Size Exclusion Chromatography Analysis Research Articles

Mechanism of CK2 Inhibition by a Ruthenium-Based Polyoxometalate.

CK2 is a Ser/Thr protein kinase involved in many cellular processes such as gene expression, cell cycle progression, cell growth and differentiation, embryogenesis, and apoptosis. Aberrantly high CK2 activity is widely documented in cancer, but the enzyme is also involved in several other pathologies, such as diabetes, inflammation, neurodegeneration, and viral infections, including COVID-19. Over the last years, a large number of small-molecules able to inhibit the CK2 activity have been reported, mostly acting with an ATP-competitive mechanism. Polyoxometalates (POMs), are metal-oxide polyanionic clusters of various structures and dimensions, with unique chemical and physical properties. POMs were identified as nanomolar CK2 inhibitors, but their mechanism of inhibition and CK2 binding site remained elusive. Here, we present the biochemical and biophysical characterizing of the interaction of CK2α with a ruthenium-based polyoxometalate, [Ru4(μ-OH)2(μ-O)4(H2O)4 (γ-SiW10O36)2]10− (Ru4POM), a potent inhibitor of CK2. Using analytical Size-Exclusion Chromatography (SEC), Isothermal Titration Calorimetry (ITC), and SAXS we were able to unravel the mechanism of inhibition of Ru4POM. Ru4POM binds to the positively-charged substrate binding region of the enzyme through electrostatic interactions, triggering the dimerization of the enzyme which consequently is inactivated. Ru4POM is the first non-peptide molecule showing a substrate-competitive mechanism of inhibition for CK2. On the basis of SAXS data, a structural model of the inactivated (CK2α)2(Ru4POM)2 complex is presented.

Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

SUMMARYGlioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

How Acinetobacter baumanniiControls Antibiotic Resistance through Phosphorylation of the BfmR Global Regulator

There is an evolving threat to our healthcare system—the rise of antibiotic‐resistant bacteria, such as Acinetobacter baumannii, and the depletion of our few useful antibiotics. These bacteria have developed an ability to fight off increasing numbers of antibiotics used to treat hospitalized patients. To combat these infections, new solutions must be discovered to target the bacteria’s ability to resist drugs and restore the efficacy of our arsenal of antibiotics. One possible therapeutic target is the BfmRS two‐protein signaling system, which activates many genes that determine antibiotic resistance and virulence in A. baumannii. The transcription factor, BfmR, is key to this gene regulation pathway. How the activity of BfmR is controlled is unknown. We hypothesize that a phosphorylation signal activates BfmR, allowing it to form an oligomeric state that binds to and enhances expression of target genes. To test this hypothesis, we developed in vitrophosphorylation conditions using purified BfmR and phosphoramidate, a small molecule phosphodonor. We used phosphate‐affinity polyacrylamide gel electrophoresis to determine that phosphorylation of the purified protein was highly efficient. Size exclusion chromatography analysis of BfmR using high‐performance liquid chromatography (HPLC) was consistent with dimerization of the protein upon phosphorylation. Next steps will include evaluating the binding affinity of the phosphorylated protein to a series of target DNA sequences associated with resistance and virulence genes, chosen from chromatin immunoprecipitation sequencing (ChiP‐Seq) data showing conserved response elements. We will employ electrophoretic mobility shift assays (EMSAs) to analyze how phosphorylation affects BfmR binding to these sequences. The information we uncover on the regulatory mechanisms of BfmR can be used to guide the development of inhibitors that counteract A. baumannii’s resistance to essential antibiotics.

A GH115 α-glucuronidase structure reveals dimerization-mediated substrate binding and a proton wire potentially important for catalysis.

Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is α-D-glucuronic acid, which is linked to the O2' position of the β-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific α-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of α-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65 Å resolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The xylohexaose moieties of the xylohexaose are recognized by residues from both protomers, thus creating a xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca2+ ions were identified in the wtsAgu115A structures. One Ca2+ ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg2+ or Mn2+ resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca2+ resulted in a decrease in activity.

Machine learning‐based phenotypic screening for postmitotic growth inducers uncover vitamin D3 metabolites as small molecule ribosome agonists

ObjectivesTo restore tissue growth without increasing the risk for cancer during aging, there is a need to identify small molecule drugs that can increase cell growth without increasing cell proliferation. While there have been numerous high‐throughput drug screens for cell proliferation, there have been few screens for post‐mitotic anabolic growth.Materials and MethodsA machine learning (ML)‐based phenotypic screening strategy was used to discover metabolites that boost muscle growth. Western blot, qRT‐PCR and immunofluorescence staining were used to evaluate myotube hypertrophy/maturation or protein synthesis. Mass spectrometry (MS)‐based thermal proteome profiling‐temperature range (TPP‐TR) technology was used to identify the protein targets that bind the metabolites. Ribo‐MEGA size exclusion chromatography (SEC) analysis was used to verify whether the ribosome proteins bound to calcitriol.ResultsWe discovered both the inactive cholecalciferol and the bioactive calcitriol are amongst the top hits that boost post‐mitotic growth. A large number of ribosomal proteins' melting curves were affected by calcitriol treatment, suggesting that calcitriol binds to the ribosome complex directly. Purified ribosomes directly bound to pure calcitriol. Moreover, we found that calcitriol could increase myosin heavy chain (MHC) protein translation and overall nascent protein synthesis in a cycloheximide‐sensitive manner, indicating that calcitriol can directly bind and enhance ribosomal activity to boost muscle growth.ConclusionThrough the combined strategy of ML‐based phenotypic screening and MS‐based omics, we have fortuitously discovered a new class of metabolite small molecules that can directly activate ribosomes to promote post‐mitotic growth.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), 19 F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66L289K and p51 were predicted to form a heterodimer while p66 and p51L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products.

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic–inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase.Compared to a reference stainless‐steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra‐ and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent.By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.

Effects of Thermal Aging on Molar Mass of Ultra-High Molar Mass Polyethylene Fibers.

Ultra-high molar mass polyethylene (UHMMPE) is commonly used for ballistic-resistant body armor applications due to the superior strength of the fibers fabricated from this material combined with its low density. However, polymeric materials are susceptible to thermally induced degradation during storage and use, which can reduce the high strength of these fibers, and, thus, negatively impact their ballistic resistance. The objective of this work is to advance the field of lightweight and soft UHMMPE inserts used in various types of ballistic resistant-body armor via elucidating the mechanisms of chemical degradation and evaluating this chemical degradation, as well as the corresponding physical changes, of the UHMMPE fibers upon thermal aging. This is the first comprehensive study on thermally aged UHMMPE fibers that measures their decrease in the average molar mass via high-temperature size exclusion chromatography (HT-SEC) analysis. The decrease in the molar mass was further supported by the presence of carbon-centered free radicals in the polyethylene that was detected using electron paramagnetic resonance (EPR) spectroscopy. These carbon-centered radicals result from a cascade of thermo-oxidative reactions that ultimately induce C–C ruptures along the backbone of the polymer. Changes in the crystalline morphology of the UHMMPE fibers were also observed through wide-angle X-ray diffraction (WAXS), showing an increase in the amorphous regions, which promotes oxygen diffusion into the material, specifically through these areas. This increase in the amorphous fraction of the highly oriented polyethylene fibers has a synergistic effect with the thermo-oxidative degradation processes and contributes significantly to the decrease in their molar mass.

The role of medium molecular weight organics on reducing disinfection by-products and fouling prevention in nanofiltration

Nanofiltration (NF) is utilized in water treatment for controlling disinfection by-products formation potential (DBPFP) and disinfection by-products (DBPs). Attention regarding NF-based technology has been paid on membrane fouling of NF and the rejection efficiency of contaminants by NF membranes. Natural organic matter (NOM) presenting in surface waters is one main removal target in drinking water treatment by NF-based technology, and is thereby a contributor to the membrane fouling of NF. In application, pretreatments of other membrane filtration (e.g., microfiltration (MF) and ultrafiltration (UF)) has been taken prior to NF, resulting in the separation of NOM of specific molecular weight. Meanwhile, it is well known that NOM is composed of organic compounds of different molecular weights. However, the effect of NOM of specific molecular weight has been seldom investigated from the aspects of membrane fouling and the resulting DBPFP after membrane filtration. By using combinations of MF and UF (molecular weight cut-off of 100K or 20K) as pretreatment prior to NF, the NOM of various molecular weight on DBPFP and DBPs in the NF-treated water were investigated. The experiments were conducted with two real-world surface water samples and one tap water sample. It was found that medium molecular weight NOM, defined as NOM that passed UF100K but did not pass UF20K in this study, reduced fouling of the NF membrane. This is supported by the excitation and emission matrix (EEM) fluorescence spectra, size exclusion chromatography (SEC) and flux analysis. In addition, the medium molecular weight NOM also reduced the DBPFP in the NF treated water and eventually the DBPs by participating in forming a protective layer on the NF surface, blocking the transfer of small molecular weight NOM into the NF filtrate, thereby reducing the DBPFP of the NF filtrate since small molecular weight NOM was the major contributor to DBPFP in this study.

Synthesis and covalent immobilization of redox-active metallopolymers for organic phase electrochemistry

Redox-active metallopolymers are promising stimuli-responsive platforms for a range of applications including sensing, energy storage, and selective separations. In particular, heterogeneously-functionalized metallopolymers can modulate the capture and release of target molecules, driven by redox electron-transfer. However, prior metallopolymer-functionalized electrodes have been fabricated by non-covalent methods, and tailored for aqueous phase applications. As such, despite the existing potential for heterogeneous applications in organic phase, there are significant constraints to the stability of metallopolymers in organic solvents, including high solubility in solvents such as chloroform or tetrahydrofuran. We propose the immobilization of thiol-functionalized redox-active metallopolymers on metallic surfaces as a facile way to enhance stability and cyclability in organic media, and thus broaden the applicability of redox-metallopolymers for organic phase applications. We explore the anionic polymerization of metal-containing monomers vinylferrocene (VFc) and ferrocenyldimethylsilane (FS), and their thiol end-functionalization by living anionic polymerization strategies. PFS and PVFc with molar masses ranging from 1800 to 49900 g mol−1 and 2900 to 6300 g mol−1 respectively were prepared with a segment of poly(ethylene sulfide), as characterized by size-exclusion chromatography, NMR spectroscopy, MALDI/ToF, thermogravimetry, and elemental analysis. Both metallopolymers were immobilized on gold substrates by a grafting-to protocol, with demonstrated redox-responsiveness by electrochemical control. In the case of immobilized PVFc, operando electrochemical testing demonstrated the stable and reversible electrochemical cycling capabilities (>74% maximum current retained after 100 oxidation/reduction cycles) in several organic solvents including chloroform, tetrahydrofuran, ethanol, methanol, acetonitrile, and acetone. Immobilized PFS was stable in chloroform, with a 83% maximum current retained after 100 oxidation/reduction cycles. We envision future applications of these covalently immobilized metallopolymers for a broad range of fields from selective separations to sensing and energy storage.

Efficient dispersion of TiO2 in water-based paint formulation using well-defined poly[oligo(ethylene oxide) methyl ether acrylate] synthesized by ICAR ATRP

In this work, a new polymeric dispersants based on poly[oligo(ethylene oxide) methyl ether acrylate] (POEOA) as hydrophilic block and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and dopamine (Dopa) as anchoring molecules, have been synthetized and successfully used as efficient tailor-made polymers for the aqueous dispersion of titanium dioxide (TiO2) particles. The controlled structure of this new class of additives was obtained by initiators for continuous activator regeneration atom transfer radical polymerization (ICAR ATRP). The proposed polymer dispersants were characterized by size exclusion chromatography (SEC) and nuclear magnetic resonance analysis (1H NMR). The dispersion efficiency was evaluated by dynamic light scattering (DLS), zeta potential (ζ), rheology and transmission electron microscopy (TEM). The influence of different stabilizing blocks bounded to the same anchoring block on the dispersion efficiency was evaluated in aqueous dispersions of TiO2 using a common water-based paint formulation (satin vinyl paint). Aiming for industrial validation, a satin paint was also formulated with the new dispersants. Higher gloss (up to 39%) and improved pigment dispersion were observed when using the tailor-made polymers, comparing to the use of a standard commercial poly(acrylic acid) (PAA) dispersing agent. The data presented suggest that POEOA-based macrostructures are highly efficient additives for the dispersion of TiO2 in water-based paint formulations.

Molecular Architecture of the Antiophidic Protein DM64 and its Binding Specificity to Myotoxin II From Bothrops asper Venom.

DM64 is a toxin-neutralizing serum glycoprotein isolated from Didelphis aurita, an ophiophagous marsupial naturally resistant to snake envenomation. This 64 kDa antitoxin targets myotoxic phospholipases A2, which account for most local tissue damage of viperid snakebites. We investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic phospholipase-like protein from Bothrops asper venom. Analytical ultracentrifugation (AUC) and size exclusion chromatography indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Attempts to crystallize native DM64 for X-ray diffraction were unsuccessful. Obtaining recombinant protein to pursue structural studies was also challenging. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a three-dimensional model of the inhibitor bound to myotoxin II. I-TASSER individually modeled the five immunoglobulin-like domains of DM64. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II, using Rosetta. AUC, small-angle X-ray scattering (SAXS), molecular modeling, and molecular dynamics simulations indicated that the DM64-myotoxin II complex is structured, shows flexibility, and has an anisotropic shape. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor’s regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II amino-terminal and beta-wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably precludes dimerization, thus interfering with toxicity, which is related to the quaternary structure of the toxin. The first domain of DM64 interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to inhibit myotoxin II toxicity by DM64 binding. The present topological characterization of this toxin-antitoxin complex constitutes an essential step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.

Characterization Techniques Coupled with Mathematical Tools for Deepening Asphaltene Structure

Asphaltenes are the heavy fraction of fossil fuels, whose characterization remains a very difficult and challenging issue due to the still-persisting uncertainties about their structure and/or composition and molecular weight. Asphaltene components are highly condensed aromatic molecules having some heteroatoms and aliphatic functionalities. Their molecular weights distribution spans in a wide range, from hundreds to millions of mass units, depending on the diagnostic used, which is mainly due to the occurrence of self-aggregation. In the present work, mass spectrometry along with size exclusion chromatography and X-ray diffraction analysis have been applied to asphaltenes for giving some further insights into their molecular weight distribution and structural characteristics. Relatively small polycyclic aromatic hydrocarbons (PAHs) with a significant degree of aliphaticity were individuated by applying fast Fourier transform (FFT) and double bond equivalent (DBE) number analysis to the mass spectra. X-ray diffraction (XRD) confirmed some aliphaticity, showing peaks specific of aliphatic functionalities. Size exclusion chromatography indicated higher molecular weight, probably due to the aliphatic substituents. Mass spectrometry at high laser power enabled observing a downward shift of molecular masses corresponding to the loss of about 10 carbon atoms, suggesting the occurrence of aryl-linked core structures assumed to feature asphaltenes along with island and archipelago structures.

Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP+-binding site. Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP+ and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP+ binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PDdef). We first solved the crystal structure for G6PDK403Q, a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP+ binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PDdef), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PDK403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PDdef and provide a foundation for future drug discovery efforts.

Experimental Evidence of Intrinsic Disorder and Amyloid Formation by the Henipavirus W Proteins.

Henipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V and W proteins which share with P their N-terminal, intrinsically disordered domain (NTD) and possess a unique C-terminal domain. Henipavirus W proteins antagonize interferon (IFN) signaling through NTD-mediated binding to STAT1 and STAT4, and prevent type I IFN expression and production of chemokines. Structural and molecular information on Henipavirus W proteins is lacking. By combining various bioinformatic approaches, we herein show that the Henipaviruses W proteins are predicted to be prevalently disordered and yet to contain short order-prone segments. Using limited proteolysis, differential scanning fluorimetry, analytical size exclusion chromatography, far-UV circular dichroism and small-angle X-ray scattering, we experimentally confirmed their overall disordered nature. In addition, using Congo red and Thioflavin T binding assays and negative-staining transmission electron microscopy, we show that the W proteins phase separate to form amyloid-like fibrils. The present study provides an additional example, among the few reported so far, of a viral protein forming amyloid-like fibrils, therefore significantly contributing to enlarge our currently limited knowledge of viral amyloids. In light of the critical role of the Henipavirus W proteins in evading the host innate immune response and of the functional role of phase separation in biology, these studies provide a conceptual asset to further investigate the functional impact of the phase separation abilities of the W proteins.

A novel approach for enumeration of extracellular vesicles from crude and purified cell culture samples.

The interest in extracellular vesicles (EVs) has been increased in recent years due to their potential application in diagnosis and therapy of severe diseases. The versatile fields of application due to the numerous possible cargos and the targeted delivery system make them a promising biopharmaceutical product. However, their broad size range as well as varied surface protein content result in challenges for the purification, characterization, and quantification. In this study a novel method, based on high‐resolution flow cytometry, was examined for the enumeration of EVs in purified as well as crude process samples. In addition to quantification, samples were characterized by dynamic light scattering, zeta potential measurement, and analytical size exclusion chromatography. It has been demonstrated that EVs were successfully enumerated with the novel method, offering great benefits for development and monitoring of EV processes.

Site-Specific Characterization of Heat-Induced Disulfide Rearrangement in Beta-Lactoglobulin by Liquid Chromatography-Mass Spectrometry.

Disulfides are important for maintaining the protein native structure, but they may undergo rearrangement in the presence of free Cys residues, especially under elevated temperatures. Disulfide rearrangement may result in protein aggregation, which is associated with in vivo pathologies in organisms and in vitro protein functionality in food systems. In a food context, it is therefore important to understand the process of disulfide rearrangement on a site-specific level in order to control aggregation. In the present study, a liquid chromatography-mass spectrometry (LC-MS)-based bottom-up site-specific proteomic approach was optimized to study disulfide rearrangements in beta-lactoglobulin (β-LG) under different heat treatments (60-90 °C). Artifactual disulfide rearrangement observed during sample preparation using a conventional protocol was detected and minimized by blocking the remaining free Cys residues with iodoacetamide in the presence of urea after heat treatment. Use of endoproteinase Glu-C for enzymatic hydrolysis allowed, for the first time, identification and comparison of the relative intensity of all theoretically possible β-LG disulfide cross-links formed by the heat treatments. Non-native disulfides were formed from heat treatment at approx. 70 °C where β-LG started to unfold, while higher levels of inter-molecular disulfide links were formed at ≥80 °C, in agreement with β-LG aggregation detected by size exclusion chromatography analysis. Collectively, the Cys residues of the surface-located native disulfide Cys66-Cys160 were proposed to be more reactive, participating in heat-induced disulfide rearrangement, compared to other Cys residues. The abundant signal of non-native disulfide bonds containing Cys66, especially Cys66-Cys66, observed after heating suggested that Cys66 is a key disulfide-linked Cys residue in β-LG participating in heat-induced inter-molecular disulfide bonds and the corresponding protein aggregation.

Characterization of DNA nanostructure stability by size exclusion chromatography.

DNA-based nanostructures (DNs) are advantageous for the design of functional materials for biology and medicine due to the nanoscale control provided by their predictable self-assembly. However, the use of DNs in vivo has been limited due to structural instability in biofluids. As the stability of a particular DN sets the scope of its potential biological applications, efficient methods to characterize stability are required. Here, we apply size exclusion chromatography (SEC) to study the stability of a tetrahedron DNA nanostructure (TDN) and demonstrate the analytical capabilities of our method in characterizing degradation by enzymes and a diluted human serum matrix. We show that SEC analysis can reliably assay TDN degradation by a nuclease through direct injection and peak integration. Furthermore, data analysis using a ratio chromatogram technique enables TDN peak deconvolution from the matrix of serum proteins. Using our method, we found that TDNs exhibit half-lives of 23.9 hours and 10.1 hours in 20% and 50% diluted human serum, respectively, which is consistent with reported stability studies in 10% fetal bovine serum. We anticipate that this method can be broadly applicable to characterize a variety of DNs and serve as an efficient technique toward analysis of the stability of new DN designs in complex biological matrixes.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

Fluorescence descriptors for algal organic matter and microalgae disintegration during ultrasonication

Ultrasonication is a potential treatment for attenuating harmful algal blooms, but it also leads to the release of algal organic matter. In this study, the effects of ultrasonication power (0.039–0.32 W/mL) for 0, 5, 10, and 20 min on microalgae disintegration were investigated, focusing on algal organic matter, using advanced organic matter characterization tools. Using the Pearson correlation analysis considering several absorption indices, only ultraviolet absorbance at 254 nm and the absorption slope index showed strong correlation with changes in algal organic matter and destroyed microalgae count. Tryptophan-like, tyrosine-like, and humic-like substances, i.e., fluorescence components were extracted from the excitation-emission matrix of algal organic matter using parallel factor analysis. A single fluorescence peak of the tryptophan-like component, at an excitation/emission = 280/380 nm, displayed significant correlation (R2 = 0.96, p < 0.05) with the disintegrated microalgae count during ultrasonication. Individual fluorescence components increased along with the power of ultrasonication; this increase was more pronounced in tryptophan-like substances. The combined approach of excitation-emission matrix-parallel factor and size-exclusion chromatography analyses revealed that the protein content in biopolymers was governed by increasing ultrasonication power. This study presents a novel approach to the use of spectroscopic descriptors while designing a probe for the real-time tracking of microalgae disintegration, and the concurrent release of algal organic matter, during the ultrasonication process.