Browsing by study line "Biochemistry and Structural Biology"

Coxsackievirus A9 (CVA9) is a member of the Picornaviridae family of viruses and Enterovirus genus. CVA9 is mostly associated with milder Enterovirus symptoms such as rashes, but there are also individual cases of more serious diseases caused by CVA9 infection, including hepatitis. The picornavirus capsid structure is largely conserved and composed of 3-4 viral proteins (VP). At the base of VP1 there is a hydrophobic pocket occupied by a lipid factor. The expulsion of that lipid factor leads to expansion of the capsid, viral uncoating and genome release into the cell. To prevent that expansion, an ongoing study has been screening and synthesizing hundreds of potential molecules to target the VP1 pocket and replace the pocket factor. One such molecule is CL300, an N-phenyl benzamide, which has an antiviral effect on CVA9. The purpose of this study was to validate the proposed mechanism of action of CL300 on CVA9 by confirming its binding location with cryogenic electron microscopy and single-particle processing methods. Through processing, 31,050 particles were narrowed down to 21,230 particles that were reconstructed into a 2.26 Å resolution density map of CVA9 with a heterogeneous density in the VP1 pocket. Due to features present in that density and the presence of multiple rotamer densities in a nearby residue, Y210, it was concluded that either CL300 or lipid factors were present in different VP1 pockets, resulting in an averaged density in the map. The final result of this study was a model of CVA9 with CL300 in the hydrophobic pocket. These results, in conjunction with the ongoing study into the antiviral effect of CL300, have implications for the potential future production of antivirals and vaccines to combat CVA9 and other Enterovirus infections where there is conservation of the binding pocket.

Intrinsically disordered proteins (IDPs) consist of charged, and polar amino acids, lacking bulky hydrophobic residues and they do not have a single well-defined 3D structure. They are found in all domains of life with higher abundance in eukaryotes, covering approximately 30% of the eukaryotic proteome. IDPs have key roles in many biological processes from cell signaling to phase-separation phenomena. Particularly, disordered protein regions serving as linkers, have been found in many multidomain proteins and they play a decisive role in the protein’s function. In the present thesis we aim to identify the correlation between sequence and rigidity disordered linkers, utilizing a synergistic method of Nuclear Magnetic Resonance (NMR) experiments and Molecular Dynamic (MD) simulations. For that purpose, glycine and proline rich disordered linkers which are widely utilized for constructing fusion proteins were used. Additionally, we aim to characterize the rigidity of the the disordered repetitive domain of the major ampullate type I dragline silk protein, using the same approach which served to connect the two terminal folded domains inside the protein. Dragline silk has been under thorough investigation due to its favorable mechanical properties and applications in material science. NMR spin relaxation times T1, T2 and hetNOE, are highly sensitive probes to motional timescales of IDPs, but they are difficult to interpret in terms of molecular dynamics. Here, we use the spin relaxation times to validate the MD simulations which in turn are set to interpret the linkers’ internal motions. Using the quality evaluation approach QEBSS, the best simulations were identified as the best description of the conformational ensemble, based on the comparison with the experimental spin relaxation times. Systematic differences in spin relaxation times correlate with systematic changes in the linkers rigidity, proving that spin relaxation times can be used to detect disordered linker rigidity. Prolines are shown to induce a comparatively expanded conformation ensemble with significantly slower dynamics whereas glycines offer flexibility. The ensemble of the repetitive domain of the silk protein showed conformations with intermediate rigidity. We also demonstrate that the synergy of NMR and MD simulations can be used for characterizing the rigidity-sequence interplay in short glycine and proline disordered linkers and silk protein systems. Being able to tune the properties of flexible and rigid linkers can be fundamental for understanding different biological systems and for protein engineering purposes. Bioengineering applications include designing and optimizing fusion protein linkers that in the long term be useful for drug design and developing protein-based biomaterials.

Plants are vital to all terrestrial ecosystems by providing ecosystem services through photosynthesis- derived compounds. Throughout the millennia, plant metabolism has diversified in the form of all plant secondary metabolites, ranging from metabolite groups such as terpenes to alkaloids to flavonoids. Many of these secondary metabolites are economically valued for their chemical, pharmaceutical and physical properties. The flavonoids are one of the largest groups and are known to provide competitional advantages and increase of survival of many plant species in extreme environments. One of the critical enzymes in the whole biosynthesis pathway of flavonoids is the dihydroflavonol 4- reductase (DFR). DFR regulates the formation of leucoanthocyanidins, predecessors of colourful anthocyanins. Anthocyanins are an economically significant group of molecules, especially for horticulturists and plant breeders, but also for nutritional and health scientists due to their potential health benefits. Dihydroflavonol 4-reductase is a much-studied enzyme due to its significant role in flavonoid biosynthesis and the economic interests of plant breeders and alike. Previous studies have expanded the knowledge of flavonoid biosynthesis and have identified several amino acid residues in the DFR structure affecting the substrate specificity of the enzyme and, consequently, the flower colours. However, only a single crystal structure model of the dihydroflavonol 4-reductase has been solved so far, originating from the grapevine Vitis vinifera. Although a single crystal structure can facilitate further structure-to-function studies associated with dihydroflavonol 4-reductase, further studies need to be carried out to shine a light on the functional basis of the enzyme. Therefore, this study aims to resolve petunia and gerbera dihydroflavonol 4-reductase crystal structures, expanding the knowledge of structural variations within the uncharted families of angiosperms, Solanaceae and Asteraceae. Several recombinant protein expression systems were utilised in my attempts to solve the crystal structure of the DFRs. These systems ranged from the bacterium Escherichia coli to yeast species such as Saccharomyces cerevisiae and Pichia pastoris, as well as the tobacco plant Nicotiana benthamiana. The genes encoding for Petunia wildtype DFRA, three mutants, and three Gerbera DFR variants were cloned to several expression vectors. Their presence and expression were identified using various genetic methodologies and enzymological assays. The expression of DFRs using an E. coli-based expression system was verified. However, the trials with E. coli were deemed unsuccessful due to the majority of the protein ending in inclusion bodies with no detectable activity. An alternative system using agroinfiltration of N. benthamiana was later utilised, as significant amounts were detected in the plant tissue extracts following the agrobacterial infiltration. Although the proteins were expressed in high quantities, no purification procedures have been established to provide plant tissue-extracted protein in crystallography-grade purity. With the protein supplied by a plant-based system and several small- scale purification steps, purified DFR enzymes could be utilised in crystallisation studies. Due to significant contamination by RuBisCO in the protein samples, alternative systems based on S. cerevisiae and Pichia pastoris were investigated, and a successful Pichia-based expression was established. Several sets of plasmids with variable expression systems were constructed in this study, facilitating future experiments into the dynamics and structure of dihydroflavonol 4-reductases. Ground-breaking techniques based on computational modelling were utilised to hypothesise the role of prior determined amino acid residues in enzyme catalysis and substrate recognition. Possible crystallisation-related issues originating from protein structure were approached using the same techniques, opening new windows and possibilities into determining the structure of Petunia hybrida and Gerbera hybrida dihydroflavonol 4-reductase structures using tools of protein engineering.

Lymphatic vascular system consists of lymphatic capillaries and collectors existing alongside a circulatory system of blood vessels. The lymphatic system is responsible of draining tissue fluids, trafficking of immune cells and intestinal absorption of dietary lipids. Most of the lymphatic networks develop during embryogenesis, but lymphangiogenesis (the growth of new lymphatic vessel, LV) occurs also in adult tissues, for example, during inflammation. Exposure to vascular endothelial growth factor C (VEGF-C) initiates lymphatic endothelial cell (LEC) proliferation and sprouting of LVs. In lymphangiogenesis, leading tip cell migrates and samples the surrounding environment while stalk cells proliferate and are responsible of LV elongation and extension. Since polarity of dividing cells and subsequent daughter cell positioning possess a key role in morphogenesis of tubular organs, such as lungs, kidney or blood vessels, a regulation of daughter LEC positioning after cell division might determine how LVs elongate and widen. The aim of this study was to investigate the LV network enlargement and daughter LEC positioning during growth of LVs and to reveal potential contributing factors guiding the cell positioning (such as cell polarity). In this study, the LV network of mouse ear pinna was used as a model tissue to investigate LV network enlargement, daughter LEC positioning and LEC polarity in growing LVs. Characterization of mitotic cells in developing LV network revealed that LEC proliferation occurs throughout the entire length of LVs in the network. To investigate LEC polarity in developing and mature LVs, I analysed Golgi and nuclear polarity of tip and stalk LECs. I found that whereas LECs during development are polarized and oriented along the long axis of LV, there is more variation in the direction of LEC polarity in relation to LV long axis in mature LV. This observation raised a question whether changes in the cell polarity were reflected to cell positioning, hence I analysed the positioning of daughter LECs by forcing LECs to the cell cycle with VEGF-C. These results indicated cell-level mechanisms that may contribute to LEC positioning in lymphangiogenesis. My finding provides an efficient tool for further research due to its suitability for monitoring proliferating LECs and studying causative factors affecting LEC proliferation and positioning. Future experiments with real-time imaging will reveal more about lymphangiogenesis process and provide insights into the role of lymphatic vasculature in conditions such as inflammation-related lymphedema or anti-tumor immunity in cancer.

The discovery and development of new antifungal drugs has lagged behind the clinical needs for effective treatments of fungal infections. Invasive fungal infections can be challenging to treat and can become life-threatening, particularly for immunosuppressed individuals. Despite the need for new and improved antifungal drugs, the pipeline for antifungal drug development has been relatively slow, with only a few new agents being approved in recent years. Many existing antifungal drugs have toxic side effects, limiting their use and highlighting the need for more targeted and effective therapeutics. The glycosylphosphatidylinositol biosynthesis pathway has been proposed as a potential new target for antifungal drugs. The glycosylphosphatidylinositol (GPI) anchor is a complex glycoconjugate that is attached to many proteins found on the surface of eukaryotic cells. GPI anchored proteins play important roles in various cellular processes, including signaling, cell adhesion, and cell recognition. The biosynthesis of GPI anchors involves multiple enzymatic steps, including the transfer of the GPI anchor to a target protein. Gpi3 is one of the key enzymes involved in the first step of GPI biosynthesis and is the catalytic subunit of the GPI GlcNAc-PI synthesis complex. The naturally occurring molecule Jawsamycin has been shown to selectively inhibit fungal Gpi3 while not interfering with its human ortholog. However, the development and research of Jawsamycin and other potential inhibitors of the GPI synthesis pathway are hampered by the lack of structural data on the proteins involved in the pathway. This thesis aimed to express and purify functionally active Gpi3 as a recombinant fusion protein using the SUMO tag expression system, with the end goal of utilizing the protein for structural studies through crystallography to better understand the function of Jawsamycin. In this thesis, Gpi3 was successfully expressed and purified as a fusion protein. However, enzymatic activity of Gpi3 was not observed, additionally, the purification and stability of the Gpi3 fusion proteins were shown to be problematic and no crystal structure of the protein of interest was acquired. These results indicate that a different approach is needed to gain structural insights into the function and interaction between Gpi3 and Jawsamycin. A likely path forward is the purification of the whole GPI GlcNAc synthesis complex which could give more insights into the organization and function of both Gpi3 and Jawsamycin.

COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has thus far claimed over six million lives. Vaccines against SARS-CoV-2 have successfully mitigated severe disease and eased the burden on healthcare systems. Neutralizing antibodies play crucial roles both in vaccine derived immunity, and as drugs widely utilized in monoclonal antibody therapy. Neutralizing antibodies primarily target the spike protein, where most of the neutralizing epitopes are located in the receptor binding domain (RBD). Elucidating the sites of vulnerability to neutralization on SARS-CoV-2 can facilitate the development of therapeutics. 7A12 is a newly-discovered IgG antigen-binding fragment (Fab) isolated from a COVID-19 patient in Finland that can neutralize SARS-CoV-2 by targeting the spike protein. In this thesis, a complex of the Fab 7A12 with SARS-CoV-2 spike ectodomain trimer was studied using cryogenic electron microscopy (cryo-EM) single-particle analysis to elucidate the epitope of 7A12 and to gain insight into the neutralization mechanism of 7A12. Cryo-EM data of the complex revealed that Fab 7A12 can bind to both “open” and “closed” conformations of RBD. Rigid-body fitting of the spike trimer and Fab 7A12 models into the cryo-EM density indicates that 7A12 recognizes an epitope in the RBD which is mainly located outside the ACE2 binding site. Together, these results suggest that the 7A12 epitope belongs to class III of SARS-CoV-2 neutralizing epitopes located in the RBD, indicating that 7A12 could neutralize by sterically hindering ACE2 and by preventing the adjacent RBD from changing to ”up” conformation. Furthermore, our results revealed an overlap of 7A12 epitope with the putative binding site of heparan sulfate, a host factor of SARS-CoV-2 infection, which might contribute to neutralization. 7A12-RBD interface mapping delineated the residues comprising the epitope, which do not include mutants found in Beta, Gamma, and Delta variants, while four mutants were found in Omicron within the epitope indicating that 7A12 is likely to neutralize Beta, Gamma, and Delta variants of concern.