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DSF protein

Aida Llauró, Pablo Guerra, Ravi Kant, Brian Bothner, Núria Verdaguer, Pedro J de Pablo
Vault particles are naturally occurring proteinaceous cages with promising application as molecular containers. The use of vaults as functional transporters requires a profound understanding of their structural stability to guarantee the protection and controlled payload delivery. Previous results performed with bulk techniques or at non-physiological conditions have suggested pH as a parameter to control vault dynamics. Here we use Atomic Force Microscopy (AFM) to monitor the structural evolution of individual vault particles while changing the pH in real time...
October 14, 2016: Scientific Reports
Jifeng Zhang, Vadim Frey, Marta Corcoran, Jian Zhang-van Enk, J Anand Subramony
Thermal stability of the CH2 domain for an IgG1 monoclonal antibody and its aggregation kinetics were systematically studied at pH 4.8, below its pI of 8.8 in individual solutions of arginine salts with acetate, glutamate (Glu(-)), chloride, and sulfate as the anion, in comparison to sodium chloride and sodium sulfate. Thermal unfolding temperature, Tm, an indicator of thermal stability, was measured by both differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF). The aggregation kinetics was determined by assessing reversibility for the CH2 domain in the DSC repetitive scans and then cross-examined by the isothermal aggregation study measured by size exclusion chromatography...
October 3, 2016: Molecular Pharmaceutics
Zhiqiang Chen, Chao Huang, Naresh Chennamsetty, Xuankuo Xu, Zheng Jian Li
Cation-exchange chromatography (CEX) of a structurally unstable Fc-fusion protein exhibited multi-peak elution profile upon a salt-step elution due to protein aggregation during intra-column buffer transition where low pH and high salt coexisted. The protein exhibited a single-peak elution behavior during a pH-step elution; nevertheless, the levels of soluble aggregates (i.e. high molecular weight species, HMW) in the CEX eluate were still found up to 12-fold higher than that for the load material. The amount of the aggregates formed upon the pH-step elution was dependent on column loading with maximum HMW achieved at intermediate loading levels, supporting the hypothesis that the aggregation was the result of both the conformational changes of the bound protein and the solution concentration of the aggregation-susceptible proteins during elution...
August 19, 2016: Journal of Chromatography. A
Dominic P Byrne, Matthias Vonderach, Samantha Ferries, Philip J Brownridge, Claire E Eyers, Patrick A Eyers
cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility-mass spectrometry (IM-MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI...
October 1, 2016: Biochemical Journal
Christoph Giese, Jonathan Eras, Anne Kern, Martin A Schärer, Guido Capitani, Rudi Glockshuber
The complex between the bacterial type 1 pilus subunit FimG and the peptide corresponding to the N-terminal extension (termed donor strand, Ds) of the partner subunit FimF (DsF) shows the strongest reported noncovalent molecular interaction, with a dissociation constant (KD ) of 1.5×10(-20)  m. However, the complex only exhibits a slow association rate of 330 m(-1)  s(-1) that limits technical applications, such as its use in affinity purification. Herein, a structure-based approach was used to design pairs of FimGt (a FimG variant lacking its own N-terminal extension) and DsF variants with enhanced electrostatic surface complementarity...
August 1, 2016: Angewandte Chemie
Umesh Yadava, Matthew W Vetting, Nawar Al Obaidi, Michael S Carter, John A Gerlt, Steven C Almo
The uptake of exogenous solutes by prokaryotes is mediated by transport systems embedded in the plasma membrane. In many cases, a solute-binding protein (SBP) is utilized to bind ligands with high affinity and deliver them to the membrane-bound components responsible for translocation into the cytoplasm. In the present study, Avi_5305, an Agrobacterium vitis SBP belonging to Pfam13407, was screened by differential scanning fluorimetry (DSF) and found to be stabilized by D-glucosamine and D-galactosamine. Avi_5305 is the first protein from Pfam13407 shown to be specific for amino sugars, and co-crystallization resulted in structures of Avi_5305 bound to D-glucosamine and D-galactosamine...
June 2016: Acta Crystallographica. Section F, Structural Biology Communications
Christine L Childers, Stuart R Green, Neal J Dawson, Kenneth B Storey
The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding (Knd), and protein unfolding by urea (I50). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity...
September 1, 2016: Analytical Biochemistry
Sujit Suklabaidya, Biswajit Das, Syed Azmal Ali, Sumeet Jain, Sharada Swaminathan, Ashok K Mohanty, Susen K Panda, Pujarini Dash, Subhankar Chakraborty, Surinder K Batra, Shantibhusan Senapati
Desmoplasia in human pancreatic cancer (PC) promotes cancer progression and hinders effective drug delivery. The objectives of this study were to characterize a homologous orthotopic model of PC in Syrian golden hamster and investigate the effect of anti-fibrotic (pirfenidone), antioxidant (N-acetyl cysteine, NAC) and anti-addiction (disulfiram, DSF) drugs on desmoplasia and tumor growth in this model. The HapT1 PC cells when implanted orthotopically into hamsters formed tumors with morphological, cellular and molecular similarities to human PC...
May 31, 2016: Oncotarget
Kara A DeSantis, Jeffrey L Reinking
Identification of small molecules that interact specifically with the ligand-binding domains (LBDs) of nuclear receptors (NRs) can be accomplished using a variety of methodologies. Here, we describe the use of differential scanning fluorimetry to identify these ligands, a technique that requires no modification or derivatization of either the protein or the ligand, and uses an instrument that is becoming increasingly affordable and common in modern molecular biology laboratories, the quantitative, or real-time, PCR machine...
2016: Methods in Molecular Biology
Erik Vernet, Gina Popa, Irina Pozdnyakova, Jakob E Rasmussen, Holger Grohganz, Lise Giehm, Malene H Jensen, Huabing Wang, Bitten Plesner, Hanne M Nielsen, Knud J Jensen, Jens Berthelsen, Michael Sundström, Marco van de Weert
PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS)...
May 2, 2016: Molecular Pharmaceutics
Wantong Song, Zhaohui Tang, Na Shen, Haiyang Yu, Yanjie Jia, Dawei Zhang, Jian Jiang, Chaoliang He, Huayu Tian, Xuesi Chen
A poly(l-glutamic acid) graft polyethylene glycol-cisplatin complex (PGA-CisPt) performs well in reducing the toxicity of free cisplatin and greatly enhances the accumulation and retention of cisplatin in solid tumors. However, there is a lack of effective treatment options for cisplatin-resistant tumors. A major reason for this is the dense PEG shell, which ensures that the PGA-CisPt maintains a long retention time in the blood that may result in it bypassing the tumor cells or failing to be endocytosed within the tumor microenvironment...
June 10, 2016: Journal of Controlled Release: Official Journal of the Controlled Release Society
Katrin Viigand, Triinu Visnapuu, Karin Mardo, Anneli Aasamets, Tiina Alamäe
Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α-methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α-glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS...
August 2016: Yeast
Yinyue Deng, Jien Wu, Wenfang Yin, Peng Li, Jianuan Zhou, Shaohua Chen, Fei He, Jun Cai, Lian-Hui Zhang
Diffusible signal factor (DSF) represents a new class of widely conserved quorum sensing signals, which regulates various biological functions through intra- or interspecies signaling. The previous studies identified that there is an antagonistic interaction between Xanthomonas and Bacillus species bacteria in natural ecosystem, but the detailed molecular mechanism of interspecies competition is not clear. This study showed that Xanthomonas campestris pv. campestris (Xcc) interfered with morphological transition and sporulation of Bacillus thuringiensis in mixed cultures, whereas abrogation of the DSF synthase RpfF reduced the interference...
May 2016: Environmental Microbiology
Lance M Hellman, Liusong Yin, Yuan Wang, Sydney J Blevins, Timothy P Riley, Orrin S Belden, Timothy T Spear, Michael I Nishimura, Lawrence J Stern, Brian M Baker
Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes...
May 2016: Journal of Immunological Methods
Lukas Hofmann, Sahil Gulati, Avery Sears, Phoebe L Stewart, Krzysztof Palczewski
Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the Kd values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT-PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL L-cystine (BFC), to overcome both of these shortcomings...
April 15, 2016: Analytical Biochemistry
Mirian Lansink, Zandrie Hofman, Stefano Genovese, Carlette H F C Rouws, Antonio Ceriello
BACKGROUND: Hyperglycemia frequently occurs in hospitalized patients receiving nutrition support. In this study, the effects of a new diabetes-specific formula (DSF) on glucose profile during 4 hours of continuous feeding and 4 hours after stopping feeding were compared with a standard formula (SF). MATERIALS AND METHODS: In this randomized, controlled, double-blind, crossover study, ambulant, nonhospitalized patients with type 2 diabetes received the DSF or an isocaloric, fiber-containing SF via a nasogastric tube...
January 29, 2016: JPEN. Journal of Parenteral and Enteral Nutrition
R Mierau
Testing for antinuclear antibodies (ANA) by the indirect immunofluorescence test (IFT) is regarded as a fundamental serological screening method for diagnosing connective tissue diseases (CTD). In the case of a negative result exclusion of certain CTDs is indicated, especially systemic lupus erythematosus, and a positive ANA result is the starting point for further tests aimed at finding disease-specific autoantibodies. The recently discovered antibodies against lens epithelium-derived growth factor (LEDGF/DSF70) deviate from the normal interpretation pattern in ANA diagnostics...
May 2016: Zeitschrift Für Rheumatologie
Amadeo B Biter, Andres H de la Peña, Roopa Thapar, Jean Z Lin, Kevin J Phillips
The Anfinsen hypothesis, the demonstration of which led to the Nobel prize in Chemistry, posits that all information required to determine a proteins' three dimensional structure is contained within its amino acid sequence. This suggests that it should be possible, in theory, to fold any protein in vitro. In practice, however, protein production by refolding is challenging because suitable refolding conditions must be empirically determined for each protein and can be painstaking. Here we demonstrate, using a variety of proteins, that differential scanning fluorimetry (DSF) can be used to determine and optimize conditions that favor proper protein folding in a rapid and high-throughput fashion...
2016: Scientific Reports
Martin Hügle, Xavier Lucas, Gerhard Weitzel, Dmytro Ostrovskyi, Bernhard Breit, Stefan Gerhardt, Oliver Einsle, Stefan Günther, Daniel Wohlwend
Several human diseases, including cancer, show altered signaling pathways resulting from changes in the activity levels of epigenetic modulators. In the past few years, small-molecule inhibitors against specific modulators, including the bromodomain and extra-terminal (BET) bromodomain family of acetylation readers, have shown early promise in the treatment of the genetically defined midline carcinoma and hematopoietic malignancies. We have recently developed a novel potent inhibitor of BET proteins, 1 (XD14[ Angew...
February 25, 2016: Journal of Medicinal Chemistry
Sujin Cho, Hookang Im, Ki-Young Lee, Jie Chen, Hae Ju Kang, Hye-Jin Yoon, Kyung Hoon Min, Kang Ro Lee, Hyun-Ju Park, Bong-Jin Lee
The crystal structure of 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDO8PS) from Helicobacter pylori (HpKDO8PS) was determined alone and within various complexes, revealing an extra helix (HE) that is absent in the structures of KDO8PS from other organisms. In contrast to the metal coordination of the KDO8PS enzyme from Aquifex aeolicus, HpKDO8PS is specifically coordinated with Cd(2+) or Zn(2+) ions, and isothermal titration calorimetry (ITC) and differential scanning fluorimetry (DSF) revealed that Cd(2+) thermally stabilizes the protein structure more efficiently than Zn(2+)...
January 27, 2016: European Journal of Medicinal Chemistry
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