Johan Isaksson

A novel discoid insert that can be positioned into a standard NMR tube using an associated plunger tool has been developed. By attaching any type of semipermeable barrier to the bottom of the insert, the detection volume of the tube can be divided into two or more chambers, separated by a barrier of choice. Slice-selective experiments are then utilized to study a plethora of transport phenomena in real time by NMR spectroscopy. The technological device is presented together with a proof of concept for three different applications: permeability studies, diffusion-controlled pH titrations, and diffusion-controlled protein titrations. The developed approach unlocks new tools for scientists working in the fields of drug discovery, organic chemistry, biochemistry, chemical biology, microbiology, or electrochemistry that will enable new types of experiments to be performed by NMR spectroscopy.

Ascidians are a group of marine invertebrates, most of which are sessile and soft-bodied. Their lack of an adaptive immune system makes them rely on innate immune responses to detect and eliminate invading microbes. Antimicrobial peptides (AMPs) play an essential part in this process. In this paper, we present the isolation, structure elucidation, and bioactivities of two new cysteine-rich peptides (CRPs) from the Arctic marine ascidian Synoicum turgens. The sequences and structures of the peptides were determined by Edman degradation sequencing, mass spectrometry, and NMR analysis. This revealed two novel 2 kDa peptides, St-CRP-1 and St-CRP-2, with neutral net charge and C-terminal amidation. St-CRP-1 consisted of 18 amino acids and displayed selective and moderate growth inhibition of two Gram-positive bacterial strains (Bacillus subtilis and Corynebacterium glutamicum) at 24.6 µM, whereas St-CRP-2 consisted of 19 amino acids and inhibited the growth of B. subtilis at 49.2 µM. St-CRP-1 had no effect on two mammalian cell lines or the brine shrimp Artemia salina at the highest concentration tested. Structural analysis of the St-CRPs indicated a Cys1–Cys6, Cys2–Cys4, and Cys3–Cys5 disulfide connectivity, which is also found in alpha-defensins. The results from this study show that Arctic marine ascidians are a rich source of novel bioactive peptides.

Marine diatoms are prolific producers of bioactive metabolites, but Arctic species remain underexplored as sources of antibacterial and antibiofilm agents. Here, seven species were grown in photobioreactors (PBRs) and systematically screened for antibacterial, antibiofilm, and cytotoxic activities. All strains inhibited Gram-positive bacteria, and four reduced Staphylococcus epidermidis biofilm formation. Porosira glacialis emerged as a lead species, combining potent antibiofilm activity with favourable traits for large-scale cultivation, and no detectable cytotoxicity. Bioactivity-guided fractionation of P. glacialis yielded two antibiofilm compounds: methyl 3-hydroxyoctadecanoate, the first time reported in diatoms and newly associated with antibiofilm bioactivity, and pheophorbide a, a chlorophyll degradation product. Both inhibited S. epidermidis biofilm formation without any observed cytotoxicity. Notably, Cylindrotheca closterium exhibited cultivation-dependent antibiofilm activity, underscoring the importance of growth conditions for metabolite production. These findings highlight the potential of Arctic diatoms as a sustainable source of antibiofilm agents and support further exploration of their metabolites for antimicrobial and industrial applications.

A novel discoid insert together with a plunger tool has been developed that can be inserted into a standard NMR tube. By attaching any type of semi-permeable barrier to the bottom of the insert, the detection volume of the tube can be divided into two or more chambers, separated by a barrier of choice. Together with slice selective experiments, a plethora of transport phenomena can be studied in real-time by NMR spectroscopy. Herein, the technological solution is presented together with three proof-of-concepts for three different potential applications: permeability studies, diffusion-controlled pH titrations and diffusion-controlled protein titrations. The technology offers a new tool to scientists working in the fields of for example organic chemistry, biochemistry, chemical biology, microbiology and electrochemistry that will enable new types of experiments to be performed by NMR spectroscopy. We look forward with excitement to what the creativity of scientists around the globe in different research disciplines will be able to achieve with this new tool at their disposal.

A marine-derived Pleosporales fungus, Uzbekistanica storfjordensis, was isolated from driftwood and described as a new species. The fungus was cultivated in liquid media and a molecular networking-driven approach was used to identify potential new secondary metabolites. The targeted compounds were isolated using preparative HPLC-MS, and through extensive spectroscopic analysis, eight new ophiobolin-type sesterterpenes, bipolarolides H-O (1-8), were identified. The absolute configurations of the compounds were determined by ECD assessment. Bipolarolide L (5), M (6), and O (8) exhibited inhibitory activity against Streptococcus agalactiae with MIC values of 86, 66, and 64 μM, respectively.

Structural- and functional heterogeneity, as well as allosteric regulation, in homo-monomeric enzymes is a highly active area of research. One such enzyme is human nuclear-associated deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase), which has emerged as an interesting drug target in combination therapy with traditional nucleotide analogue treatment of cancer. We report, for the first time, a full structural dynamics study of human dUTPase by NMR. dUTPase has been investigated in terms of structural dynamics in its apo form, in complex with the modified substrate resistant to hydrolysis, 2’-deoxyuridine 5’-α,β-imido-triphosphate (dUpNHpp), as well as the product, 2’-deoxy-uridine-monophosphate (dUMP). The apo form of the enzyme displayed slow dynamics in the milli- to microsecond regime in relaxation dispersion experiments, which was further slowed down to observable heterogeneity upon substrate-analogue binding. The results suggest that the non-hydrolysable substrate-analogue traps the enzyme in the conformational isomerization step that has been previously suggested to be part of the enzyme catalysis kinetics cycle. The observed heterogeneity fits well with the pattern expected to emerge from the suggested kinetic model, and no evidence for homotropic allosterism was found. The heatmaps of the slow dynamics, chemical shift perturbation upon substrate binding and conserved regions of the enzyme sequence all displayed a similar pattern, which suggests that the structural dynamics is finely tuned and important for the biological function of the enzyme for binding, conformational shift, catalysis and substrate release.

Cationic, antimicrobial peptides (AMPs) are abundantly present in nature as host-defensive peptides, forming the backbone of the natural defence of many organisms. Their primary interaction with bacterial membranes is thought to be a binding to the outer layer, followed by a disruption of the lipid membrane. Investigating the mode-of-action of AMPs is an ever-growing field, where a plethora of different biophysical experiments are used to investigate this. Here, we explore the experimental limitation in NMR spectroscopy as we migrate towards increasingly realistic but larger membrane model systems with the aim to scout which data we could realistically extract from AMPs interacting with self-aligning nanodiscs. A set of cyclic hexapeptides were used as model compounds, in water, DMSO, and SDS micelles. The quality of isotropic and anisotropic NMR parameters decreased as the result of faster relaxation due to size and dynamics, as well as the need for water suppression and buffer salts. Chemical shifts, scalar couplings, NOE derived distances, and simulated annealing suggested that the backbone conformation of both peptides remained mostly rigid, forming two β-turns, while the sidechains remain flexible in all environments. The difference between Lys or Arg did not change the backbone conformation, even though the two peptides have different MIC concentrations, indicating that amino acid composition is more important than conformation for antimicrobial activity. Theoretical structures in an explicit lipid bilayer were simulated showing similar structures. Pilot experiments on SMA-QA nanodiscs indicated that they are viable as membrane model for investigating the interaction between AMPs and lipid membranes. It was shown that signal can be obtained from bound peptide, but further investigations with isotopically labelled peptides are needed to extract relevant structural data under conditions where the nanodisc is not over-saturated by peptides.

Structural- and functional heterogeneity, as well as allosteric regulation, in homo-monomeric enzymes is a highly active area of research. One such enzyme is human nuclear-associated deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), which has emerged as an interesting drug target in combination therapy with traditional nucleotide analogue treatment of cancer. We report, for the first time, a full structural dynamics study of human dUTPase by NMR. dUTPase has been investigated in terms of structural dynamics in its apo form, in complex with the modified substrate resistant to hydrolysis, 2'-deoxyuridine 5'-a,b-imido-triphosphate (dUpNHpp), as well as the product, 2'-deoxy-uridine-monophosphate (UMP). The apo form of the enzyme displayed slow dynamics in the milli- to microsecond regime in relaxation dispersion experiments, which was further slowed down to observable heterogeneity upon substrate-analogue binding. The results suggest that the non-hydrolysable substrate-analogue traps the enzyme in the conformational isomerization step that has been previously suggested to be part of the enzyme catalysis kinetics cycle. The observed heterogeneity fits well with the pattern expected to emerge from the suggested kinetic model, and no evidence for homotropic allosterism was found. The heatmaps of the slow dynamics, chemical shift perturbation upon substrate binding and conserved regions of the enzyme sequence all displayed a similar pattern, which suggests that the structural dynamics is finely tuned and important for the biological function of the enzyme for binding, conformational shift, catalysis and substrate release.

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point need to either cross or just interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions with novel molecules -both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing relevant synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Bacterial extracellular vesicles (bEVs) are naturally secreted by Gram-negative and Gram-positive bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. bEVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work, we have isolated and characterized bEVs from one Escherichia coli mutant and three clinical strains of the ESKAPE pathogens Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The bEVs were shown to be representative models for the bacterial membrane in terms of lipid composition with speciesstrain specific variations. The bEVs were further used to probe the interactions between bEV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that bEVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect induced by antibiotics, or the response to host-pathogen interactions.

One strategy to combat antimicrobial resistance is the discovery of new classes of antibiotics. Most antibiotics will at some point interact with the bacterial membrane to either interfere with its integrity or to cross it. Reliable and efficient tools for determining the dissociation constant for membrane binding (K_D) and the partitioning coefficient between the aqueous- and membrane phases (K_P) are therefore important tools for discovering and optimizing antimicrobial hits. Here we demonstrate that microscale thermophoresis (MST) can be used for label-free measurement of K_D by utilising the intrinsic fluorescence of tryptophan and thereby removing the need for chromophore labelling. As proof of principle, we have used the method to measure the binding of a set of small cyclic AMPs to large unilamellar vesicles (LUVs) and two types of lipid nanodiscs assembled by styrene maleic acid (SMA) and quaternary ammonium SMA (SMA-QA). The measured K_D values correlate well with the corresponding measurements using surface plasmon resonance (SPR), also broadly reflecting the tested AMPs’ minimal inhibition concentration (MIC) towards S. aureus and E. coli. We conclude that MST is a promising method for fast and cost-efficient detection of peptide-lipid interactions or mapping of sample conditions in preparation for more advanced studies that rely on expensive sample preparation, labelling and/or instrument time.