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How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers which provide the scaffold for ribosome assembly but also prevent protein aggregation as part of the cellular stress response. Examining the relationship between the self-assembly and chaperone activity of NPM1, we find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning and cryo-electron microscopy reveal fuzzy interactions between the disordered region and the C-terminal nucleotide-binding domain that cross-link NPM1 pentamers into oligomers. Ribosomal peptides mediate in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. We conclude that NPM1 uses a “grappling hook” interaction to form a network-like structure whose chaperone activity is tuned by basic proteins, suggesting a regulatory mechanism for the nucleolar stress response.

R-phycoerythrin is a fluorescent red protein-pigment complex present in red algae. It belongs to the light-harvesting phycobiliprotein family with phycocyanin and allophycocyanin, which together make up the phycobilisome. The protein is most commonly used for its fluorescence properties in biochemistry. To determine the highest R-phycoerythrin yielding red algal species, a reliable quantification method is required. Calculating R-phycoerythrin yield from the crude extract’s absorption values has been the quickest and most widely used technique but at the same time, often misleading approach when impurities are present. Small phycoerythrin, which is often present in red algal suspensions, alters the absorption curve and leads to erroneous results. In the given study, we utilized an HPLC-SEC method with fluorescence and photo-diode array detectors, which is capable of separating the phycobiliproteins from interfering compounds. Also, the technique can detect and quantitate R-phycoerythrin yields reliably. A total of nine red algal species from Estonian and Japanese waters were tested, and small phycoerythrin was detected from most of the samples. For two of the tested species, the spectrophotometric quantification methods overestimated the yields by more than 200%.

Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion-ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.

Pure Na+ forms of furcellaran (β/κ-hybrid carrageenan), κ-carrageenan, κ/ι-hybrid carrageenan, ι-carrageenan and a hybrid carrageenan mix (made up of κ-, ι-, λ- and ν-carrageenans) were tested as beer wort fining agents. To find out which of the used carrageenans are the best beer wort clarifiers and whether they are prone to overdosage, polysaccharides with varying concentrations and molecular weights were tested. The polysaccharides were characterized by 1H NMR spectroscopy and size-exclusion chromatography. The fining efficiency of the tested polysaccharides was measured nephelometrically. Ultrasonicated and alkali treated carrageenan preparations were tested as beer wort fining agents and compared to the native samples. Undegraded furcellaran, κ-carrageenan and κ/ι-hybrids were efficient in lowering the turbidity of various beer worts, but the addition of ι-carrageenan and the carrageenan mix caused the turbidity to rise above the unfined sample value. Compared to the degraded samples, native galactans were more effective in lowering the beer wort turbidity. Higher concentrations of ultrasonicated carrageenans are needed to clarify the beer wort to similar turbidity values. Alkali treated carrageenans were more efficient beer wort fining agents compared to the native samples.

Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT* domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT*. In summary, we demonstrate how inducing co-translational folding via a molecular “spindle and thread” mechanism can overcome poor translation efficiency of partially disordered proteins.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder hamper the development of cancer therapeutics. Here, we probe the role of N-terminal disorder in p53 by fusing the protein to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Electron microscopy and mass spectrometry reveal that the protein adopts a compact conformation. Molecular dynamics simulations and NMR measurements show that the disordered transactivation domain of p53 is wrapped around the NT* domain, resulting in a pseudo-globular structure. We find that expression of partially disordered cancer targets is similarly enhanced by fusion to NT*. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT

Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring‐shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light‐harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.

Furcellaran (β/κ-carrageenan hybrid) samples were extracted from the drifting (n = 36) and attached (n = 46) forms of red algae Furcellaria lumbricalis, collected from the Estonian waters in the Baltic Sea. The extracted polysaccharides were characterised with 1H NMR and size-exclusion chromatography. Additionally, the gelling points, storage moduli and melting points of furcellaran samples were determined with a dynamic rheometer. Results showed that the extracted furcellaran samples contained a sulphate ester group per every 3.3 to 4.5 monosaccharide residues. On average, the furcellarans had a sulphate group for every 3.9 monomers, indicating nearly equimolar amounts of β- and κ-carrageenans in the tested polysaccharide samples. The estimated peak average molecular weights of the samples were higher for the furcellarans extracted from attached specimens. Furcellaran gels exhibited a three-time decrease in storage moduli (from ~30000 Pa to ~9300 Pa) when KCl concentrations were reduced from 40 mM to 10 mM. Also, on average, the furcellaran gels (1.5% w/w) in 40 mM KCl solutions exhibited a degree hysteresis of 31 °C.

Phycobiliproteins, red phycoerythrins and blue (allo)phycocyanins, are one of the most highly valued components extracted from cyanobacteria and red algae. In the given study, 95 Furcellaria lumbricalis and 92 Coccotylus truncatus samples were collected from different locations and depths from Kassari Bay, Estonia. Firstly, a strong, positive exponential correlation between the R-phycoerythrin and allophycocyanin concentrations and the collection depth (R > 0.75 and R > 0.61, respectively) was found. Secondly, a preparative scale purification was performed, and the proteins were further characterised by spectrophotometry, fluorimetry and native mass spectrometry. The obtained purity indices (A566/A280) for the R-phycoerythrins were 1.6 for F. lumbricalis and 2.6 for C. truncatus, thus making them suitable for cosmetics and food industries. Purified R-phycoerythrins from the two algal species had subtle differences in absorption and fluorescence maxima. Based on their slightly different absorption characteristics, R-phycoerythrin from F. lumbricalis would categorise as type III (RIII-PE) and R-phycoerythrin from C. truncatus, alongside with the Sigma 52412 standard, as type II (RII-PE). The native mass spectrometry analysis supported the results by showing different intact complexes with distinct γ-subunits for the R-phycoerythrin types. The R-phycoerythrin hexamers linked to various γ-subunits, which are in turn coupled to different chromophores, could explain the similarities (analytical standard and C. truncatus) and differences (F. lumbricalis) in absorption and fluorescence characteristics among the tested species.

Red algal galactans are sulfated polysaccharides, which usually have a linear backbone built up of alternating 3-linked β-D-galactopyranose and 4-linked α-galactopyranose residues. The β-galactose residues always belong to D-series, whereas the α-galactose residues are D in carrageenans and L in agars. A substantial part of α-galactose may exist in the form of a 3,6-anhydro derivative. Various hydroxy groups may be substituted with ester sulfate, methyl groups, pyruvic acid acetal, and sometimes with additional monosaccharide residues. The gelling properties characteristic to some algal polysaccharides (e.g. kappa carrageenan) make them valuable as food ingredients. Chondrus elatus Holmes is endemic species to Japan and belongs together with 10 other species to the Chondrus genus which represents a group of thoroughly studied economically important marine seaweeds. Although used as a gelatinous food or food additive known as kanten in Japan, the composition, structure and rheological properties of the polysaccharides from C. elatus have not been described in the literature. In this work the galactans extracted from C. elatus were characterized by 1H-NMR, 13C-NMR, size-exclusion chromatography, gas chromatography and thermogravimetric methods. By varying the extraction medium (water or phosphate buffer) and temperature, concentration of salt in alcohol used for precipitation of the galactans, separation of polysaccharides on the basis of molecular weight and chemical structure was possible. Coexistence of at least three chemically different polysaccharide fractions in the seaweed was observed, including an abundant cold water soluble fraction with high amounts of lambda and mu carrageenans and hot water soluble fraction containing predominantly kappa carrageenan moieties. Altogether more than six different disaccharide repeating units were determined in the summary polysaccharide from C. elatus, responsible for the characteristic rheological properties of the polysaccharide mixture.