Characterization of Rubisco plays a crucial role when it comes to the development and understanding of carbon sequestration in plants. This project took place at BMC in Uppsala, in the Gunn lab, and aimed to structure three Rubisco structures and analyze these with regard to the assembly pathway of the biogenesis of Rubisco but also how fast the reaction of binding of atmospheric carbon dioxide takes place with regard to different isoforms of the small subunit. The structural regulations led to the conclusion that an additional step in the assembly pathway would be added when one side of Rubisco had the chaperone BSD2 bound while the other side of Rubisco had the small subunit bound.The different subunits are believed to effect the structure of the LSu. The result also indicate that when the SSu are binding to the LSu octomer the interactions between the BSD2 and the LSu changes. This indicats that the SSu could indirectly facilitate the binding of the SSu on the other side by affecting the interactions of the LSu and the BSD2. Therefore the cooperative binding of the different subunits would be interesting to further evaluate. The NtL8B4(S-T1)4 , which is the first model for this structure to be determined, and therefore extended the assembly pathway for the biogenesis of higher plants, had the CABP bound, indicating that this intermediate structure could be analytically competent. This hypothesis is only based on the analyses of the structural determination, therefore further studies are needed to determine whether this is legitimate. Teknisk-naturvetenskapliga fakulteten, Uppsala universitet. Utgivni

Glutamine synthetase (GS) plays an important role in nitrogen metabolism, where it catalyzes the ATP-dependent condensation of glutamate and ammonia to yield glutamine. Recent studies showed the importance of M. tuberculosis GS (MtGS) for growth and survival of the bacterium, and demonstrated its potential as a drug target. This thesis presents structural studies of MtGS and mammalian GSs, which are aimed at identifying and developing novel inhibitors against the mycobacterial target. The structure of MtGS was solved in complex with a phosphorylated form of the inhibitor methionine sulfoximine, magnesium and ADP. The complex structure provides a detailed picture of the active site, offering several insights into catalysis and inhibition, as well as forming a solid basis for structure-based drug design. The apo canine GS and liganded human GS structures described in this thesis represent the first structures of the mammalian enzymes. Comparison of the structures revealed substrate-induced conformational changes. Inspection of the nucleotide-binding site showed that it differs from that of MtGS, thus offering good opportunities to design specific and selective inhibitors of the mycobacterial enzyme. The amino acid-binding site of MtGS was evaluated as a target for inhibition, using a combination of a literature survey, structure-based virtual screening and the synthesis of a small library of compounds. As a result, several new inhibitors of MtGS could be identified. Finally, the structural basis for inhibition of MtGS by a purine analogue (PA) is provided. PA, an analogue of a class of compounds found to inhibit MtGS in a high-throughput screening assay, targets the nucleotide-binding site. The architecture of the HsGS nucleotide-binding site indicates that PA would not be able to bind to the human enzyme, offering good prospects for selective inhibition of MtGS.

The actin cytoskeleton is a complex structure that performs a wide range of cellular functions including: cell locomotion, cytokinesis, chemotaxis, signal transduction and apoptosis. The coordinated assembly and disassembly of actin filaments is controlled by a multitude of proteins (ABPs) in the cell. There are over 160 actin-binding proteins known, which with actin, account for approximately 25% of cellular protein. ABPs are classified to several major groups based on their sequence identity and functions. In this work, we have elucidated the crystal structure of ATP bound gelsolin. We have shown that ATP binding involves the two halves of gelsolin through forming numerous polar and hydrophobic contacts. Amino acid residues that form the ATP-binding sites in inactive gelsolin are widely dispersed in the activated molecule, and hence, ATP binding is disrupted on gelsolin activation. This suggests that binding of ATP may modulate the sensitivity of gelsolin to calcium ions. The structure of human gelsolin domains 1-3 bound to actin revealed a calcium ion bound to domain 2. Here, we demonstrated that only two calcium ions are needed to activate geloslin. We speculate that this domain 2 calcium ion and the one in domain 6 participate in the initial activation of gelsolin. The crystal structure of the activated adseverin C-terminus is highly similar to that of the C-terminus of gelsolin. Comparative analysis suggests that, like the gelsolin C-terminus, adseverin will also contact actin through domain 4 and domain 6. Biochemical experiments, presented here, show that a minimum of one calcium is required for adseverin to depolymerizing actin filaments compared to two calcium for gelsolin. We speculate that this is due to the lack of the C-terminal extension in adseverin. We undertook a comparative analysis of four members of the gelsolin family proteins, gelsolin, adseverin, villin and capG, in the aspects of their calcium binding, pH activation and ATP binding. The results show that only gelsolin and adseverin are able to depolymerize actin filaments at pH < 6 in the absence of calcium ions and only gelsolin bind to ATP.

Endophilin B1 (EnB1) is a BAR protein located in the cytosol that controls membrane dynamics of different organelles such as the mitochondria and the Golgi, as well as autophagosomes. It has been suggested that this protein coordinates membrane remodeling events during essential cell death processes. For instance, previous studies show that knockdown of EnB1 leads to dysregulation of mitochondrial dynamics and inhibition of apoptosis. This protein could thereby have a critical tumor suppressor role in the cell. Despite the important role of EnB1 in many intracellular signaling processes, some parts of its underlying mechanisms of function are still unknown, more specifically, what drives the protein to bind to the membrane and what the protein structure looks like when bound.  Since EnB1 plays an important role in many intracellular trafficking events, it is of interest to obtain more information about this protein, both about its structure and membrane binding interactions. New information on this subject will contribute to a better understanding of how EnB1 modulates intracellular membranes to control several critical trafficking processes that contribute to neuron degradation and carcinogenesis.  This specific project aims at designing membrane templates that support EnB1 membrane- binding and bending for evaluation of binding capacity and for structural characterization by cryo-EM, and other associated methods.  To study the binding interactions of EnB1, different protein constructs were first expressed and purified. Membrane templates (liposomes and nanodiscs) were then created to enable structural characterization as well as studying the binding capacity of EnB1 to different lipids. A lipid binding assay with multiple variants of liposomes were created to study the biding capacity of EnB1, and negative stain transmission electron microscopy, as well as cryo-electron microscopy was used for visualization of the templates. By analyzing the data from the lipid binding assay, it can be concluded that both lipid composition and membrane curvature affects EnB1 membrane binding. The cryo-EM visualization also confirm that EnB1 is involved in membrane remodeling.

The bacterium Saccharopolyspora erythraea is utilized to produce the widely used antibiotic erythromycin. With the increasing problem of antibiotic resistance, the development of derivatives of the drug is of great importance. To produce antibiotics biosynthetically, it is crucial to understand the metabolic pathways utilized by the bacterium. It is key to clarify the behaviour of the proteins that sense the environment and regulate the gene expression accordingly. The iron-dependent regulator IdeR utilizes iron as a cofactor and is the main transcriptional regulator acting on genes crucial for iron homeostasis. Its main function is to act as a repressor for genes that are necessary for iron uptake, but it also functions as an activator for genes that promote iron storage. The role of the protein’s SH3-like domain in gene regulation is not understood. However, it might serve as a crosslinker between dimers bound to adjacent binding sites and be necessary for proper gene regulation. The aim of this paper was to examine this hypothesis. Cysteine residues with high probability of forming disulfide bonds between the SH3-like domain and the rest of the protein were introduced to IdeR and the constructed variants characterized. Methods used included electrophoretic mobility shift assay (EMSA), circular dichroism (CD) spectroscopy, and small-angle X-ray scattering (SAXS). Attempts to characterize the proteins using differential scanning fluorimetry (DSF) and X-ray crystallography were also performed. Four IdeR variants were produced and purified. The EMSA analysis indicated that theiraffinities for the consensus DNA sequence was similar to or a bit lower than that of the wild-type (wt). The CD spectroscopy measurements showed that the variants had, similar to the wt, mainly α-helical secondary structure. One of the variants had, however, less secondary structure content than the wt and was probably less stable. The secondary structure content of the wt did not change upon metal binding. The variants’ melting temperatures under oxidizing conditions were similar to or lower than that of the wt. The same experiment in reducing environment showed a loss of cooperativity for all examined variants. No data was gained from the DSF experiment and the IdeR variants did not crystallize under the investigated conditions during the project timeline. The results from the SAXS experiment show that the IdeR variants had the same overall structure as the wt as dimers and when bound to DNA. Conclusions that can be drawn from the experiments are that the introduced mutations did not affect the overall structure nor the thermal stability of the protein. However, since it cannot be concluded from the performed experiments if the crosslink between the protein domains formed as intended, another attempt to get crystal structures of the proteins is required.

No description available.

The zinc finger protein, zBED6, is a transcriptional regulator of IGF2 along with hundreds of other genes relating to development and growth. Studies on the growth of commercially bred pigs discovered a single nucleotide substitution in the third intron of IGF2 which disrupts the binding of zBED6 and is responsible for the three-fold upregulation of IGF2 in skeletal muscle. The mutation is linked to decreased subcutaneous fat deposition, larger organ size, and increased skeletal muscle mass. Three different constructs of the zBED6 protein made by Björklund 2018 were expressed and purified to characterize their binding affinity, where one contained both zinc finger domains and two of the constructs contained only one zinc finger domain each. Electrophoretic mobility shift assay protocol was optimized to determine the apparent Kd (= 210 ± 31nM) for the full-length construct C13 and to determine which zinc finger domain was sensitive to the mutation in the IGF2 gene. The first zinc finger domain seems to be more specific in its binding target. Preliminary microscale thermophoresis results were highly variable, needing further optimization of the protocol in order to obtain a full binding curve. The next steps involve site directed mutagenesis of residues binding DNA to determine which interactions are the most significant and possibly crystallization studies as well.

More conservation programs directed towards insects are needed in order to prevent the continue decline in which many insect species in the world are heading to. New ways to use the remaining microhabitats are needed to mitigate the fragmentation and degradation that urbanization and modern agriculture have contributed to in the southern Swedish landscapes. This study focusses on how microhabitats like road-verges can help the conservation projects of pollinating insects. To increase the knowledge about three groups of pollinators (Apidae, Rhopalocera and Syrphidae) and their distribution between two different microhabitats, a malaise trap was set up in road verges and in sample locations further away from the road-verges. The result from this study shows that road-verges are a microhabitat that could work as mitigation to the decline of some pollinating species, if right actions are taken. This study shows that Syrphidae was most acceptant to the hostile environment close to the roads. If these actions are going to help the most treated species is still unknown.

PP2A is a major phosphatase in the cell that participates in multiple cell signaling pathways. It is a heterotrimer of a core dimer and variable regulatory subunits. Details of its structure, function and regulation are slowly emerging. Here, the structure of two regulators of PP2A are de-scribed; PTPA and B56γ. PTPA is a highly conserved enzyme that plays a crucial role in PP2A activity but whose biochemical function is still unclear. B56γ is a PP2A regulatory subunit linked to cancer and the structure presented here of B56γ in its free form is particularly valuable in light of the recent structures of the PP2A holoenzyme and core dimer. Protein production is a major bottleneck in structural genomic projects. Here, we describe two novel methods for improved protein production. The first is a colony based screening method where any DNA library can be screened for soluble expression of recombinant proteins in E.coli. The second method involves improvements of the well established IMAC purification method. We have seen that a low molecular weight component of E.coli lysate decreases the binding capacity of IMAC columns and by removing the low molecular weight components, recombinant proteins only present at low levels in E.coli lysate can be purified, which has previously been believed to be unfeasible.

The hereditary transthyretin (TTR) amyloidoses are rare, and in severe cases, fatal disorders caused by mutations in the TTR gene. The clinical picture is diverse, involving neuropathies and myopathies, and mainly depends on the causative mutation and the sites and rates of amyloid deposition. The ultimate aim of the field of research presented in this thesis is to prevent TTR amyloid disease. To reach this ambitious goal, a thorough understanding of the normal as well as the pathological properties of the protein is essential. Here, comparisons between TTR from humans and other species may provide valuable information. The three-dimensional structure of TTR from Gilthead sea bream (Sparus aurata) was determined at 1.75 Å resolution by X-ray crystallography, and was found to be structurally similar to human TTR. However, significant differences were observed in the area at and around β-strand D, an area believed to dissociate from the structure prior to amyloid formation, thereby allowing the β-strands A and B to participate in polymerization. During evolution, the preference of TTR for the thyroid hormones, 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4), has shifted. While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles, where T3 is the main ligand. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution, as well as the complex of human TTR with T3. A significantly wider entrance and narrower thyroid hormone binding channel suggest a structural explanation to the differences in thyroid hormone preference between human and piscine TTR. The Tyr114Cys substitution in TTR is associated with severe systemic amyloidosis. The mutation introduces a second cysteinyl group in the TTR monomer, and has been shown to inhibit the formation of fibril formation in vitro, promoting the formation of disulfide-bonded amorphous aggregates. To deduce the role of intermolecular disulfide bonds in fibril formation we characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular disulfide bond at position 114 enhances the exposure of Cys10, which promotes the formation of additional intermolecular disulfide-linked assemblies. Also, we were able to isolate a disulfide-linked dimeric form of this mutant that formed protofibrils in vitro, suggesting the architecture of TTR amyloid may be the result of different underlying structures rather than that of a highly stringent assembly. We have also been able to successfully adapt a method of protein pre-heating to enable crystallization, thereby succeeding in a particularly problematic protein crystallization experiment. By heating the protein solution, we succeeded in separating several forms of protein micro-heterogeneities from the properly folded protein species, thereby allowing the growth of well diffracting crystals. / Ärftlig transthyretinamyloidos är en ovanlig och i allvarliga fall dödlig proteininlagringssjukdom som orsakas av mutationer i genen för transthyretin. Den kliniska bilden är huvudsakligen beroende av den bakomliggande genförändringen samt amyloidlokaliseringen och -depositionshastigheten och omfattar vanligen neuropatier och myopatier av varierande grad. Det slutgiltiga målet med forskningsfältet som presenteras i denna avhandling är att förhindra eller bota transthyretinamyloidos. En förutsättning för att lyckas med detta ambitiösa mål är en ingående förståelse för proteinets grundläggande egenskaper, såväl i normalfallet som i de patologiska processerna, bland annat genom jämförande studier av humant och icke-humant transthyretin (TTR). Den tredimensionella röntgenkristallografiska strukturen av TTR från fisken guldsparid (Sparus aurata) bestämdes till en upplösning på 1,75Å och befanns vara strukturellt snarlik humant TTR. Signifikanta skillnader observerades emellertid i och kring β-sträng D, en region som tros dissociera från huvudstrukturen innan själva bildningen av amyloid. Enligt denna hypotes leder D-strängsdissociationen till exponering av β-strängarna A och B, vilka därmed kan delta i de reaktioner som bildar amyloid. Under evolutionen har bindningspreferenserna för thyroideahormonerna T3 (3,5,3’-trijod-L-thyronin) och T4 (3,5,3’,5’-tetrajod-L-thyronin) hos TTR ändrats. Humant TTR har högre affinitet för T4 än för T3, medan det motsatta förhållandet gäller för lägre vertebrater, t ex fisk, där T3 är den huvudsakliga liganden. Strukturerna bestämdes för guldsparid i komplex med T4 och med T3 till 1,9 Å upplösning, samt för humant TTR i komplex med T3 till 1,7 Å upplösning. Jämförande analyser visade på signifikanta skillnader i thyroideahormonbindningskanalen, vilken var vidare och grundare i fisk än i människa. Dessa strukturella skillnader kan delvis förklara olikheterna i hormonbindning mellan högre och lägre vertebrater. Substitutionen Tyr114Cys i TTR är kopplad till en allvarlig form av systemisk transthyretinamyloidos. Mutationen introducerar en andra cysteinylgrupp i TTR-monomererna, vilket förhindrar fibrillbildning in vitro, men gynnar bildningen av amorfa disulfidbundna aggregat. För att närmare studera betydelsen av disulfidbindningar vid fibrillbildning av detta protein så karakteriserades dubbelmutanten TTR Cys10Ala/Tyr114Cys. Baserat på våra resultat föreslår vi att intermolekylära disulfidbindningar i position 114 ökar exponeringen av Cys10, vilket förstärker tendensen att bilda ytterligare disulfidbundna aggregat. Vi isolerade även en disulfidbunden dimerisk form av dubbelmutanten som kan bilda protofibriller in vitro. Baserat på denna observation föreslår vi att transthyretinamyloids underliggande arkitektur är sammansatt och kan nås genom sammanfogning av olika substrukturer snarare än genom en strikt ordnad uppbyggnad. Vi har också modifierat och anpassat en metod för uppvärmning av proteiner för att möjliggöra kristallisation i ett synnerligen problematiskt proteinkristallisations-experiment. Genom uppvärmning av proteinlösningen lyckades vi separera olika former av mikroheterogeniteter från det rättveckade proteinet, som sedan bildade kristaller av god röntgendiffraktiv kvalitet.