| 1. |
- de Lichtenberg, Casper, 1989-
(författare)
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Time-resolved Structural and Mechanistic Studies of Water Oxidation in Photosystem II : water here, water there, water everywhere
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxygenic photosynthesis is undisputedly one of the most important chemical processes for human life on earth as it not only fills the atmosphere with the oxygen that we need to breathe, but also sustains the accumulation of biomass, which is not only used as nourishment but is also present in almost every aspect of our lives as building material, textiles in clothes and furniture, or even as living decorations to name a few.The photosynthetic water-splitting mechanism is catalyzed by a water:plastoquinone oxido-reductase by the name of photosystem II (PSII), which is embedded in the thylakoid membranes of plants, algae and cyanobacteria. As it is excited by light, charge separation occurs in the reaction center of the protein and an electron is extracted by oxidation of Mn4Ca-cluster, that constitutes the active site for the water splitting reaction in PSII. When the Mn4Ca-cluster has been oxidized 4 times, it forms an oxygen-oxygen bond between two water derived ligands bound to the Mn4Ca-cluster and returns to the lowest oxidation state of the catalytic cycle. Understanding what ligands of the cluster that are used in the water splitting reaction is the key to unlocking the underlying chemical mechanism.In this thesis I describe investigations, with room temperature X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) on PSII microcrystals, of how the active site looks in all the stable intermediate oxidation states. Furthermore I describe how we uncovered the sequence of events that lead to insertion of an additional water ligand in the S2-S3 state transition of the catalytic cycle.Furthermore, through time-resolved membrane-inlet mass spectrometry (TR-MIMS) measurements of the isotopic equilibration of the substrate waters with the bulk in conditions that induce different electron magnetic resonance (EPR) spectroscopic signatures, I present evidence that the exchange of the slowly exchanging substrate water Ws is controlled by a dynamic equilibrium between conformations in the S2-state that give rise to either the low-spin multiline (LS-ML) signal or the high-spin (HS) signal. Based on the crystal structures and litterature suggestions for the conformation of the HS state different scenarios were presented for the assignment of Ws and how it exchanges. This analysis is discussed in the context of all semi-stable intermediate oxidation states in the Kok cycle.To further the understanding of this equilibrium, I also studied a selection of mutants positioned at strategic places in the vicinity of the different proposed substrates and at points that were suggested to be critical for substrate entry. With the combination of TR-MIMS and EPR, I reached the conclusion that by mutating valine 185 to asparagine, the water bound A-type conformation was stabilized, meanwhile in the mutant where aspartate 61 was mutated to alanine I observed that the barrier of the equilibrium between the exchanging conformations was so high that the interchange between them was arrested at room temperature. Additionally the retardation of the substrate exchange rates in the S3-states fit best with D61 being in the vicinity of the fast exchanging water. With this information we found the data best explained in a scenario where the water insertion of the S2-S3 transition was determining the if O-O bond formation occurred between the waters that were W2 and W3 or W2 and O5 in the S2 state. In addition, by mutation of glutamate 189 to glutamine that this residue is not important for the exchange of substrate waters in the S2 or the S3 states.Finally I use a combination of substrate labelling with TR-MIMS and time resolved labelling of the waters that ligate the Mn4Ca-cluster to show that the briding oxygen O5 is exchanging with a near identical rate to Ws, further supporting the assignment that Ws=O5.In conclusion, O-O bond formation most likely occurs between W2 (Wf) and O5 (Ws) via an oxo-oxyl radical coupling mechanism. The newly inserted water thus represents the slow exchanging water of the following S-state cycle.
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| 2. |
- Mohammadi, Nasibeh, 1981-
(författare)
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Determining the role of guanylate-binding proteins for host defense against Francisella tularensis
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Francisella tularensis is a highly virulent, intracellular bacterium and the causative agent of the human disease tularemia. This is a zoonotic, often vector-borne disease. Due to its intracellular nature, F. tularensis can infect many cell types, but of special relevance is its ability to infect monocytic cells and avoid their otherwise potent antimicrobial effects. Monocytic cells can; however, control infection after activation with IFN-γ, but the molecular mechanisms behind this control are not well understood. Recently, guanylate-binding proteins (GBPs) have been identified as crucial for the control of intracellular F. tularensis and many other bacteria, viruses, and parasites. They represent a vast family of interferon-inducible proteins, but it is incompletely understood how their ubiquitous abilities to control diverse types of infections are executed. The overall aim of the thesis was to obtain a better understanding of how GBPs execute the control of infection caused by Francisella and how the bacterium counteracts the bactericidal effects of the GBPs and of other immune mediators. To this end, the responses of bone marrow-derived murine macrophages (BMDM) to Francisella was one model investigated and the other employed a co-culture system whereby BMDM were infected and to the cultures immune cells from vaccinated mice were added. To comprehensively understand the host-pathogen interaction, a variety of Francisella strains were utilized; the highly virulent SCHU S4 strain, the human live vaccine strain (LVS), and the widely used surrogate for F. tularensis, the low virulent F. novicida. All strains have similar capability of intracellular multiplication in BMDM, however, activation of the microbicidal ability of BMDM with IFN-γ, significant control of infection was observed for the LVS and F. novicida strains, whereas there was no control of the SCHU S4 infection. The control of the former strains was GBP-dependent, despite that no differences in GBP transcription or translation were observed in the infected cell cultures. Patterns of 18 cytokines very clearly discriminated the different types of infections and high levels were generally observed in F. novicida-infected cultures and very low levels in SCHU S4-infected cultures. Co-infection with F. novicida and SCHU S4 led to significant control of both strains and in these cultures, a majority of cytokines showed intermediate or high levels. A critical component in the immune recognition of Francisella is AIM2, which is a core constituent of a special form of inflammasome, a cytoplasmic multimeric complex. We determined that AIM2-deficient BMDM, despite the central role of AIM2 for immune recognition of F. novicida and LVS, still controlled infection with either of the two strains after activation with IFN-γ. Again, no control of the virulent strain SCHU S4 was observed. The co-culture system revealed further complexity beyond that of the BMDM model. Utilizing splenocytes obtained from immunized C57BL/6 mice as effectors in cultures with BMDM infected with either of the three Francisella strains, we observed that regardless of strain, significant control of replication occurred with wild-type macrophages and immune splenocytes, even for the highly virulent SCHU S4 strain, but not in cultures with immune splenocytes and GBP-deficient macrophages. Supernatants from the cultures demonstrated very distinct patterns for each of the three infections. Thus, the co-culture assay identified, as for the BMDM model, a crucial role of GBPs for the control of intracellular replication of Francisella, however, in contrast to the BMDM model, the co-culture conferred significant control of SCHU S4 infection.Collectively, our studies demonstrate a very important role of GBPs for the IFN-γ-dependent control of Francisella infection, with the notable exception of the highly virulent strain SCHU S4. A GBP-mediated control of SCHU S4 was; however, observed in the co-culture system, thereby identifying additional bactericidal mechanisms, besides those that are IFN-γ-dependent. We also demonstrate that the inflammatory potential of Francisella strains is correlated to their virulence, most notable is the almost complete lack of inflammatory response during infection with the highly virulent SCHU S4 strain, but this anti-inflammatory capacity was counteracted by the strong pro-inflammatory property of F. novicida during co-infection.
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| 3. |
- Tomás Graça, André, 1994-
(författare)
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Light’EM up! : structural characterization of light-driven membrane protein complexes by cryogenic electron microscopy
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Photosynthesis is probably the most important process for allowing life to develop into the diverse forms we see today. In this process, solar radiation is used to convert CO2 into biomass. From this process, we obtain oxygen to breathe, sources of food (plant biomass), and the potential for clean and sustainable energy. Photosystem II (PSII) – a key enzyme in photosynthesis –, is a protein complex located in the thylakoid membrane of photosynthetic organisms. PSII and its light-harvesting antennae capture light energy, driving a charge separation process, which leads to the extraction of electrons from water molecules, forming and releasing molecular oxygen. A PSII dimer is composed of more than 20 unique proteins and hundreds of cofactors which fine-tune the mechanisms of light-harvesting and water oxidation, and stabilize the whole complex. While the arrangement of most (but not all!) of these proteins and cofactors is known, their dynamics and individual contributions are not yet fully understood.In my thesis work, I took on the challenge of resolving the structure of large protein complexes, such as PSII complexes from various photosynthetic organisms, using a technique called cryogenic electron microscopy (cryo-EM). This PhD dissertation focuses on structurally describing these macromolecular assemblies and how their components (protein, cofactors, and substrate) interact with each other or with their immediate cellular environment.Among the several outcomes of my research on PSII, I would like to highlight the following findings: 1) the usage of digitonin as a detergent to solubilize PSII destroys the catalytic activity and changes LHCII pigment content, among other consequences; 2) PSII does not seem to incorporate chlorophyll (Chl) a molecules with a farnesyl tail, and the Chl tails’ flexibility justifies not resolving the full-length of some of these molecules in PSII structures. We concluded that flexibility may be an advantage to PSII function; 3) cryo-EM is a technique with the potential to reveal information about electron/proton transfer processes within PSII, and provided us with data, for instance, to suggest a pathway for the protonation of QB, the final electron acceptor in PSII.In another project, also using cryo-EM, I studied the structure of the S-layer Deinoxanthin Binding Complex (SDBC), a membrane protein complex from Deinococcus radiodurans. This complex is an essential part of the cell envelope, the outermost barrier of this bacterium, and it is known to bind a carotenoid called deinoxanthin, which has significant spectroscopic and antioxidant properties. Additionally, we studied the function of this complex and showed that the SDBC is a quencher of UVC-UVB radiation and reactive oxygen species, with superoxide dismutase activity. This complex has an α-β coiled-coil stalk long enough to reach the inner membrane of the cell envelope.In summary, visualizing the structural organization and chemistry within these complexes allowed us to gain a new understanding of their function and diversity. Furthermore, this work demonstrates the potential of cryo-EM as a method to render complementary information at resolution superior to state-of-the-art X-ray diffraction methods.
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| 4. |
- Shiryaeva, Liudmila, 1970-
(författare)
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Proteomics and metabolomics in biological and medical applications
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological processes in living organisms consist of a vast number of different molecular networks and interactions, which are complex and often hidden from our understanding. This work is focused on recovery of such details for two quite distant examples: acclimation to extreme freezing tolerance in Siberian spruce (Picea obovata) and detection of proteins associated with prostate cancer.The first biological system in the study, upon P. obovata, is interesting by this species ability to adapt and sustain extremely low temperatures, such as -60⁰C or below. Despite decades of investigations, the essential features and mechanisms of the amazing ability of this species still remains unclear. To enhance knowledge about extreme freezing tolerance, the metabolome and proteome of P. obovata’s needles were collected during the tree’s acclimation period, ranging from mid August to January, and have been analyzed.The second system within this study is the plasma proteome analysis of high risk prostate cancer (PCa) patients, with and without bone metastases. PCa is one of the most common cancers among Swedish men, which can abruptly develop into an aggressive, lethal disease. The diagnostic tools, including PSA-tests, are insufficient in predicting the disease’s aggressiveness and novel prognostic markers are urgently required.Both biological systems have been analyzed following similar steps: by two-dimensional difference gel electrophoresis (2D-DIGE) techniques, followed by protein identification using mass spectrometry (MS) analysis and multivariate methods. Data processing has been utilized for searching for proteins that serve as unique indicators for characterizing the status of the systems. In addition, the gas chromatography-mass spectrometry (GC-MS) study of the metabolic content of P.obovata’s needles, from the extended observation period, has been performed. The studies of both systems, combined with thorough statistical analysis of experimental outcomes, have resulted in novel insights and features for both P. obovata and prostate cancer. In particular, it has been shown that dehydrins, Hsp70s, AAA+ ATPases, lipocalin and several proteins involved in cellular metabolism etc., can be uniquely associated with acclimation to extreme freezing in conifers. Metabolomic analysis of P. obovata needles has revealed systematic metabolic changes in carbohydrate and lipid metabolism. Substantial increase of raffinose, accumulation of desaturated fatty acids, sugar acids, sugar alcohols, amino acids and polyamines that may act as compatible solutes or cryoprotectants have all been observed during the acclimation process.Relevant proteins for prostate cancer progression and aggressiveness have been identified in the plasma proteome study, for patients with and without bone metastasis. Proteins associated with lipid transport, coagulation, inflammation and immune response have been found among them.
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