Oral Presentation Abstracts 2023

Areali Antonia¹

¹ NTNU

Secretion systems in bacteria are of high scientific importance, as various molecules can be secreted from bacteria to respond in different processes. Many factors can affect bacterial secretion systems and the export of proteins, therefore understanding of those factors is the key for a proper biotechnological exploitation of microbial secretion systems. Currently, several bacteria workhorses like Escherichia coli have been extensively studied and engineered to produce high value proteins. The purpose of this research is to study, engineer, optimize and test for secretion limitations regarding protein size in the flagellar secretion systems T3SS which is one of the most important secretion systems in bacteria and has shown a remarkably fast exportation of proteins to the medium. As proof-of-concept, within this project we will establish biotechnological production of high value proteins as, for instance, protein-based fish vaccines at different production scales.  

Diego S. Reyes-Weiss¹, Nanna Rhein-Knudsen¹, Bjørge Westereng¹, Svein J. Horn¹

¹ Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway

Fucoidans are a heterogeneous group of sulfated polysaccharides found in the cell wall of brown seaweeds. Fucoidans have attractive valorisation prospects due to several reported biological activities (Fitton et al. 2019), and recent studies suggest that they play an important role in carbon sequestration (Buck-Wiese et al. 2023). Typically, fucoidans are composed of a linear backbone of α-L-fucose units arranged as α-1,3-, or alternating α-1,3;α-1,4- linkages, with varying degree of sulfation at carbon 2, 3, and 4.  Additionally, they can be branched, acetylated, and contain galactose, xylose, mannose, rhamnose, glucose, and uronic acids units. The complex and largely undefined structure of fucoidans, together with their large environmental variability, represent a major challenge in the understanding of their bioactivities and for their processing. Fucoidan-active enzymes, found in many marine bacteria, are classified into different glycosyl hydrolase (GH) families within the carbohydrate-active enzyme database (CAZy). Different types of enzymatic activities have been described, including endo- and exo-fucoidanases, sulfatases, carbohydrate esterases, and lyases, all potential enzymatic tools for processing of fucoidans and for structural elucidation. In this study, we identified a new putative endo-1,3-fucoidanase (FunA50) by blasting the sequence of FunA (Shen et al. 2020), the only characterized member from family GH168, against the genome of Lentimonas sp. CC4, a marine bacterium highly specialized in fucoidan degradation (Sichert et al. 2020). FunA50 was recombinantly produced in E. coli and activity on a fucoidan from Fucus vesiculosus was detected by screening against a collection of fucoidans from 13 brown seaweed species using size-exclusion chromatography. Optimal reaction conditions (35 °C, pH 5.6, and 320 mM NaCl) were determined using the colorimetric PAHBAH method for quantification of reducing ends. Current work is being carried out to elucidate the specificity of FunA50.

References:

(1) Buck-Wiese et al. (2023). Fucoid brown algae inject fucoidan carbon into the ocean. Proc. Nat. Acad. Sci. U.S.A., 120, e2210561119 doi: 10.1073/pnas.2210561119
(2) Fitton et al. (2019). Therapies from fucoidan: New developments. Mar. Drugs, 17, 571. doi: 0.3390/md17100571.
(3) Shen et al. (2020). Discovery and characterization of an endo-1,3-fucanase from marine bacterium Wenyingzhuangia fucanilytica: A novel glycoside hydrolase family. Front. Microbiol., 11:1674. doi: 10.3389/fmicb.2020.01674
(4) Sichert et al. (2020). Verrucomicrobia use hundreds of enzymes to digest the algal polysaccharide fucoidan. Nat. Microbiol., 5, 1026-1039. doi: 10.1038/s41564-020-0720-2.   

Gutiérrez-Fernández, J.¹, Hammerstad, M.², Hersleth, H.P.²

¹ Structural Biology Team, Norway Center for Molecular Medicine, University of Oslo, NO-0318 Oslo, Norway
² Department of Biosciences, University of Oslo, Section for Biochemistry and Molecular Biology, NO-0316 Oslo, Norway

Low molecular thiols are involved in many processes in all organisms playing a protective role against reactive oxygen, chlorine and electrophilic species, heavy metals, toxins and antibiotics. Not only they maintain the reduced state of cytosolic proteins but also act as cofactors of many oxidoreductases. This is the case of the mycothiol disulphide reductase (Mtr), an oxidoreductase of Actinobacteria that is able to reduce mycothiol disulphide (MSSM) to mycothiol (MSH) which could be oxidized again by reactive species, thus contributing to the redox homeostasis. To catalyze the reduction of MSSM, Mtr oxidizes NADP+H⁺ into NADP⁺ through a flavin cofactor (FADH₂ to FAD). In this work we show crystal structures of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases (MsMtr and ReMtr respectively) to unveil their mechanism of action, the binding of mycothiol disulphide and to analyze the increased affinity ReMtr shows by certain additional substrates, such as the telluride oxyanion tellurite (TeO₃^2-). This information will also allow us to design potential compounds targeting Mtr with therapeutic purposes.

References:

(1) Hammerstad, M. et al. (2020) The Crystal Structures of Bacillithiol Disulfide Reductase Bdr (YpdA) Provide Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductase. Biochemistry, 59, 4793-4798.
(2) Arenas-Salinas, M. et al. (2016) Flavoprotein-Mediated Tellurite Reduction: Structural Basis and Applications to the Synthesis of Tellurium-Containing Nanostructures. Front. Microbiol. 7, 1160.
(3) Kumar, A. et al. (2017) Structural and mechanistic insights into Mycothiol Disulphide Reductase and the Mycoredoxin-1-alkylhydroperoxide reductase E assembly of Mycobacterium tuberculosis. BBA, 1861, 9, 2354-2366.

Juliana M Tatara¹, Amanda Moraes¹, Walter O. Beys-da-Silva^1,2, Lucélia Santi^1,2

¹ Post-Graduation Program in Cellular and Molecular Biology, UFRGS. Porto Alegre, RS. Brazil.
² Center of Experimental Research, Clinical Hospital of Porto Alegre. Porto Alegre, RS. Brazil

Klebsiella pneumoniae is a gram-negative encapsulated bacterium part of the ESKAPE group, which comprises six highly virulent and antibiotic resistant bacterial pathogens [1]. While most microorganisms are unable to resist the microbicidal effect of human serum, some serum-resistant bacteria have evolved and developed mechanisms to subvert this barrier [2-3]. Although efforts have been made to understand the mechanisms involved in serum resistance, the molecular mechanisms are still poorly understood. Herein, we use proteomics (MALDI-TOF mass spectrometry) to evaluate the molecular alterations in a clinical isolated strain of K. pneumoniae (resistant to 16 antibiotics) triggered after active and heat-inactivated serum (control) exposure for 1 and 4 hours, and its potential antibacterial targets. Cell viability assay indicates K. pneumoniae to be resistant to active serum. After one hour of active serum exposure, “thiamine biosynthetic process” was the third most significant impacted biological process and KEGG analysis pointed to a modulation of “thiamine metabolism”, highlighting 4 upregulated proteins in this metabolic pathway. This suggests a higher production of thiamine diphosphate/pyrophosphate (TPP), an essential cofactor for several cellular processes [4] which is also involved in the pathogenesis of Pseudomonas aeruginosa [5]. In situations of nutritional deprivation or a stressful environment, Escherichia coli can accumulate thiamine as a defence mechanism [6]. When we added exogenous thiamine to active serum, bacteria presented increased resistance. Overall, our data suggest that K. pneumoniae changes its metabolism to evade the host immune system, as can be observed in the predicted activation of the “thiamine metabolism”, specifically the production of TPP metabolite. Further experiments must be conducted to explore this relationship with serum resistance, as well as to develop novel therapeutic targets, such as inactivation of some enzymes within the Thiamine Metabolism pathway.

References:

(1) De Oliveira, David M P et al. “Antimicrobial Resistance in ESKAPE Pathogens.” Clinical microbiology reviews vol. 33,3 e00181-19. 13 May. 2020, doi:10.1128/CMR.00181-19
(2) Samant, Shalaka et al. “Nucleotide biosynthesis is critical for growth of bacteria in human blood.” PLoS pathogens vol. 4,2 (2008): e37. doi:10.1371/journal.ppat.0040037
(3) Sheldon, Jessica R et al. “Iron Acquisition Strategies of Bacterial Pathogens.” Microbiology spectrum vol. 4,2 (2016): 10.1128/microbiolspec.VMBF-0010-2015. doi:10.1128/microbiolspec.VMBF-0010-2015
(4) Bettendorff, Lucien et al. “Thiamine triphosphate: a ubiquitous molecule in search of a physiological role.” Metabolic brain disease vol. 29,4 (2014): 1069-82. doi:10.1007/s11011-014-9509-4
(5) Kim, Hyung Jun et al. “The ThiL enzyme is a valid antibacterial target essential for both thiamine biosynthesis and salvage pathways in Pseudomonas aeruginosa.” The Journal of biological chemistry vol. 295,29 (2020): 10081-10091. doi:10.1074/jbc.RA120.013295
(6) Lakaye, Bernard et al. “Thiamine triphosphate, a new signal required for optimal growth of Escherichia coli during amino acid starvation.” The Journal of biological chemistry vol. 279,17 (2004): 17142-7. doi:10.1074/jbc.M313569200

Lisa Reinmuth¹, Mar Trinidad Fernandez¹, Petri Kursula² and Jan Haavik¹

¹ Department of Biomedicine, University of Bergen, Bergen, Norway
² Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Oulu, Finland

Neurological disorders are an increasing issue in our society, getting recognized more frequently and especially burdening the aging population. Treatment options are often limited, only effective against specific symptoms or just slowing the disease progression. Additionally, neurological issues often appear clustered in individuals, meaning the chances of having multiple disorders are heightened. This is due to converging mechanisms across the wide variety of neurological and neurodevelopmental pathologies, like Alzheimer’s or autism.

One of these converging pathways is mediated through CRMP2, a cytosolic protein involved in signal transmission in pathologies like Alzheimer’s, chronic pain and epilepsy. CRMP2 regulates neurite outgrowth, membrane trafficking and neuronal excitability through protein-protein interactions. This, in turn, is modulated by post-translational modifications, which prime specific pathways.

In our work, we are screening ligands selective for certain pathways and trying to elucidate the protein-ligand interaction. This can then be the base for improved ligands targeting the same pathway, or ligands adapted to target multiple pathways at a time to treat patients suffering from multiple diseases with one compound.

One key prerequisite for this is the oligomeric state of the protein. Purified CRMP2 can exist as monomer, dimer, tetramer and several higher order oligomers. The tetrameric structure is well known, this state has been crystalized and was long considered to be the natural state of the protein. However, in this form the protein-protein interface is largely inaccessible due to the tight packing, with the unordered C-terminus potentially wrapping around the complex.

One hypothesis is that post-translational modifications on this tail can open up the complex to allow specific interactions. In chronic pain signalling for example, the protein must be phosphorylated on S522 before K374 can be SUMOylated, which in turn regulates membrane trafficking of the sodium channel NaV1.7, modulating the ability to generate action potentials.

Using a full length and truncated protein construct, which is missing the C-terminus after residue 490, we are optimizing conditions to get specific oligomeric states. Co-crystals combined with assays to analyse ligand binding kinetics and interaction effects are beginning to shed light on what is needed of a successful drug candidate.

Mina Bathen¹, Åsmund Haatveit¹, Anders Vik¹ and Trond Vidar Hansen¹

¹ Department of Pharmacy, Section for Pharmaceutical Chemistry, University of Oslo, PO Box 1068 Blindern, N-0316 Oslo, Norway

Epoxy fatty acids (EpFAs) are biosynthesised from dietary polyunsaturated fatty acids  (PUFAs) by the enzyme cytochrome P450 (CYP450).¹ The EpFAs are lipid mediators with potent anti-inflammatory² and pain relief effects.³ The most important degradation pathway for the EpFAs is hydrolysis by the enzyme soluble epoxide hydrolase (sEH), which gives the corresponding dihydroxy fatty acids.

The PUFA-product 4S,5S-dihydroxy docosapentaenoic acid (1) could be the product of such an enzymatic process. This PUFA has been reported from various human samples⁴ and has not yet been the target of stereoselective total synthesis. This poster presents our current work on a stereoselective semi-synthesis of 1 from docosahexaenoic acid (DHA).

References:

(1) Morisseau, C.; Hammock, B. D. Impact of Soluble Epoxide Hydrolase and Epoxyeicosanoids on Human Health. Annu. Rev. Pharmacol. Toxicol. 2013, 53 (1), 37-58.
(2) Wagner, K. M.; McReynolds, C. B.; Schmidt, W. K.; Hammock, B. D. Soluble epoxide hydrolase as a therapeutic target for pain, inflammatory and neurodegenerative diseases. Pharmacol. Ther. 2017, 180, 62-76.
(3) Wagner, K.; Inceoglu, B.; Hammock, B. D. Soluble epoxide hydrolase inhibition, epoxygenated fatty acids and nociception. Prostaglandins Other Lipid Mediat. 2011, 96 (1-4), 76-83.
(4) (a) Lundström, S. L.; Yang, J.; Brannan, J. D.; Haeggström, J. Z.; Hammock, B. D.; Nair, P.; O’Byrne, P.; Dahlén, S.-E.; Wheelock, C. E. Lipid mediator serum profiles in asthmatics significantly shift following dietary supplementation with omega-3 fatty acids. Mol. Nutr. Food Res. 2013, 57 (8), 1378-1389. (b) Schuchardt, J. P.; Schneider, I.; Willenberg, I.; Yang, J.; Hammock, B. D.; Hahn, A.; Schebb, N. H. Increase of EPA-derived hydroxy, epoxy and dihydroxy fatty acid levels in human plasma after a single dose of long-chain omega-3 PUFA. Prostaglandins Other Lipid Mediat. 2014, 109-111, 23-31.

Philipp Garbers¹, Sara M. Gaber², Hans A. Brandal¹, Aksel V. Skeie¹, Svein H. Knutsen² Catrin Tyl¹, and Bjørge Westereng¹

¹ Norwegian University of Life Science (NMBU), Ås, Norway
² Nofima AS, Ås, Norway

Pulses like peas or faba beans are interesting in the shift towards a more sustainable food ecosystem and healthier diets due to the plants ability to fixate nitrogen as well as their constituents with favourable nutritional effects, most notably protein and dietary fibre. However, they also contain constituents of potential concern such as fermentable oligo-, di- and monosaccharides and polyols (FODMAPs), most significantly the raffinose family oligosaccharides (RFOs).

The aim of this work is therefore to develop a sustainable and industrially feasible process for the extraction of RFOs from protein concentrates and residuals derived from pulses. At the same time, the aim is to also utilize the extracted oligosaccharides to produce new and potentially value-added food products or ingredients. To extract the oligosaccharides from the milled plant material, a water-based pilot-scale process was designed that utilizes solubilization, separation, and filtration technology to produce a concentrated RFO extract as well as a protein enriched fraction. The fractions were freeze-dried and characterized. The RFO extract was furthermore used as carbon source in fermentation studies with food-grade microorganisms, for example as a possibility to enhance the growth of lactic acid bacteria in modern sour beer production as previously demonstrated with wood derived oligosaccharides. Furthermore, the potential enzymatic modifications of RFOs are researched through recombinant enzyme production and activity screenings.

The developed process allowed for kilogram-scale extraction of high purity RFOs from pea protein concentrates. The residual material after extraction had on the other hand increased protein and decreased oligosaccharide content in comparison to the starting material. The freeze-dried extract was shown to be a suitable carbon source for a variety of food-grade microorganisms and was successfully applied as an additional carbon source for these organisms in the production of sour beer. In a first step towards enzymatic modifications, activity of pure enzymes could be shown on the obtained RFO extract as well.

References:

(1) Green Technology for Plant Based Food, Nofima AS, 2023 – nofima.com/projects/gree-technology-for-plant-based-food/
(2) Nyyssölä et al., 2020 – Reduction of FODMAP content by bioprocessing –https://doi.org/10.1016/j.tifs.2020.03.004
(3) Dysvik et al., 2019 – Secondary Lactic Acid Bacteria Fermentation with Wood-Derived Xylooligosaccharides as a Tool to Expedite Sour Beer Production – https://doi.org/10.1021/acs.jafc.9b05459

Szymon Mikołaj Szostak,¹ Reidar Lund,¹ Alessia Battigelli²

¹Department of Chemistry, University of Oslo, Oslo, Norway
²Department of Chemistry, University of Maine, Orono, United States

Peptoids, first synthesized in 1992,¹ are a class of compounds consisting of sequence-defined oligomers of N-substituted glycine monomers that mimic the structure of peptides. Recently, they are gaining more and more attention due to their variety of side chains, biocompatibility, protease stability, and low cytotoxicity, which make them a great candidate for drug delivery platform.^2–4 One of the most promising classes is block amphiphilic peptoids that can self-assemble into nanotubes and nanoparticles. A lipophilic core can be designed to complex the drug inside the carrier, whereas a hydrophilic shell provides solubility of the carrier in the water and allow the attachment of the targeting agent.⁵ This project aims to design, synthesize and characterize several peptoids that could potentially self-assemble into nanoparticles, micelles, or vesicles.⁶ After the preparation of well-established self-assembling peptoids, attempts to incorporate the drug into the structures will be carried out.

References:

(1) Simon, R. J. et al. Peptoids: a modular approach to drug discovery. Proc. Natl. Acad. Sci. 89, 9367–9371 (1992).
(2) Chan, B. A. et al. Polypeptoid polymers: Synthesis, characterization, and properties. Biopolymers 109, (2018).
(3) Xuan, S. & Zuckermann, R. N. Diblock copolypeptoids: A review of phase separation, crystallization, self-assembly and biological applications. J. Mater. Chem. B 8, 5380–5394 (2020).
(4) Xuan, S. et al. Thermoreversible and Injectable ABC Polypeptoid Hydrogels: Controlling the Hydrogel Properties through Molecular Design. Chem. Mater. 28, 727–737 (2016).
(5) Battigelli, A. Design and preparation of organic nanomaterials using self-assembled peptoids. Biopolymers 110, e23265 (2019).
(6) Ding, S. et al. Protein-based nanomaterials and nanosystems for biomedical applications: A review. Mater. Today 43, 166–184 (2021).

Yomkippur Perez^1,2, Erik Agner¹, and Magne Olav Sydnes²

¹ Polypure
² University of Stavanger

Protein and peptide purification has been a major bottleneck in both the pharmaceutical industry and a significant portion of life science research. In all cases, target peptides and proteins must be separated from components of the host cell system or other impurities in chemical synthesis. The well-established gradient chromatography still has issues with column binding and loading capacity. More efficient purification methods are therefore needed.

Displacement chromatography offers an alternative which allows for significantly larger sample loading and more efficient binding. Since the same sample can be processed several times in the column, this method also delivers lower losses. DC has been extensively used in polymer purification and can be translated into peptides and proteins.

Amanda K. Votvik¹, Åsmund K. Røhr¹, Bastien Bissaro², Anton A. Stepnov¹, Morten Sørlie¹, Vincent G. H. Eijsink¹ and Zarah Forsberg¹

¹ Faculty of Chemistry, Biotechnology, and Food Science, The Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway.
² INRAE, Aix Marseille University, UMR1163 Biodiversité et Biotechnologie Fongiques, 13009, Marseille, France

Bacterial lytic polysaccharide monooxygenases (LPMOs) oxidize abundant and recalcitrant polymers such as chitin and cellulose. Interestingly, the genome of the model actinomycete Streptomyces coelicolor A3(2) encodes seven putative LPMOs. Phylogenetic analysis shows that four of these putative LPMOs group with typical chitin-active LPMOs, two with typical cellulose-active LPMOs, whereas one stands out by being part of a novel subclade of non-characterized enzymes. The latter enzyme, called ScLPMO10D, stands out among S. coelicolor LPMOs as the only featuring a C-terminal extension with a cell wall sorting signal (CWSS), which is expected to covalently anchor the enzyme to the cell wall peptidoglycan layer. Here, we have produced a truncated version of ScLPMO10D without the CWSS and determined its crystal structure, EPR spectrum, and various functional properties. Although showing several structural and functional features typical for cellulose-active LPMO10s, ScLPMO10D showed oxidative activity towards chitin only. Comparison with two other chitin-active LPMO10s of bacterial origin revealed interesting functional differences related to the reactivity of the catalytic copper ion. The characterization of this unique LPMO sheds light on structure-function relationships in phylogenetically distant LPMOs with similar substrate specificities and adds to our understanding of how structural variation contributes to fine-tuning of LPMO function.  

Arne Raasakka¹ and Petri Kursula^1,2

¹ Department of Biomedicine, University of Bergen, Norway
² Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Finland

Nerve impulse propagation is greatly accelerated by the lipid-rich cellular structure called myelin, which ensheathes axons in the central and peripheral nervous systems. The electric insulation of myelin is enabled by a repetitive stack of plasma membranes that are adhered together in a process called myelin compaction. Myelin is highly enriched in peripheral and integral membrane proteins that are crucial for the correct formation and stability of myelin. Disturbances in the expression levels of myelin proteins, as well as truncations and missense mutations, result in incurable demyelinating and dysmyelinating diseases that significantly deteriorate the quality of life of affected individuals. Such conditions include Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease, Guillain-Barré syndrome, and multiple sclerosis. A major problem with all of these diseases is that they are difficult to treat, mostly due to inherent heterogeneity within each disease as well as the general lack of information in the structure-function relationships of myelin proteins.

How do myelin proteins drive myelin compaction? What is the conformation of proteins between the membrane sheets? In our group, we strive to understand the structure-function relationships of myelin proteins in solution and in their native state between the myelin membranes. A remarkable attribute of many myelin proteins is that they spontaneously form stacked membrane structures when mixed with lipid vesicles or bicelles in vitro, often undergoing a disorder-to-order transition in the process. We study these proteins in complex with lipids using an array of biophysical methods, such as synchrotron radiation circular dichroism spectroscopy, small-angle X-ray diffraction, surface plasmon resonance, vesicle aggregation experiments, atomic force microscopy, electron microscopy, and neutron scattering methods. We also study the structures of myelin proteins, including structure-based and disease-associated mutant variants, using X-ray crystallography and complementary methods. We also develop tools for studying myelin proteins using biophysical methods as well as in cell culture and in vivo systems.

Flore Kersten^1,2, Gabriele Cordara², Stefanie Schmieder³, Markus Künzler³, Ute Krengel²

¹ NCMM, University of Oslo, Norway
² Department of Chemistry, University of Oslo, Norway
³ Institute of Microbiology, Department of Biology, ETH Zürich, Switzerland

CCTX2 is an 89 kDa chimerolectin expressed in the vegetative mycelium of the fungus Coprinopsis cinerea (grey shag) upon challenge with the nematode Aphelenchus avenae [1]. The protein acts as defense protein against fungivorous nematodes. CCTX2 contains four ricin B chain-like domains, two of which show lectin activity and selective binding of LacdiNAc-containing glycoepitopes. While the ricin B domains are required for cell entry, the nematotoxic activity is associated to a fifth domain, with no known orthologues outside the realm of fungi. The domain displays an “RDQ motif”, a signature sequence found in Poly-ADP-Ribose Polymerases (PARPs) [2]. However, the lack of significant sequence conservation with other PARPs outside of the RDQ-motif and the unknown fold of the fifth domain have so far prevented the unambiguous identification of its possible enzymatic activity, and the full understanding of the toxicity mechanism. Here we present the first high-resolution cryo-EM structure of CCTX2, showing that the structure of the fifth domain does not match any of the folds found the in the PDB database.

References:

(1) Plaza, D.F., Schmieder, S.S., Lipzen, A., Lindquist, E., and Künzler, M. (2015). Identification of a novel nematotoxic protein by challenging the model mushroom Coprinopsis cinerea with a fungivorous nematode. G3 (Bethesda) 6, 87-98.
(2) Schmieder, S.S. (2015). The spatiotemporal regulation and mechanism of action of fungal nematotoxic defense effectors. PhD thesis, doi: 10.3929/ethz-a-010607472.

Greta Daae Sandsdalen¹, Bjarte Aarmo Lund¹, Maryam Imam², Ole Morten Seternes³, Adele Williamson⁴ and Hanna-Kirsti Schrøder Leiros¹

¹ Biomolecular and Structural Chemistry, Department of Chemistry, UiT The Arctic University of Norway
² The Norwegian College of Fishery Science, UiT The Arctic University of Norway
³ Department of Phamacy, UiT The Arctic University of Norway
⁴ The University of Waikato, New Zealand

The CRISPR-Cas genome editing system has revolutionized molecular biology, providing an array of biotechnological tools for carrying out precision genome modification and regulation in eukaryotic cells. One limitation of the system at present is that most available tools are developed from and optimized for mesophilic organisms, which limits their utility in extremophilic organisms. Although thermophilic Cas homologs have been developed and verified for use at high temperatures, less attention has been given to the low-temperature end of the spectrum. This paucity of available knowledge on psychrophilic CRISPR-Cas systems and affiliated genome editing tools is particularly problematic for researchers of cold-blooded eukaryotes such as fish biologists, as both the current enzymes and fish cells lose viability at elevated temperatures.

This project is one of three interdisciplinary components of the UiT Strategic-Funded ‘FISH&CRISPR Innovative strategies to improve salmon health’ which aims to establish a platform for the development of a low-temperature CRISPR-Cas genome editing system optimized for salmonids.
In this project, the main goal is to discover and develop one or more CRISPR-associated endonucleases for effective and precise genome editing at low temperatures. A second aim is to investigate the abundance of CRISPR-systems across cold-adapted bacteria through bioinformatics analysis.

Through the analysis of 952 genomes of cold-adapted bacteria, it has been found that CRISPR-Cas systems are present in low abundance, with only 20% of the genomes containing them. Among the identified CRISPR-Cas systems, Class 1 is the most frequently observed. However, Type II-C with the Cas9 endonuclease is the dominant Class 2 CRISPR-Cas system. From five unique psychrophilic bacteria, five Cas endonucleases has been expressed recombinantly in soluble form. Out of the five, three has been purified at low concentrations and is currently being characterized.

Markus Miettinen¹

¹ University of Bergen, Bergen, Norway

Although molecular dynamics (MD) simulations are a widely used tool in (bio)sciences, they often actually fail to provide a veritable description (i.e. matching NMR data within experimental accuracy) of the conformations and the dynamics of the molecules simulated—a crucial prerequisite for using MD to intuitively interpret experiments. These failures can largely be attributed to the quality of the underlying MD models (force fields), improvement of which is hindered by the lack of comprehensive comparison to experiments, and outdated approaches, such as hand-tuning parameters.

The High-fidelity Biomolecular Modelling Group employs open-science approaches to test and data-driven optimization methods to improve biomolecular force fields. We strive to make such force-field-optimized MD a tool for Integrative Structural Biology: Combining data from multiple experimental techniques to generate physics-based high-fidelity structural models with realistic structure and dynamics. I argue that such approach could eventually evolve MD into a bona fide Ångström-resolution microscope.

Mateu Montserrat-Canals^1,2, Kilian Schnelle³, Vilde Leipart⁴, Øyvind Halskau⁵, Gro Amdam^4,6, Arne Moeller³, Harmut Luecke⁷ and Eva Cunha

¹ Department of Chemistry, University of Oslo, 0315 Oslo, Norway; Norwegian Centre for Molecular
² Medicine, University of Oslo, 0318 Oslo, Norway; Department of Structural Biology, Osnabrück
³ University, 49076 Osnabrück, Germany;
⁴ Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, Aas, Norway; Department of Biological Sciences, University of Bergen, Norway
⁵ School of Life Sciences, Arizona State University, Tempe, AZ, United State
⁶ NOVA School of Science and Technology, Lisbon, Portugal

Vitellogenin (Vg) is the main yolk precursor protein in almost all egg laying animals. In addition, along its evolutionary history, Vg has developed a range of new functions in different taxa. In the honey bee, Vg has functions related to immunity, antioxidant protection, social behaviour and longevity. However, the molecular mechanisms underlying Vg functionalities are still poorly understood. Here, we report our cryo-EM studies on Vg purified directly from the haemolymph of honey bees.

Rannei Skaali, Dag Ekeberg, Hanne M. Devle, and Morten Sørlie

Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ås, Norway

Lignin is abundant in nature and can be biorefined by reductive catalytic fractionation (RCF) for industrial use. Monolignols such as 4-propylphenol, 4-propylguaiacol, and 4-propylsyringol are obtained from lignin in this process. The commercial value of monolignols is increased by monolignol oxyfunctionalization that can be used in a sustainable green industry. Using synthetic chemicals to oxidize monolignols is not sustainable due to their environmental impact, costs, and low selectivity. However, enzymes are a green alternative and unlike synthetic chemicals, can catalyze selective oxyfunctionalization. Preferable positions for monolignol oxidation are the C_a and C_g producing the corresponding benzoic acid and alcohol, aldehyde, or carboxylic acid, respectively (Fig. 1). Here, we present a validated method for the identification and quantification of native and oxidized monolignols by direct infusion electrospray ionization mass spectrometry (DI-ESI-MS).

Figure 1: Native and oxidized monolignols. Monolignols such as 4-propylphenol (R = R’ = H), 4-propylguaiacol (R = H, R’ = OCH3), and 4-propylsyringol (R = R’ = OCH3) are obtained from lignin. Pseudomolecular ions are observed in a total ion chromatogram (MS) which can be differentiated from one another by fragmentation using collision-induced dissociation (MS²).

Robin Jeske¹

¹ Arctic University of Norway, Department of Chemistry, Norstruct

Peptide therapeutics have gained significant attention in recent years due to their potential as effective drugs for treating a variety of diseases caused by druggable targets^[1].

Recent advances in peptide design and computational methods have improved our ability to model and manipulate peptide-mediated interactions^[2]. Combining binding site prediction with peptide docking has been shown to be effective in modeling peptide-protein interactions^[3].

To take advantage of this advancement to design novel peptides with therapeutic application, understanding of the binding pathway is important.

Flexible protein segments and bound peptides or ligands can populate several microstates in thermodynamic equilibrium, indicating intermediate flexibility. A peptide, for example, can significantly populate multiple microstates in thermodynamic equilibrium^[4].

Induced fit cannot generate the arbitrary conformational states required for the biological function of many enzymes. Instead conformational selection accesses various conformational states, and each binding event takes place via conformational selection coupled to a population shift, which allosterically guides the next binding event^[1]. Therefore, binding is viewed as a game of induced fit and conformational selection, with one partner’s conformation serving as an environment or set of preconditions for the other partner

References:

(1) K. Fosgerau and T. Hoffmann, “Peptide therapeutics: current status and future directions,” Drug Discovery Today, vol. 20, pp. 122–128, jan 2015.
(2) G. Weng, J. Gao, Z. Wang, E. Wang, X. Hu, X. Yao, D. Cao, and T. Hou, “Comprehensive evaluation of fourteen docking programs on protein–peptide complexes,” vol. 16, pp. 3959–3969, apr 2020.
(3) O. Schueler-Furman and N. London, eds., Modeling Peptide-Protein Interactions. Springer New York, 2017.
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