Size Exclusion Chromatography Experiments Research Articles

A Biochemical and Biophysical Analysis of the Interaction of nsp9 with nsp12 from SARS-CoV-2-Implications for Future Drug Discovery Efforts.

The ongoing global pandemic of the coronavirus 2019 (COVID-19) disease is caused by the virus SARS-CoV-2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9-nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS-CoV-2 and any newly emerging coronaviruses.

Investigation of the Reaction between a Homemade PEEK Oligomer and an Epoxy Prepolymer: Optimisation of Critical Parameters Using Physico-Chemical Methods.

Several researchers have examined the interest in using a thermoplastic to increase thermoset polymers' shock resistance. However, fewer studies have examined the nature of the mechanisms involved between both kinds of polymers. This was the objective of our work, which was carried out using a gradual approach. First, we describe the synthesis of a poly(ether ether ketone) oligomer (oPEEK) with hydroxyl terminations from the reaction of hydroquinone and 4,4'-difluorobenzophenone in N-methyl-2-pyrrolidone. Then, the main physicochemical properties of this oligomer were determined using different thermal analyses (i.e., differential scanning calorimetry (DSC), thermogravimetric (ATG), and thermomechanical analyses) to isolate its response alone. The chemical characterisation of this compound using conventional analytical chemistry techniques was more complex due to its insolubility. To this end, it was sulfonated, according to a well-known process, to make it soluble and enable nuclear magnetic resonance (NMR) and size exclusion chromatography (SEC) experiments. Additional information about the structural and chemical characteristics of the oligomer and its average molecular weight could thus be obtained. The synthesis of an oligoPEEK with α,ω-hydroxyl end-groups and a molecular weight of around 5070 g/mol was thus confirmed by NMR. This value was in accordance with that determined by SEC analysis. Next, the reaction of oPEEK with an epoxy prepolymer was demonstrated using DSC and dynamic rheometry. To this end, uncured mixtures of epoxy prepolymer (DGEBA) with different proportions of oPEEK (3, 5, 10 and 25%) were prepared and characterised by both techniques. Ultimately, the epoxy-oPEEK mixture was cured with isophorone diamine. Finally, topological analyses were performed by atomic force microscopy (AFM) in tapping mode to investigate the interface quality between the epoxy matrix and the oPEEK particles indirectly. No defects, such as decohesion areas, microvoids, or cracks, were observed between both systems.

Tanc1/2 TPR domain interacts with Myo18a C-terminus and undergoes liquid-liquid phase separation

Tanc1 and its homologous protein Tanc2 are critical synaptic scaffold proteins which regulate synaptic spine densities and excitatory synapse strength. Recent studies indicated TANC1 and TANC2 are candidate genes of several neurodevelopmental disorders (NDDs). In this study, we identified and characterized a novel interaction between Tanc1/2 and Myo18a, mediated by the Tanc1/2 TPR domains and Myo18a coiled-coil domain and C-extension (CCex). Sequence analysis and size exclusion chromatography experiments reveal that high salt disrupts the interaction between Myo18a and Tanc1/2, indicating that the interaction is primarily driven by charge-charge interactions. More importantly, we found that the Tanc1-TPR/Myo18a CCex interaction could undergo liquid-liquid phase separation (LLPS) in both cultured cells and test tubes, which provides the biochemical basis and potential molecular mechanisms for the LLPS-mediated interactions between Myo18a and Tanc1/2.

S100A6 inhibits MDM2 to suppress breast cancer growth and enhance sensitivity to chemotherapy

BackgroundS100A6 and murine double minute 2 (MDM2) are important cancer-related molecules. A previous study identified an interaction between S100A6 and MDM2 by size exclusion chromatography and surface plasmon resonance experiments. The present study investigated whether S100A6 could bind to MDM2 in vivo and further explored its functional implication.MethodsCo-immunoprecipitation, glutathione-S-transferase pull-down assay, and immunofluorescence were performed to determine the in vivo interaction between S100A6 and MDM2. Cycloheximide pulse-chase assay and ubiquitination assay were performed to clarify the mechanism by which S100A6 downregulated MDM2. In addition, clonogenic assay, WST-1 assay, and flow cytometry of apoptosis and the cell cycle were performed and a xenograft model was established to evaluate the effects of the S100A6/MDM2 interaction on growth and paclitaxel-induced chemosensitivity of breast cancer. The expressions of S100A6 and MDM2 in patients with invasive breast cancer were analyzed by immunohistochemistry. In addition, the correlation between the expression of S100A6 and the response to neoadjuvant chemotherapy was statistically analyzed.ResultsS100A6 promoted the MDM2 translocation from nucleus to cytoplasm, in which the S100A6 bound to the binding site of the herpesvirus-associated ubiquitin-specific protease (HAUSP) in MDM2, disrupted the MDM2–HAUSP–DAXX interactions, and induced the MDM2 self-ubiquitination and degradation. Furthermore, the S100A6-mediated MDM2 degradation suppressed the growth of breast cancer and enhanced its sensitivity to paclitaxel both in vitro and in vivo. For patients with invasive breast cancer who received epirubicin and cyclophosphamide followed by docetaxel (EC-T), expressions of S100A6 and MDM2 were negatively correlated, and high expression of S100A6 suggested a higher rate of pathologic complete response (pCR). Univariate and multivariate analyses showed that the high expression of S100A6 was an independent predictor of pCR.ConclusionThese results reveal a novel function for S100A6 in downregulating MDM2, which directly enhances sensitivity to chemotherapy.

Mutations to the BTN2A1 Linker Region Impact Its Homodimerization and Its Cytoplasmic Interaction with Phospho-Antigen-Bound BTN3A1.

Intracellular binding of small-molecule phospho-Ags to the HMBPP receptor complex in infected cells leads to extracellular detection by T cells expressing the Vγ9Vδ2 TCR, a noncanonical method of Ag detection. The butyrophilin proteins BTN2A1 and BTN3A1 are part of the complex; however, their precise roles are unclear. We suspected that BTN2A1 and BTN3A1 form a tetrameric (dimer of dimers) structure, and we wanted to probe the importance of the BTN2A1 homodimer. We analyzed mutations to human BTN2A1, using internal domain or full-length BTN2A1 constructs, expressed in Escherichia coli or human K562 cells, that might disrupt its structure and/or function. Although BTN2A1 is a disulfide-linked homodimer, mutation of cysteine residues C247 and C265 did not affect the ability to stimulate T cell IFN-γ production by ELISA. Two mutations of the juxtamembrane region (at EKE282) failed to impact BTN2A1 function. In contrast, single point mutations (L318G and L325G) near the BTN2A1 B30.2 domain blocked phospho-Ag response. Size exclusion chromatography and nuclear magnetic resonance (NMR) experiments showed that the isolated BTN2A1 B30.2 domain is a homodimer, even in the absence of its extracellular and transmembrane region. [31P]-NMR experiments confirmed that HMBPP binds to BTN3A1 but not BTN2A1, and binding abrogates signals from both phosphorus atoms. Furthermore, the BTN2A1 L325G mutation but not the L318G mutation prevents both homodimerization of BTN2A1 internal domain constructs in size exclusion chromatography (and NMR) experiments and their binding to HMBPP-bound BTN3A1 in isothermal titration calorimetry experiments. Together, these findings support the importance of homodimerization within the BTN2A1 internal domain for phospho-Ag detection.

The human phosphorylated pathway: a multienzyme metabolic assembly for l-serine biosynthesis.

De novo l-serine biosynthesis in the mammalian astrocytes proceeds via a linear, three-step pathway (the phosphorylated pathway) catalysed by 3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP). The first reaction, catalysed by PHGDH and using the glycolytic intermediate 3-phosphoglycerate, is strongly shifted towards the reagents, and coupling to the following step by PSAT is required to push the equilibrium towards l-serine formation; the last step, catalysed by PSP, is virtually irreversible and inhibited by the final product l-serine. Very little is known about the regulation of the human phosphorylated pathway and the ability of the three enzymes to organise in a complex with potential regulatory functions. Here, the complex formation was investigated in differentiated human astrocytes, by proximity ligation assay, and in vitro on the human recombinant enzymes. The results indicate that the three enzymes co-localise in cytoplasmic clusters that more stably engage PSAT and PSP. Although in vitro analyses based on native PAGE, size exclusion chromatography and cross-linking experiments do not show the formation of a stable complex, kinetic studies of the reconstituted pathway using physiological enzyme and substrate concentrations support cluster formation and indicate that PHGDH catalyses the rate-limiting step while PSP reaction is the driving force for the whole pathway. The enzyme agglomerate assembly of the phosphorylated pathway (the putative 'serinosome') delivers a relevant level of sophistication to the control of l-serine biosynthesis in human cells, a process strictly related to the modulation of the brain levels of d-serine and glycine, the main co-agonists of N-methyl-d-aspartate receptors and various pathological states.

Free-Radical Propagation Rate Coefficients of Diethyl Itaconate and Di-n-Propyl Itaconate Obtained via PLP-SEC.

The propagation step is one of the key reactions in radical polymerization and knowledge about its kinetics is often vital for understanding and designing polymerization processes leading to new materials or optimizing technical processes. Arrhenius expressions for the propagation step in free-radical polymerization of diethyl itaconate (DEI) as well as di-n-propyl itaconate (DnPI) in bulk, for which propagation kinetics was yet unexplored, were thus determined via pulsed-laser polymerization in conjunction with size-exclusion chromatography (PLP-SEC) experiments in the temperature range of 20 to 70 °C. For DEI, the experimental data was complemented by quantum chemical calculation. The obtained Arrhenius parameters are A = 1.1 L·mol-1·s-1 and Ea = 17.5 kJ·mol-1 for DEI and A = 1.0 L·mol-1·s-1 and Ea = 17.5 kJ·mol-1 for DnPI.

Cooperative Regulation of Flagellar Synthesis by Two EAL-Like Proteins upon Salmonella Entry into Host Cells.

When Salmonella enters host cells, the synthesis of flagella is quickly turned off to escape the host immune system. In this study, we investigated the cooperative regulatory mechanism of flagellar synthesis by two EAL-like proteins, STM1344 and STM1697, in Salmonella. We found that Salmonella upregulated the expression of both STM1344 and STM1697 to various degrees upon invading host cells. Importantly, deletion of STM1697 or STM1344 led to failure of Salmonella flagellar control within host cells, suggesting that the two factors are not redundant but indispensable. STM1697 was shown to modulate Salmonella flagellar biogenesis by preventing the flagellar master protein FlhDC from recruiting RNA polymerase. However, STM1344 was identified as a bifunctional factor that inhibits RNA polymerase recruitment of FlhDC at low molar concentrations and the DNA binding activity of FlhDC at high molar concentrations. Structural analysis demonstrated that STM1344-FlhD binds more tightly than STM1697-FlhD, and size exclusion chromatography (SEC) experiments showed that STM1344 could replace STM1697 in a STM1697-FlhDC complex. Our data suggest that STM1697 might be a temporary flagellar control factor upon Salmonella entry into the host cell, while STM1344 plays a more critical role in persistent flagellar control when Salmonella organisms survive and colonize host cells for a long period of time. Our study provides a more comprehensive understanding of the complex flagellar regulatory mechanism of Salmonella based on regulation at the protein level of FlhDC. IMPORTANCE Salmonella infection kills more than 300,000 people every year. After infection, Salmonella mainly parasitizes host cells, as it prevents host cell pyroptosis by turning off the synthesis of flagellar antigen. Previous studies have determined that there are two EAL-like proteins, STM1344 and STM1697, encoded in the Salmonella genome, both of which inhibit flagellar synthesis by interacting with the flagellar master protein FlhDC. However, the expression order and simultaneous mechanism of STM1344 and STM1697 are not clear. In this study, we determined the expression profiles of the two proteins after Salmonella infection and demonstrated the cooperative mechanism of STM1344 and STM1697 interaction with FlhDC. We found that STM1344 might play a more lasting regulatory role than STM1697. Our results reveal a comprehensive flagellar control process after Salmonella entry into host cells.

Regulation of futile ligation during early steps of BER in M. tuberculosis is carried out by a β-clamp-XthA-LigA tri-component complex

The Class-II AP-endonuclease (XthA) is a mycobacterial DNA base excision repair (BER) pathway enzyme that functions in the initial steps. It acts on DNA substrates that contain abasic sites to create nicks with 3′-hydroxyl (OH) and 5′-deoxyribose phosphate (5′-dRP) moieties. The NAD+-dependent DNA ligase (LigA) is the terminal player in mycobacterial BER and seals such nicks efficiently. Here, we demonstrate that the Mtbβ-clamp-MtbXthA complex that exists in the initial steps of BER engages with MtbLigA to form a novel tri-component BER complex. Size exclusion chromatography (SEC) experiments analysis show that the three proteins interact with equimolar stoichiometry. Small angle X-ray scattering (SAXS) analysis and associated studies reveal that the apo tri-component BER-complex adopts an extended conformation where MtbXthA is sandwiched between the Mtbβ-clamp and MtbLigA. The studies support that in the apo-complex MtbXthA binds subsite-I of Mtbβ-clamp through 239QLRFPKK245 motif and to MtbLigA by 104DGQPSWSGKP113 motif simultaneously. However, the complex adopts a less-extended conformation in the presence of substrate DNA, where MtbXthA interactions switch from predominantly subsite-I to subsite-II of the Mtbβ-clamp. Overall, the novel tri-component complex prevents futile ligation activity of MtbLigA on the product of MtbXthA and ensures forward progression of the pathway and productive mycobacterial BER interactions.

Inflammasome sensor NLRP1 disease variant M1184V promotes autoproteolysis and DPP9 complex formation by stabilizing the FIIND domain

The inflammasome sensor NLRP1 (nucleotide-binding oligomerization domain-like receptor containing a pyrin domain 1) detects a variety of pathogen-derived molecular patterns to induce an inflammatory immune response by triggering pyroptosis and cytokine release. A number of mutations and polymorphisms of NLRP1 are known to cause autoinflammatory diseases, the functional characterization of which contributes to a better understanding of NLRP1 regulation. Here, we assessed the effect of the common NLRP1 variant M1184V, associated with asthma, inflammatory bowel disease, and diabetes, on the protein level. Our size-exclusion chromatography experiments show that M1184V stabilizes the "function-to-find" domain (FIIND) in a monomeric conformation. This effect is independent of autoproteolysis. In addition, molecular dynamics simulations reveal that the methionine residue increases flexibility within the ZU5 domain, whereas valine decreases flexibility, potentially indirectly stabilizing the catalytic triad responsible for autocleavage. By keeping the FIIND domain monomeric, formation of a multimer of full-length NLRP1 is promoted. We found that the stabilizing effect of the valine further leads to improved dipeptidyl peptidase 9 (DPP9)-binding capacities for the FIIND domain as well as the full-length protein as determined by surface plasmon resonance. Moreover, our immunoprecipitation experiments confirmed increased DPP9 binding for the M1184V protein in cells, consistent with improved formation of an autoinhibited complex with DPP9 in activity assays. Collectively, our study establishes a molecular rationale for the dichotomous involvement of the NLRP1 variant M1184V in autoimmune syndromes.

Development of a Sensitive ELISA for Gastric Intrinsic Factor and Detection of Intrinsic Factor Immunoreactivity in Human Serum.

Gastric Intrinsic Factor (IF) is produced by the parietal cells of the stomach and secreted into the gastrointestinal tract where it ensures the active absorption of vitamin B12. We hypothesized that a small amount of IF ends up in the circulation and can be measured in serum. The aim of this study was to develop an assay for measuring human IF and to demonstrate its presence in serum. We designed a sensitive ELISA for measurement of human IF using a commercial monoclonal antibody and an in-house polyclonal antibody as capture and detecting antibody, respectively. Imprecision, accuracy, and linearity of the assay were examined. We established a reference interval based on serum samples from 240 healthy donors, and explored the daily IF fluctuations in 20 healthy subjects. Employing a prototype IF ELISA and size exclusion chromatography experiments, we demonstrated the presence of IF in human serum. In its final design, the IF ELISA has a measurement range of 0.2 to 50 pmol/L. The intra-assay and total imprecision were 7.9% and 15%, respectively. The 95% reference interval (18–65 years) was 1.7–11.6 pmol/L. No diurnal fluctuation or notable sex differences were observed. Our results suggest that the assay is capable of detecting and quantifying human IF in the circulation and may prove useful in the characterization of patients with impaired IF production.

Vaccine target and carrier molecule nontypeable Haemophilus influenzae protein D dimerizes like the close Escherichia coli GlpQ homolog but unlike other known homolog dimers.

We have determined the 1.8Å X-ray crystal structure of nonlipidated (i.e., N-terminally truncated) nontypeable Haemophilus influenzae (NTHi; H. influenzae) protein D. Protein D exists on outer membranes of H. influenzae strains and acts as a virulence factor that helps invade human cells. Protein D is a proven successful antigen in animal models to treat obstructive pulmonary disease (COPD) and otitis media (OM), and when conjugated to polysaccharides also has been used as a carrier molecule for human vaccines, for example in GlaxoSmithKline Synflorix™. NTHi protein D shares high sequence and structural identify to the Escherichia coli (E. coli) glpQ gene product (GlpQ). E. coli GlpQ is a glycerophosphodiester phosphodiesterase (GDPD) with a known dimeric structure in the Protein Structural Database, albeit without an associated publication. We show here that both structures exhibit similar homodimer organization despite slightly different crystal lattices. Additionally, we have observed both the presence of weak dimerization and the lack of dimerization in solution during size exclusion chromatography (SEC) experiments yet have distinctly observed dimerization in native mass spectrometry analyses. Comparison of NTHi protein D and E. coli GlpQ with other homologous homodimers and monomers shows that the E. coli and NTHi homodimer interfaces are distinct. Despite this distinction, NTHi protein D and E. coli GlpQ possess a triose-phosphate isomerase (TIM) barrel domain seen in many of the other homologs. The active site of NTHi protein D is located near the center of this TIM barrel. A putative glycerol moiety was modeled in two different conformations (occupancies) in the active site of our NTHi protein D structure and we compared this to ligands modeled in homologous structures. Our structural analysis should aid in future efforts to determine structures of protein D bound to substrates, analog intermediates, and products, to fully appreciate this reaction scheme and aiding in future inhibitor design.

Conformational Changes of α-Crystallin Proteins Induced by Heat Stress.

α-crystallin is a major structural protein in the eye lenses of vertebrates that is composed of two relative subunits, αA and αB crystallin, which function in maintaining lens transparency. As a member of the small heat-shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent the misfolding or aggregation of critical proteins in the lens, which is associated with cataract disease. In this study, high-purity αA and αB crystallin proteins were expressed from E. coli and purified by affinity and size-exclusion chromatography. The size-exclusion chromatography experiment showed that both αA and αB crystallins exhibited oligomeric complexes in solution. Here, we present the structural characteristics of α-crystallin proteins from low to high temperature by combining circular dichroism (CD) and small-angle X-ray scattering (SAXS). Not only the CD data, but also SAXS data show that α-crystallin proteins exhibit transition behavior on conformation with temperature increasing. Although their protein sequences are highly conserved, the analysis of their thermal stability showed different properties in αA and αB crystallin. In this study, taken together, the data discussed were provided to demonstrate more insights into the chaperone-like activity of α-crystallin proteins.

Cationic Polyelectrolytes Containing Perfluorinated Groups: Synthesis and Self-Assembly Properties in Aqueous Solutions

In this work, the synthesis and solution self-assembly of partially fluorinated cationic polyelectrolytes based on post-polymerization-modified poly(2-(dimethylamino) ethyl methacrylate), incorporating hydrophobic fluorinated groups along the polymer chain, is reported. The post-polymerization modification aims to establish amphiphilic characteristics and strong polyelectrolyte properties to the polymer. Therefore, the self-organization characteristics in aqueous media are expected to be affected. The poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) homopolymer precursor is synthesized using RAFT polymerization. A quaternization reaction on the amine side groups using heptadecafluoro-10-iododecane results in the poly((2-dimethylamino) ethyl methacrylate-co-quaternized heptadecafluoro-10-iododecane (2-dimethylamino) ethyl methacrylate) (PQFD) amphiphilic statistical copolymers. We intentionally study statistical copolymers in our research of DMAEMA-based polyelectrolytes as there are not many studies about such materials. We have also chosen the incorporation of highly hydrophobic perfluorinated groups to study the changes in the solution properties of the initial hydrophilic homopolymer. The successful synthesis is supported by size-exclusion chromatography (SEC), 1H-NMR, and FTIR experiments. The self-assembly of the copolymers in aqueous solutions along with the dependence of the properties of the resulting aggregates on the pH, temperature, and ionic strength of the solution are studied using light scattering methods (DLS, ELS) and fluorescence spectroscopy (FS). The structural characteristics of the aggregates depend on composition, solution preparation protocol and solution pH, and temperature.

Structural and dynamic studies of the human RNA binding protein RBM3 reveals the molecular basis of its oligomerization and RNA recognition.

Human RNA-binding motif 3 protein (RBM3) is a cold-shock protein which functions in various aspects of global protein synthesis, cell proliferation and apoptosis by interacting with the components of basal translational machinery. RBM3 plays important roles in tumour progression and cancer metastasis, and also has been shown to be involved in neuroprotection and endoplasmic reticulum stress response. Here, we have solved the solution NMR structure of the N-terminal 84 residue RNA recognition motif (RRM) of RBM3. The remaining residues are rich in RGG and YGG motifs and are disordered. The RRM domain adopts a βαββαβ topology, which is found in many RNA-binding proteins. NMR-monitored titration experiments and molecular dynamic simulations show that the beta-sheet and two loops form the RNA-binding interface. Hydrogen bond, pi-pi and pi-cation are the key interactions between the RNA and the RRM domain. NMR, size exclusion chromatography and chemical cross-linking experiments show that RBM3 forms oligomers in solution, which is favoured by decrease in temperature, thus, potentially linking it to its function as a cold-shock protein. Temperature-dependent NMR studies revealed that oligomerization of the RRM domain occurs via nonspecific interactions. Overall, this study provides the detailed structural analysis of RRM domain of RBM3, its interaction with RNA and the molecular basis of its temperature-dependent oligomerization.

Crystal Structure and Molecular Mechanism of Phosphotransbutyrylase from Clostridium acetobutylicum.

Acetone-butanol-ethanol (ABE) fermentation by the anaerobic bacterium Clostridium acetobutylicum has been considered a promising process of industrial biofuel production. Phosphotransbutyrylase (phosphate butyryltransferase, PTB) plays a crucial role in butyrate metabolism by catalyzing the reversible conversion of butyryl-CoA into butyryl phosphate. Here, we report the crystal structure of PTB from the Clostridial host for ABE fermentation, C. acetobutylicum, (CaPTB) at a 2.9 Å resolution. The overall structure of the CaPTB monomer is quite similar to those of other acyltransferases, with some regional structural differences. The monomeric structure of CaPTB consists of two distinct domains, the N- and C-terminal domains. The active site cleft was formed at the interface between the two domains. Interestingly, the crystal structure of CaPTB contained eight molecules per asymmetric unit, forming an octamer, and the size-exclusion chromatography experiment also suggested that the enzyme exists as an octamer in solution. The structural analysis of CaPTB identifies the substrate binding mode of the enzyme and comparisons with other acyltransferase structures lead us to speculate that the enzyme undergoes a conformational change upon binding of its substrate.

Mutational analysis of TlyA from Brachyspira hampsonii reveals two key residues conserved in pathogenic bacteria responsible for oligomerization and hemolytic activity

BackgroundTlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis. MethodsTlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis. ResultsPurified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments. ConclusionsOligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence. General significanceOur results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.

Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells.

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

Characterization of a macroporous epoxy-polymer based resin for the ion-exchange chromatography of therapeutic proteins

This study investigated the adsorption capacity and mass transfer properties of a novel macroporous epoxy-polymer-based anion-exchanger, MPR Q, for the efficient separation of therapeutic proteins. MPR Q resin was prepared by phase separation based on spinodal decomposition followed by dextran grafting and ligand conjugation. Under static conditions, MPR Q exhibited a binding capacity of 49.8 mg-IgG/cm3-resin at pH 10, whereas the fastest adsorption was observed among the anion-exchanger resins tested. Inverse size-exclusion chromatography (iSEC) experiments revealed that the apparent pore diameter of MPR Q was approximately 90 nm, which was sufficiently large for the penetration of human IgG and bovine IgM. Moreover, the reduced height equivalent to a theoretical plate, h, of human IgG, determined using the linear gradient elution method was 65.8 and was not significantly changed in the range of linear velocities from 20.37 to 50.93 cm/min. The dynamic binding capacity at 10% breakthrough of MPR Q, determined by frontal analysis, exhibited a capacity of 43.8 mg/cm3 at 5.09 cm/min and 58% of DBC10% was maintained even though the linear velocity was increased to 50.93 cm/min. Furthermore, a resolution for separation of IgG and BSA by MPR Q was 1.06 at 5.09 cm/min, while it was higher than that for the conventional resin at all linear velocities from 5.09 cm/min to 50.93 cm/min. Thus, it was suggested that the MPR Q developed in this study is a promising resin that can efficiently separate large biomacromolecules such as human IgG at higher velocities.

Biochemical properties of VGLL4 from Homo sapiens and Tgi from Drosophila melanogaster and possible biological implications.

The TEAD (Sd in drosophila) transcription factors are essential for the Hippo pathway. Human VGLL4 and drosophila Tgi bind to TEAD/Sd via two distinct binding sites. These two regions are separated by few amino acids in VGLL4 but they are very distant from each other in Tgi. This difference prompted us to study whether it influences the interaction with TEAD4/Sd. We show that the full-length VGLL4/Tgi proteins behave as intrinsically disordered proteins. They have a similar affinity for TEAD4/Sd revealing that the length of the region between the two binding sites has little effect on the interaction. One of their two binding sites (high-affinity site) binds to TEAD4/Sd 100 times more tightly than to the other site, and size exclusion chromatography experiments reveal that VGLL4/Tgi only form trimeric complexes with TEAD4/Sd at high protein concentrations. In solution, therefore, VGLL4/Tgi may predominantly interact with TEAD4/Sd via their high-affinity site to create dimeric complexes. In contrast, when TEAD4/Sd molecules are immobilized on sensor chips used in Surface Plasmon Resonance experiments, one VGLL4/Tgi molecule can bind simultaneously with an enhanced affinity to two immobilized molecules. This effect, due to a local increase in protein concentration triggered by the proximity of the immobilized TEAD4/Sd molecules, suggests that in vivo VGLL4/Tgi could bind with an enhanced affinity to two nearby TEAD/Sd molecules bound to DNA. The presence of two binding sites in VGLL4/Tgi might only be required for the function of these proteins when they interact with TEAD/Sd bound to DNA.