the correct localisation of p150Glued at spindle poles and for accurate chromosome segregation. Furthermore, we explore interactions between two other CCT subunits and two regulatory components of mitosis, the centromeric protein Mis18BP1 and the outer kinetochore component KNL1. Taken together the work in this thesis extends the understanding of both oligomeric and monomeric functions of CCT beyond folding.
Emerging roles and functional mechanisms of dynein light chain DYNLRB1 and DYNLRB2
(2024-10-28) shuwen, he
Dyneins are a class of molecular ATPase motors that generate force and movement on microtubules in a wealth of biological processes, including organelle distribution, cell division, ciliary beating, and flagella motility, and intracellular transport. DYNLRB1 and DYNLRB2 are the light chains subunits identified both in cytoplasmic dynein and axonemal dynein, but how these two light chains contribute to dynein function remains poorly understood. In our research, DYNLRB2 was found to be specifically upregulated in mouse testis and DYNLRB2 depletion in meiotic spermatogenesis resulted in frequent metaphase arrest with the defects in bipolar spindle formation, spindle assembly checkpoint regulation, chromosome alignment, spindle organization and orientation. DYNLRB2 maintains male meiotic spindle bipolarity by preventing pericentriolar material (PCM) fragmentation through targeting nuclear mitotic apparatus protein (NUMA) to the spindle poles and suppressing premature centriole disengagement. Emerging roles of DYNLRB2 were characterized from ciliogenesis to fertility. DYNLRB2 deficiency caused a series of ciliopathies both in female and male, as well as dysfunctional spermiogenesis with abnormal manchette removal, thereby contributing to male infertility, while DYNLRB2 KO female mice were fertile with normal follicle development and ovulation. In contrast, the mitotic DYNLRB1 regulated bipolar spindle formation by targeting NUMA and suppressing centriole overduplication in mitotic cells. DYNLRB1 KD phenotypes are rectified by ectopic overexpression of DYNLRB2, supporting the notion that DYNLRB1 and DYNLRB2 have interchangeable roles in mitosis. Correspondingly, downregulation of DYNLRB1 during mouse oocyte meiosis by siRNA injection resulted in several defects on decrease of germinal vesicle breakdown rate, spindle organization, formation of actin cap and cortical granule-free domain (CGFD) and reduced polar body extrusion rate with abnormal cytokinesis. In summary, the results defined the distinct roles and functional mechanisms of DYNLRB1 and DYNLRB2.
Toward the synthesis and characterization of bioactive peptides: Engineering of transmembrane peptide conjugates for targeted drug delivery
(2024-10-09) Abujubara, Helal
Under det senaste decenniet har intresset för terapeutiska peptider inom läkemedelsindustrin växt stort. Idag finns det upp åt 60 olika peptider som är godkända och används globalt på kliniker. Detta tack vare nya produktions-, modifierings- och analytiska teknologier som förändrat status-quo i upptäckten av peptidterapier. Peptider har producerats med både kemiska och biologiska metoder, tillsammans med nya strukturella modifieringar och leveransstrategier, som har hjälpt till att förbättra peptiders bristande farmakokinetik och har möjliggjort fortsatta framsteg inom just detta område. Denna avhandling fokuserar på att utveckla nya modifierade peptider som täcker flera terapeutiska områden. Resultaten som uppnåddes i artikel I rapporterar syntes och karakterisering av hydrofoba transmembrana (TM) peptider, med en sekvens som motsvarar TM-domänet för den humana epidermala tillväxtfaktorreceptorn 2 (ErbB2), för att hämma cancercelltillväxt. Dessutom introducerar artikeln konstruktionen av två nya ytaktiva joniska flytande nanobärare för att leverera TM-peptiderna som en metod för att förbättra deras anticanceraktivitet. Denna synergiska anticanceraktiviteten etablerar en solid plattform för framtida design av mer effektiva terapeutiska modaliteter med minskad toxicitet för hälsosamma celler. Artikel II beskriver en effektiv syntetisk strategi som optimerar pseudoproliner för syntes av holinpeptidvarianter som är 44 till 68 aminosyror långa. Holinerna är små hydrofoba proteiner som produceras av bakteriofager. Holinpeptiderna kan aggregera och bilda hål i cellmembranet som hjälper till vid klyvningen av den bakteriella värdcellens cellvägg. Platsspecifika konjugationsmetoder är av stor betydelse för upptäckten av nya läkemedel. Artikel III introducerar så kallade ionic liquids, eller jonvätskor, som bas för konjugeringsmetoder för funktionalisering av tiol-föreningar. Metoden använder jonvätskor som lösningsmedel och reagens för konjugeringsreaktionen. Denna studie lägger grunden för ytterligare undersökningar av användningen av aktiva jonvätskor för att modifiera, märka eller konjugera större och mer komplexa terapeutiska modaliteter såsom proteiner och antikroppar. Det bakteriella transpeptidaset Sortase A (SrtA) är en kritisk virulensfaktor, som det finns rikt av i grampositiva bakterier, av vilka många är patogena. Genom att rikta in sig på virulensfaktorer kan effektiv behandling uppnås, utan att inducera antibiotikaresistens. Artikel IV, rapporterar flera lovande peptidomimetiska hämmare av SrtA som är analyserade in vitro med IC50-värden under 200 μM. Några av dem har potential att gå vidare till ytterligare klinisk avancering.
The Impact of Arsenic on Protein Homeostasis and Aggregation in Saccharomyces cerevisiae
(2024-09-30) Lorentzon, Emma
Arsenic and cadmium are two toxic heavy metals that occur naturally in bedrock. Arsenic is found in high concentrations in certain areas and can contaminate groundwater, leading to exposure through drinking water and crop irrigation for the local population. Cadmium is primarily dispersed in the environment with fertilizers and as a byproduct of the electronics industry, and it is absorbed by the body through food and cigarette smoke. Long-term exposure to these heavy metals is associated with cardiovascular diseases, cancer, diabetes and neurodegenerative diseases. One of the primary reasons for their toxicity is their ability to interact with proteins in cells, essential for normal cellular function. This leads to protein misfolding and aggregation, and disruption of cellular processes. We used the yeast Saccharomyces cerevisiae to better understand how these toxic substances affect cells. The first article demonstrates how yeast cells mobilize specific control pathways to varying degrees to manage protein homeostasis and eliminate arsenic stress. The second article focuses on the roles of chaperones and ubiquitin ligases in maintaining protein balance under arsenic stress. We show that the ubiquitin-proteasome pathway is the major player in preventing and eliminating arsenite-induced protein aggregates. The third article shows that arsenite and cadmium alter the formation and structure of alpha-synuclein amyloid fibers, as well as cause changes in the protein's cellular localization. The final study provides a proteome-wide analysis of arsenic-binding proteins and demonstrates that nuclear transport is a direct target of arsenite-induced proteotoxicity. Together, these studies offer a comprehensive insight into the mechanisms by which arsenite disrupts protein homeostasis - from interactions with proteins to aggregate management mechanisms. This dissertation aims to deepen our understanding of cellular responses to heavy metal exposure, hopefully with implications for future therapeutic strategies against metal-related diseases.
Unveiling Latent Variables Affecting Protein Interactions Using Survivin as a Model Protein
(2024-09-17) Anindya, Atsarina Larasati
Proteins are typically viewed as a sequence of amino acids, where the sequence dictates folding into specific shapes to carry out distinct biological tasks by interacting with specific partners as a consequence of their complementary interfaces. This tidy sequence-to-function model interpreted through protein 3D structure, however, does not tell the whole story. Some proteins are moonlighters, juggling multiple, seemingly unrelated jobs, possibly aided by their ability to change shapes, move around the cell, and team up with different partners. The delicate dance of forming and breaking protein complexes hinges on two main factors: stability and specificity, both of which are fine-tuned by the balance between the solvent environment and weak atomic interactions. While we often think of protein interactions as straightforward, step-by-step processes, there is a growing recognition that a more dynamic approach, like using energy landscapes, might better capture the complexity—especially when it comes to these moonlighting proteins. The focus of this work is to take a part in unraveling protein moonlighting behavior by using Bayesian approach to analyze interaction kinetics and developing survivin binding prediction models to enhance our understanding of specificity, using survivin as the model protein due to its remarkable functional versatility. Bayesian progress curve analysis applied on survivin ligand-target interaction and survivin dimerization experiments using biolayer interferometry and microscale thermophoresis (MST) reduces selection bias and reveals interesting kinetic processes which otherwise can be potentially ignored. The binding prediction model shows that chemical group compositions contained in amino acids is a major decision factor for survivin interaction partner recognition, even without taking sequence or structure into account. This work also describes how the prediction model can be applied on the human proteome. The roles of specific amino acid residues on survivin dimerization are further explored with MST and small-angle X-ray scattering experiments.

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