Taylor Dispersion-Induced Phase Separation for the Efficient Characterisation of Protein Condensate Formation.

Biomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid-liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion-induced phase separation (TDIPS), a method to robustly measure condensation phenomena using a commercially available microfluidic platform. It uses only nanoliters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates in vivo, PGL-3 and Ddx4. Uniquely accessible to this method, we find an unexpected re-entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both α-synuclein and for lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS-modifying compounds can be screened highly efficiently.

1H, 13C, 15N resonance assignment of the enzyme KdgF from Bacteroides eggerthii.

To fully utilize carbohydrates from seaweed biomass, the degradation of the family of polysaccharides known as alginates must be understood. A step in the degradation of alginate is the conversion of 4,5-unsaturated monouronates to 4-deoxy-L-erythro-5-hexoseulose catalysed by the enzyme KdgF. In this study BeKdgF from Bacteroides eggerthii from the human gut microbiota has been produced isotopically labelled in Escherichia coli. Here the 1H, 13C, and 15N NMR chemical shift assignment for BeKdgF is reported.

Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation.

Liquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

Binding Sites for Oligosaccharide Repeats from Lactic Acid Bacteria Exopolysaccharides on Bovine ß-Lactoglobulin Identified by NMR Spectroscopy.

Lactic acid bacterial exopolysaccharides (EPS) are used in the food industry to improve the stability and rheological properties of fermented dairy products. ß-Lactoglobulin (BLG), the dominant whey protein in bovine milk, is well known to bind small molecules such as fatty acids, vitamins, and flavors, and to interact with neutral and anionic polysaccharides used in food and pharmaceuticals. While sparse data are available on the affinity of EPS-milk protein interactions, structural information on BLG-EPS complexes, including the EPS binding sites, is completely lacking. Here, binding sites on BLG variant A (BLGA), for oligosaccharides prepared by mild acid hydrolysis of two EPS produced by Streptococcus thermophilus LY03 and Lactobacillus delbrueckii ssp. bulgaricus CNRZ 1187, respectively, are identified by NMR spectroscopy and supplemented by isothermal titration calorimetry (ITC) and molecular docking of complexes. Evidence of two binding sites (site 1 and site 2) on the surface of BLGA is achieved for both oligosaccharides (LY03-OS and 1187-OS) through NMR chemical shift perturbations, revealing multivalency of BLGA for EPS. The affinities of LY03-OS and 1187-OS for BLGA gave K D values in the mM range obtained by both NMR (pH 2.65) and ITC (pH 4.0). Molecular docking suggested that the BLGA and EPS complexes depend on hydrogen bonds and hydrophobic interactions. The findings provide insights into how BLGA engages structurally different EPS-derived oligosaccharides, which may facilitate the design of BLG-EPS complexation, of relevance for formulation of dairy products and improve understanding of BLGA coacervation.

Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus.

Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked ß-d-mannuronic acid (M) and α-l-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel ß-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel ß-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.

Alginate Trisaccharide Binding Sites on the Surface of ß-Lactoglobulin Identified by NMR Spectroscopy: Implications for Molecular Network Formation.

ß-lactoglobulin (BLG) is a promiscuous protein in terms of ligand interactions, having several binding sites reported for hydrophobic biomolecules such as fatty acids, lipids, and vitamins as well as detergents. BLG also interacts with neutral and anionic oligo- and polysaccharides for which the binding sites remain to be identified. The multivalency offered by these carbohydrate ligands is expected to facilitate coacervation, an electrostatically driven liquid-liquid phase separation. Using heteronuclear single quantum coherence NMR spectroscopy and monitoring chemical shift perturbations, we observed specific binding sites of modest affinity for alginate oligosaccharides (AOSs) prepared by alginate lyase degradation. Two different AOS binding sites (site 1 and site 2) centered around K75 and K101 were identified for monomeric BLG isoform A (BLGA) at pH 2.65. In contrast, only site 1 around K75 was observed for dimeric BLGA at pH 4.0. The data suggest a pH-dependent mechanism whereby both the BLGA dimer-monomer equilibrium and electrostatic interactions are exploited. This variability allows for control of coacervation and particle formation of BLGA/alginate mixtures via directed polysaccharide bridging of AOS binding sites and has implication for molecular network formation. The results are valuable for design of polyelectrolyte-based BLG particles and coacervates for carrying nutraceuticals and modulating viscosity in dairy products by use of alginates.

Isoenergic modification of whey protein structure by denaturation and crosslinking using transglutaminase.

Transglutaminase (TG) catalyzes formation of covalent bonds between lysine and glutamine side chains and has applications in manipulation of food structure. Physical properties of a whey protein mixture (SPC) denatured either at elevated pH or by heat-treatment and followed by TG catalyzed crosslinking, have been characterised using dynamic light scattering, size exclusion chromatography, flourescence spectroscopy and atomic force microscopy. The degree of enzymatic crosslinking appeared higher for pH- than for heat-denatured SPC. The hydrophobic surface properties depended on the treatment, thus heating caused the largest exposure of the hydrophobic core of SPC proteins, which was decreased by crosslinking. The particle size of the treated SPC samples increased upon crosslinking by TG. Moreover, the particle morphology depended on the type of denaturing treatment, thus heat-treated SPC contained fibrillar structures, while pH-denatured SPC remained globular as documented by using atomic force microscopy. Finally, the in vitro digestability of the different SPC samples was assessed under simulated gastric and intestinal conditions. Notably heat-treatment was found to lower the gastric digestion rate and enzymatic crosslinking reduced both the gastric and the intestinal rate of digestion. These characteristics of the various SPC samples provide a useful basis for design of isoenergic model foods applicable in animal and human studies on how food structure affects satiety.

Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.

Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis transcription activation factor), cup-shaped cotyledon] TFs shows that the domains are present in similar average pre-molten or molten globule-like states, but have different patterns of order/disorder and MoRFs (molecular recognition features). ANAC046 (Arabidopsis NAC 046) was selected for further studies because of its simple MoRF pattern and its ability to interact with RCD1 (radical-induced cell death 1). Experiments in yeast and thermodynamic characterization suggest that its single MoRF region is sufficient for both transcriptional activation and interaction with RCD1. The remainder of the large regulatory domain is unlikely to contribute to the interaction, since the domain and truncations thereof have similar affinities for RCD1, which are also similar for ANAC013-RCD1 interactions. However, different enthalpic and entropic contributions to binding were revealed for ANAC046 and ANAC013, suggestive of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation does not involve folding-upon-binding, but rather fuzziness or an unknown structure in ANAC046. We suggest that the ANAC046 regulatory domain functions as an entropic chain with a terminal hot spot interacting with RCD1. RCD1, a cellular hub, may be able to interact with many different TFs by exploiting their ID-based flexibility, as demonstrated for its interactions with ANAC046 and ANAC013.