Nicotinamide adenine dinucleotide (NAD+) is the substrate used for the introduction of the ubiquitous and highly dynamic PTM in which either one or multiple adenosine diphosphate ribose (ADPr)... Show moreNicotinamide adenine dinucleotide (NAD+) is the substrate used for the introduction of the ubiquitous and highly dynamic PTM in which either one or multiple adenosine diphosphate ribose (ADPr) moieties are covalently attached to a nucleophilic side chain of an specific amino acid in the target protein to regulate cellular pathways including adipogenesis, DNA damage repair and gene expression. A significant fraction of the nucleophilic amino acid functionalities, most recently histidine and tyrosine, have been identified as ADPr-acceptor sites. In this thesis, new methodologies have been developed to synthesize peptide fragments carrying an ADPr modification to investigate ADP-ribosylation on histidine. Show less
The endocannabinoid receptors CB1R and CB2R are involved in a plethora of processes, and consequently are involved in many pathological conditions. Their wide distribution makes the CBRs both an... Show moreThe endocannabinoid receptors CB1R and CB2R are involved in a plethora of processes, and consequently are involved in many pathological conditions. Their wide distribution makes the CBRs both an interesting therapeutic target and hard to study. Additional chemical tools are required to study and understand the function and mechanism of CB1R and CB2R. This thesis describes the development of several such tools to improve our insight in the (pathological) roles of the receptors in order to develop novel and improved therapeutics. First evaluation of three dimensional ligand-CB2R complexes made and analysed with Cryo-EM are described. Hotspots that potentially generate selectivity between CB1R and CB2R are evaluated with point-mutations in vitro. Consequently describes the development of the first tools, two-step bifunctional probes based on LEI-121 and LEI 102, is described. As two-step probes are not compatible with every assay, the toolbox is expanded with a one-step fluorescent probe. Briefly touching upon CB1R, ligands were designed with negatively charged phosphonium groups that are potentially selective for mtCB1R. Show less
Diacylglycerol lipases (DAGLα and DAGLβ) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain and peripheral tissues. Selective DAGLβ inhibitors... Show moreDiacylglycerol lipases (DAGLα and DAGLβ) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain and peripheral tissues. Selective DAGLβ inhibitors have been proposed as a potential treatment for inflammatory diseases with reduced potential for central nervous system (CNS) mediated side effects, but they are currently lacking. To develop DAGLβ selective inhibitors, a fluorescent biochemical assay was optimized and applied in a high-throughput screening (HTS) for DAGLβ. During the HTS, eight hits classified into four distinct chemotypes were identified. Subsequent structure-activity relationship (SAR) studies, focusing on hit 1 and its modifications, revealed a specific group as the modification hotspot crucial for achieving selectivity towards DAGLβ. Through an extensive SAR investigation, focusing on modifying this group, the first-in-class DAGLβ selective inhibitors, LEI-130 and LEI-131, were discovered. Following their discovery, LEI-130 and LEI-131 underwent comprehensive in vitro and in situ profiling studies. These investigations confirmed that LEI-130 and LEI-131 are selective and noncompetitive inhibitors of DAGLβ, effectively reducing inflammation. Show less
Toll-like receptor 2 (TLR2) belongs to the TLR protein family that plays an important role in the immune and inflammation response system. While TLR2 is predominantly expressed in immune cells, its... Show moreToll-like receptor 2 (TLR2) belongs to the TLR protein family that plays an important role in the immune and inflammation response system. While TLR2 is predominantly expressed in immune cells, its expression has also been detected in the brain, specifically in microglia and astrocytes. Recent studies indicate that genomic deletion of TLR2 can result in impaired neurobehavioural function. It is currently not clear if the genomic deletion of TLR2 leads to any alterations in the microstructural features of the brain. In the current study, we noninvasively assess microstructural changes in the brain of TLR2-deficient (tlr2-/-) zebrafish using state-of-the art magnetic resonance imaging (MRI) methods at ultrahigh magnetic field strength (17.6 T). A significant increase in cortical thickness and an overall trend towards increased brain volumes were observed in young tlr2-/- zebrafish. An elevated T2 relaxation time and significantly reduced apparent diffusion coefficient (ADC) unveil brain-wide microstructural alterations, potentially indicative of cytotoxic oedema and astrogliosis in the tlr2-/- zebrafish. Multicomponent analysis of the ADC diffusivity signal by the phasor approach shows an increase in the slow ADC component associated with restricted diffusion. Diffusion tensor imaging and diffusion kurtosis imaging analysis revealed diminished diffusivity and enhanced kurtosis in various white matter tracks in tlr2-/- compared with control zebrafish, identifying the microstructural underpinnings associated with compromised white matter integrity and axonal degeneration. Taken together, our findings demonstrate that the genomic deletion of TLR2 results in severe alterations to the microstructural features of the zebrafish brain. This study also highlights the potential of ultrahigh field diffusion MRI techniques in discerning exceptionally fine microstructural details within the small zebrafish brain, offering potential for investigating microstructural changes in zebrafish models of various brain diseases. Show less
Observations of low-lying dark states in several photosynthetic complexes challenge our understanding of the mechanisms behind their efficient energy transfer processes. Computational models are... Show moreObservations of low-lying dark states in several photosynthetic complexes challenge our understanding of the mechanisms behind their efficient energy transfer processes. Computational models are necessary for providing novel insights into the nature and function of dark states, especially since these are not directly accessible in spectroscopy experiments. Here, we will focus on signatures of dark-type states in chlorosomes, a light-harvesting complex from green sulfur bacteria well-known for uniting a broad absorption band with very efficient energy transfer. In agreement with experiments, our simulations of two-dimensional electronic spectra capture the ultrafast exciton transfer occurring in 100s of femtoseconds within a single chlorosome cylinder. The sub-100 fs process corresponds to relaxation within the single-excitation manifold in a single chlorosome tube, where all initially created populations in the bright exciton states are quickly transferred to dark-type exciton states. Structural inhomogeneities on the local scale cause a redistribution of the oscillator strength, leading to the emergence of these dark-type exciton states, which dominate ultrafast energy transfer. The presence of the dark-type exciton states suppresses energy loss from an isolated chlorosome via fluorescence quenching, as observed experimentally. Our results further question whether relaxation to dark-exciton states is a leading process or merely competes with transfer to the baseplate within the photosynthetic apparatus of green sulfur bacteria. Show less
This thesis introduces the concept of "physics-based inverse design", working on the notion that the physical driving forces governing functionality are inherently encoded in independently... Show moreThis thesis introduces the concept of "physics-based inverse design", working on the notion that the physical driving forces governing functionality are inherently encoded in independently parameterized energy functions, which can be resolved through the use of inverse design strategies.The thesis describes the development of EVO-MD, a Python-based implementation of the physics-based inverse design concept. EVO-MD is capable of automatically setting-up, performing, and analyzing molecular dynamics simulations, allowing for the evolutionary optimization of complex and dynamic features in peptides. Examples of such applications include the optimization of lipid composition and curvature sensors, and the development of peptides with antiviral properties. Show less
Paus, L.V. de; Yu, A.; Janssen, A.P.A.; Berg, R.J.B.H.N. van den; Heitman, L.H.; Stelt, M. van der 2024
The cannabinoid receptor type 2 (CB2R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor... Show moreThe cannabinoid receptor type 2 (CB2R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI-121 that stabilized the inactive conformation of the CB2R. Here, we report the structure-based design of a novel bifunctional probe that captures the active conformation of the CB2R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click-chemistry with fluorophore-azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function. Show less
Miloslavina, Y.; Thomas, B.; Reus, M.; Sai Sankar Gupta, K.B.; Oostergetel, G.T.; Andreas, L.B.; ... ; Groot, H.J.M. de 2024
The largest light-harvesting antenna in nature, the chlorosome, is a heterogeneous helical BChl self-assembly that has evolved in green bacteria to harvest light for performing photosynthesis in... Show moreThe largest light-harvesting antenna in nature, the chlorosome, is a heterogeneous helical BChl self-assembly that has evolved in green bacteria to harvest light for performing photosynthesis in low-light environments. Guided by NMR chemical shifts and distance constraints for Chlorobaculum tepidum wild-type chlorosomes, the two contrasting packing modes for syn-anti parallel stacks of BChl c to form polar 2D arrays, with dipole moments adding up, are explored. Layered assemblies were optimized using local orbital density functional and plane wave pseudopotential methods. The packing mode with the lowest energy contains syn-anti and anti-syn H-bonding between stacks. It can accommodate R and S epimers, and side chain variability. For this packing, a match with the available EM data on the subunit axial repeat and optical data is obtained with multiple concentric cylinders for a rolling vector with the stacks running at an angle of 21° to the cylinder axis and with the BChl dipole moments running at an angle ß ∼ 55° to the tube axis, in accordance with optical data. A packing mode involving alternating syn and anti parallel stacks that is at variance with EM appears higher in energy. A weak cross-peak at -6 ppm in the MAS NMR with 50 kHz spinning, assigned to C-181, matches the shift of antiparallel dimers, which possibly reflects a minor impurity-type fraction in the self-assembled BChl c. Show less
Pol, R.W.I. van der; Brinkmann, B.W.; Sevink, G.J.A. 2024
In the current global context, there is a pressing need to address sustainable energy supplies to safeguard our Planet and its ecosystems. The choices made by human society have a significant... Show moreIn the current global context, there is a pressing need to address sustainable energy supplies to safeguard our Planet and its ecosystems. The choices made by human society have a significant impact on genetic evolution and climate. To ensure a better future for all, it is crucial to exercise foresight, foster collaboration across various sectors, and reach agreements based on fair and ethical principles. Science plays a pivotal role in advancing energy conversion, offering the potential for significant scientific breakthroughs that contribute to the protection and respect of our World. Specifically, the development of solar-to-fuel devices holds promise for achieving this transition to green energy. This Ph.D. dissertation centers on the development and functionalization of 2D membranes and materials, which constitute integral components of these conversion devices. The optimization of functionalized 2D materials necessitates a comprehensive computational design approach. This involves the adoption of a multiscale computational framework for the thorough design of these materials and the precise prediction and understanding of molecular processes, encompassing molecular self-assembly, ion transport, and catalytic surface reactions. Show less
Light is required for photosynthesis, but plants are often exposed to excess light, which can lead to photodamage and eventually cell death. To prevent this, they evolved photoprotective feedback... Show moreLight is required for photosynthesis, but plants are often exposed to excess light, which can lead to photodamage and eventually cell death. To prevent this, they evolved photoprotective feedback mechanisms that regulate photosynthesis and trigger processes that dissipate light energy as heat, called non-photochemical quenching (NPQ). In excess light conditions, the light reaction and activity of Photosystem II (PSII) generates acidification of the thylakoid lumen, which is sensed by special pH-sensitive proteins called Photosystem II Subunit S (PsbS), actuating a photoprotective "switch" in the light-harvesting antenna. Despite its central role in regulating photosynthetic energy conversion, the molecular mechanism of PsbS as well as its interaction with partner proteins are not well understood. This review summarizes the current knowledge on the molecular structure and mechanistic aspects of the light-stress sensor PsbS and addresses open questions and challenges in the field regarding a full understanding of its functional mechanism and role in NPQ. Show less
The research presented in this thesis explores the chemotherapeutic potential of metal-based compounds as chemotherapy agents, with an initial focus on the synthesis and DNA interaction studies of... Show moreThe research presented in this thesis explores the chemotherapeutic potential of metal-based compounds as chemotherapy agents, with an initial focus on the synthesis and DNA interaction studies of platinum and palladium compounds utilizing the [Pt(bapbpy)]2+ scaffold. The study identifies intercalation as the primary mechanism of action for these complexes. Furthermore, it provides a detailed structure-activity relationship analysis, highlighting the critical role of the complex's protonation state in influencing its biological activity and efficacy. Subsequently, the study delves into photoactivated chemotherapy (PACT) using ruthenium (II) complexes, where light activation of ruthenium complexes enables targeted drug delivery to tumor cells, thereby reducing adverse effects. This research emphasizes the development of ruthenium-based compounds that can photorelease a DNA repair inhibitor, specifically targeting the RAD51 protein, essential for Homologous Recombination (HR). By disrupting the DNA repair mechanisms in cancer cells, this approach seeks to enhance the cytotoxicity of the therapy and address drug resistance. Show less
Kim Y.; Alia, A.; Kurle-Tucholski, P.; Wiebeler, C.; Matysik, J. 2024
A fundamental understanding of proton transport through graphene nanopores, defects, and vacancies is essential for advancing two-dimensional proton exchange membranes (PEMs). This study employs... Show moreA fundamental understanding of proton transport through graphene nanopores, defects, and vacancies is essential for advancing two-dimensional proton exchange membranes (PEMs). This study employs ReaxFF molecular dynamics, metadynamics, and density functional theory to investigate the enhanced proton transport through a graphene nanopore. Covalently functionalizing the nanopore with a benzenesulfonic group yields consistent improvements in proton permeability, with a lower activation barrier (≈0.15 eV) and increased proton selectivity over sodium cations. The benzenesulfonic functionality acts as a dynamic proton shuttle, establishing a favorable hydrogen-bonding network and an efficient proton transport channel. The model reveals an optimal balance between proton permeability and selectivity, which is essential for effective proton exchange membranes. Notably, the benzenesulfonic-functionalized graphene nanopore system achieves a theoretically estimated proton diffusion coefficient comparable to or higher than the current state-of-the-art PEM, Nafion. Ergo, the benzenesulfonic functionalization of graphene nanopores, firmly holds promise for future graphene-based membrane development in energy conversion devices. Show less
The research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study.... Show moreThe research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study. Enzymes inherently embody a delicate balance between activity and stability, and the acquisition of new enzymatic functions is often accompanied by trade-offs, such as decreased stability or reduction of the original activity. Probing evolutionary adaptability of BlaC with laboratory evolution in combination with structural characterization can provide information about the mechanisms of rapid adaptations observed for β-lactamases in the clinic. The role of temperature as a conventional selection pressure in such evolutionary adaptation is unclear. The cooperative nature of enzyme unfolding over a narrow temperature trajectory raises the question whether evolution at temperatures well below the melting point is influenced by temperature. The approach used in this work to answer these questions is by simulating evolution under different selection pressures and characterize the variant enzymes in terms of activity, structure, dynamics and melting temperature. The research makes clear how enzyme kinetics and dynamics vary with different selection pressures and maps the evolutionary path that enzymes may take. The underlying structural mechanisms are established to provide a rationale for the observed effects. Show less
This work describes the use of click-to-release chemistry to get spatiotemporal control over immunocytokine activity. Until now, immunocytokines (cytokines coupled to a tumor-targeting-moiety)... Show moreThis work describes the use of click-to-release chemistry to get spatiotemporal control over immunocytokine activity. Until now, immunocytokines (cytokines coupled to a tumor-targeting-moiety) remained active throughout the body, being able to bind their respective receptors, causing mild to severe side-effects in cancer patients. Attempts have been made to improve the specific action of these immunocytokines, but these solutions remained very cytokine-specific and toxicity was not reduced significantly. Click-to-release chemistry allows us to inactivate a cytokine by blocking its free amines, present in lysines. This prevents the cytokine, IL-1β and TNF-α in particular, from binding its receptor. Removal of the blocking agent using a tetrazine restores the native amine and for IL-1β also its activity. By coupling the blocked cytokine to a targeting moiety allows for transport to the target, the tumor(-environment) upon which the unblocking or decaging can take place. This blocking-unblocking or caging-decaging was assessed using various cell-based assay. This technique can provide new opportunities in the immunocytokine field, as it is not cytokine-specific, and thereby opportunities in cancer therapy development. Show less