Jannik Nedergaard Pedersen

The N‐terminus of α‐synuclein is essential for both monomeric and oligomeric interactions with membranes

The intrinsically disordered protein α‐synuclein (αSN) is linked to Parkinsons Disease and forms both oligomeric species and amyloid fibrils. The N‐terminal part of monomeric αSN interacts strongly with membranes and αSN cytotoxicity has been attributed to oligomers’ ability to interact with and perturb membranes. We show that membrane folding of monomeric wt αSN and N‐terminally truncated variants correlates with membrane permeabilization. Further, the first 11 N‐terminal residues are crucial for monomers’ and oligomers’ interactions with and permeabilization of membranes. We attribute oligomer permeabilization both to cooperative electrostatic interactions through the N‐terminus and interactions mediated by hydrophobic regions in the oligomer.

Generic Structures of Cytotoxic Liprotides: Nano‐Sized Complexes with Oleic Acid Cores and Shells of Disordered Proteins

The cytotoxic complex formed between α‐lactalbumin and oleic acid (OA) has inspired many studies on protein–fatty acid complexes, but structural insight remains sparse. After having used small‐angle X‐ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein–oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core–shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off‐loading of the OA into cell membranes.

Using protein-fatty acid complexes to improve vitamin D stability

Liprotides are complexes between lipids and partially denatured proteins in which the protein forms a stabilizing shell around a fatty acid micelle core. We have previously shown that liprotides stabilize small aliphatic molecules such as retinal and tocopherol by sequestering these molecules in the fatty acid core. This opens up the use of liprotides to formulate food additives. Here, we expand our investigations to the large and bulky molecule vitamin D3 (vitD), motivated by the population-wide occurrence of vitD deficiency. We prepared liprotides using different proteins and fatty acids and evaluated their ability to protect vitD upon exposure to heating or intense UV light. Additionally, we determined the stability of liprotides toward pH, Ca(2+), and BSA. The best results were obtained with liprotides made from α-lactalbumin and oleate. These liprotides were able to completely solubilize vitD, increase the stability toward UV light 9-fold, and increase the long-term stability at 37°C up to 1,000-fold. Native α-lactalbumin binds Ca(2+), making Ca(2+) potentially disruptive toward liprotides. However, liprotides prepared by incubation at 80°C were stable toward Ca(2+), in contrast to those made at 20°C. Nevertheless, the fatty acid binding protein BSA reduced the ability of both liprotides to protect vitD; the amount of vitD remianing after 20d at 20°C decreased from 79±3% in the absence of BSA to 49±4 and 23±3% in the presence of BSA for liprotides made at 80 and 20°C, respectively. Both classes of liprotides were able to release their vitD content, as demonstrated by the transfer of vitD encapsulated in liprotides to phospholipid vesicles. Importantly, liprotides were not stable at pH 6 and below, limiting the useful pH range of the liprotides to >pH 6. Our results indicate that vitD may be encapsulated and stabilized for enrichment of clear beverages at neutral pH to improve the intake and bioavailability of vitD.

Glycolipid Biosurfactants Activate, Dimerize, and Stabilize Thermomyces lanuginosus Lipase in a pH-Dependent Fashion

We present a study of the interactions between the lipase from Thermomyces lanuginosus (TlL) and the two microbially produced biosurfactants (BSs), rhamnolipid (RL) and sophorolipid (SL). Both RL and SL are glycolipids; however, RL is anionic, while SL is a mixture of anionic and non-ionic species. We investigate the interactions of RL and SL with TlL at pH 6 and 8 and observe different effects at the two pH values. At pH 8, neither RL nor SL had any major effect on TlL stability or activity. At pH 6, in contrast, both surfactants increase TlLs thermal stability and fluorescence and activity measurements indicate interfacial activation of TlL, resulting in 3- and 6-fold improved activity in SL and RL, respectively. Nevertheless, isothermal titration calorimetry reveals binding of only a few BS molecules per lipase. Size-exclusion chromatography and small-angle X-ray scattering suggest formation of TlL dimers with binding of small amounts of either RL or SL at the dimeric interface, forming an elongated complex. We conclude that RL and SL are compatible with TlL and constitute promising green alternatives to traditional surfactants.

Role of charge and hydrophobicity in liprotide formation : A molecular dynamics study with experimental constraints

Bovine α‐lactalbumin (aLA) and oleate (OA) form a complex that has been intensively studied for its tumoricidal activity. Small‐angle X‐ray scattering (SAXS) has revealed that this complex consists of a lipid core surrounded by partially unfolded protein. We call this type of complex a liprotide. Little is known of the molecular interactions between OA and aLA, and no technique has so far provided any high‐resolution structure of a liprotide. Here we have used coarse‐grained (CG) molecular dynamics (MD) simulations, isothermal titration calorimetry (ITC) and SAXS to investigate the interactions between aLA and OA during the process of liprotide formation. With ITC we found that the strongest enthalpic interactions occurred at a molar ratio of 12.0±1.4:1 OA/aLA. Liprotides formed between OA and aLA at several OA/aLA ratios in silico were stable both in CG and in all‐atom simulations. From the simulated structures we calculated SAXS spectra that show good agreement with experimentally measured patterns of matching liprotides. The simulations showed that aLA assumes a molten globular (MG) state, exposing several hydrophobic patches involved in interactions with OA. Initial binding of aLA to OA occurs in an area of aLA in which a high amount of positive charge is located, and only later do hydrophobic interactions become important. The results reveal how unfolding of aLA to expose hydrophobic residues is important for complex formation between aLA and OA. Our findings suggest a general mechanism for liprotide formation and might explain the ability of a large number of proteins to form liprotides with OA.

α-Synucleins from Animal Species Show Low Fibrillation Propensities and Weak Oligomer Membrane Disruption

The intrinsically disordered protein α-synuclein (aSN) forms insoluble aggregates in the brains of Parkinsons disease (PD) patients. Cytotoxicity is attributed to a soluble aSN oligomeric species that permeabilizes membranes significantly more than monomers and fibrils. In humans, the A53T mutation induces early onset PD and increases the level of aSN oligomerization and fibrillation propensity, but Thr53 occurs naturally in aSNs of most animals. We compared aSNs from elephant, bowhead whale, and pig with human aSN. While all three animal aSNs showed significantly weakened fibrillation, elephant aSN formed much more oligomer, and pig aSN much less, than human aSN did. However, all animal aSN oligomers showed weakened permeabilization toward anionic lipid vesicles, indicative of decreased cytotoxicity. These animal aSNs share three substitutions compared to human aSN: A53T, G68E, and V95G. We analyzed aggregation and membrane binding of all eight mutants combining these three mutations. While the G68E mutation is particularly important in weakening fibrillation and possible toxicity, the strongest effect is seen when all three mutations are present. Thus, a small number of mutations can significantly decrease aSN toxicity.

Tailoring thermal treatment to form liprotide complexes between oleic acid and different proteins

Liprotides are protein-lipid complexes in which the fatty acids form a micelle-like core surrounded by a shell of partially unfolded protein molecules. These complexes can be formed in different ways. The simplest approach is a thermal treatment where protein and fatty acid are mixed and then incubated at elevated temperatures. Using this approach we here demonstrate that we can monitor liprotide formation in real time using Small-Angle X-ray Scattering (SAXS). Optimal conditions for liprotide formation, i.e. temperature and incubation times, as well as liprotide stability and structure, vary for different proteins. The apo form of α-lactalbumin (aLA) forms liprotides at room temperature, however, Ovalbumin (Ova) and Bovine Serum Albumin (BSA) require elevated temperatures (≥60°C) to form liprotides, and in addition, they need to be returned to lower temperatures to remain stable; repeated cycles of heating and cooling gradually dissociate the liprotides in parallel with the formation of disulfide-bonded aggregates. Real-time tracking of the formation of liprotides of BSA or Ova with OA at 60-65°C showed that liprotide formation takes place within a period of 12-18min and is preceded by a loss of secondary structure of the protein and binding of OA to the protein. Our SAXS-based approach provides a straightforward strategy to optimize liprotide formation for a wide range of different proteins.

Insight into the molecular mechanism behind PEG-mediated stabilization of biofluid lipases

Bioconjugates established between anionic polyethylene glycol (PEG) based polymers and cationic proteins have proven to be a promising strategy to engineer thermostable biocatalysts. However, the enzyme activity of these bioconjugates is very low and the mechanism of non-covalent PEG-stabilization is yet to be understood. This work presents experimental and molecular dynamics simulation studies, using lipase-polymer surfactant nanoconjugates from mesophile Rhizomucor miehei (RML), performed to evaluate the effect of PEG on enzyme stability and activity. Results demonstrated that the number of hydrogen bonds between the cationized RML and PEG chain correlates with enzyme thermostability. In addition, an increase of both the number of PEG-polymers units and cationization degree of the enzyme leads to a decrease of enzyme activity. Modelling with SAXS data of aqueous solutions of the biofluid lipases agrees with previous hypothesis that these enzymes contain a core constituted of folded protein confined by a shell of surfactants. Together results provide valuable insight into the mechanism of non-covalent PEG mediated protein stabilization relevant for engineering active and thermostable biofluids. Furthermore, the first biofluids RML with activity comparable to their cationized counterpart are presented.

Stabilizing vitamin D3 using the molten globule state of α-lactalbumin

α-Lactalbumin (α-LA) is the second most abundant bovine whey protein. It has been intensively studied because of its readiness to populate the molten globular (MG) state, a partially folded state with native levels of secondary structure but loss of tertiary structure. The MG state of α-LA exposes a significant number of hydrophobic patches that could be used to bind and stabilize small hydrophobic molecules such as vitamin D3 (vitD). Accordingly, we tested the ability of α-LA to stabilize vitD in a pH interval from 7.4 to 2; over this pH interval, α-LA transitions from the folded state to the MG state. The MG state stabilized vitD better than the folded state and was superior to the major bovine whey protein β-lactoglobulin (β-LG), which is known to stabilize vitD. At pH 7.4, β-LG and α-LA stabilized vitD to the same extent. Tryptophan fluorescence quenching measurements indicated that α-LA has one binding site at pH 7.4 but acquires an additional binding site when the pH is lowered to pH 2 to 4. Stability measurements of the vitD in the α-LA-vitD complex at different temperatures suggest that UHT processing would lead to little loss of vitD. This study demonstrates the potential of α-LA as a component in vitD fortification, particularly for low pH applications.