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MicroScale Thermophoresis

André Reinhard, Thorsten Nürnberger
Dissecting the functional basis of pathogenicity and resistance in the context of plant innate immunity benefits greatly from detailed knowledge about biomolecular interactions, as both resistance and virulence depend on specific interactions between pathogen and host biomolecules. While in vivo systems provide biological context to host-pathogen interactions, these experiments typically cannot provide quantitative biochemical characterization of biomolecular interactions. However, in many cases, the biological function does not only depend on whether an interaction occurs at all, but rather on the "intensity" of the interaction, as quantified by affinity...
2017: Methods in Molecular Biology
Clemens Entzian, Thomas Schubert
Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera)...
January 7, 2017: Journal of Visualized Experiments: JoVE
Qingshan Liu, Ran Deng, Qingfang Yan, Lin Cheng, Yongming Luo, Li Keqin, Xiaoying Yin, Xiaoyan Qin
In order to efficiently screen and isolate β-tubulin inhibitors, β-tubulin was immobilized on core-shell PMMA/CS (poly(methyl methacrylate)/Chitosan) nanoparticles to produce a new type of immobilized affinity material named β-tubulin immobilized nanoparticles (β-TIN). The selectivity and adsorption performance of β-TIN were characterizedusing various control drugs. The β-TIN, the paclitaxel molecularly imprinted ploymers (MIP) , and the C18 adsorbing material were compared for selectivity and enrichment ratio...
January 23, 2017: ACS Applied Materials & Interfaces
Tiphaine Rogez-Florent, Catherine Foulon, Anne-Sophie Drucbert, Nadège Schifano, Perrine Six, Stéphanie Devassine, Patrick Depreux, Pierre-Marie Danzé, Laurence Goossens, Cécile Danel, Jean-François Goossens
The aim of this study was to develop a method combining chiral separation and biophysical techniques to evaluate the enantioselective affinity of original sulfonamide derivatives towards their therapeutic target, the human carbonic anhydrase II (hACII). The first step consisted in the preparation of the enantiomers by chromatographic separation. The performances of HPLC and Supercritical Fluid Chromatography (SFC) were studied at the analytical scale by optimization of various experimental conditions using adsorbed polysaccharide chiral stationary phases (amylose AD-H and cellulose OD-H)...
January 9, 2017: Journal of Pharmaceutical and Biomedical Analysis
Christian Nienberg, Claudia Garmann, Andreas Gratz, Andre Bollacke, Claudia Götz, Joachim Jose
Human protein kinase CK2 has emerged as promising target for the treatment of neoplastic diseases. The vast majority of kinase inhibitors known today target the ATP binding site, which is highly conserved among kinases and hence leads to limited selectivity. In order to identify non-ATP competitive inhibitors, a 12-mer peptide library of 6 × 10⁵ variants was displayed on the surface of E. coli by autodisplay. Screening of this peptide library on variants with affinity to CK2 was performed by fluorophore-conjugated CK2 and subsequent flow cytometry...
January 5, 2017: Pharmaceuticals
Gunther Zimmermann, Yizhou Li, Ulrike Rieder, Martin Mattarella, Dario Neri, Jörg Scheuermann
DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article, we present novel methods, based on the hybridization of oligonucleotide conjugates with fluorescently-labeled complementary oligonucleotides, which facilitate the determination of affinity constants and kinetic dissociation constants...
January 9, 2017: Chembiochem: a European Journal of Chemical Biology
Sophie Dobiasch, Szilard Szanyi, Aleko Kjaev, Jens Werner, Albert Strauss, Christian Weis, Lars Grenacher, Katya Kapilov-Buchman, Liron-Limor Israel, Jean-Paul Lellouche, Erica Locatelli, Mauro Comes Franchini, Jennifer Vandooren, Ghislain Opdenakker, Klaus Felix
BACKGROUND: Functionalized nanoparticles (NPs) are one promising tool for detecting specific molecular targets and combine molecular biology and nanotechnology aiming at modern imaging. We aimed at ligand-directed delivery with a suitable target-biomarker to detect early pancreatic ductal adenocarcinoma (PDAC). Promising targets are galectins (Gal), due to their strong expression in and on PDAC-cells and occurrence at early stages in cancer precursor lesions, but not in adjacent normal tissues...
December 19, 2016: Journal of Nanobiotechnology
Si-Ting Chen, Ning-Yu He, Juan-Hua Chen, Fang-Qing Guo
Photosystem II (PSII) is the most thermolabile photosynthetic complex. Physiological evidence suggests that the small chloroplast heat-shock protein 21 (HSP 21) is involved in plant thermotolerance but the molecular mechanism of its action remains largely unknown. Here, we have provided genetic and biochemical evidence that HSP 21 is activated by the GUN5-dependent retrograde signaling pathway and stabilizes PSII by directly binding to its core subunits such as D1 and D2 proteins under heat stress. We further demonstrate that the constitutive expression of HSP21 sufficiently rescues the thermo-sensitive stability of PSII and survival defects of the gun5 mutant with dramatically improving granal stacking under heat stress, indicating that HSP21 is a master chaperone protein in maintaining the integrity of thylakoid membrane system under heat stress...
December 10, 2016: Plant Journal: for Cell and Molecular Biology
David Ulbricht, Kathrin Oertwig, Kristin Arnsburg, Anja Saalbach, Jan Pippel, Norbert Sträter, John T Heiker
Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules...
January 20, 2017: Journal of Biological Chemistry
Flavia A Mercurio, Daniela Marasco, Concetta Di Natale, Luciano Pirone, Susan Costantini, Emilia M Pedone, Marilisa Leone
The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam-Sam interactions. Because EphA2-Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues...
October 20, 2016: Chembiochem: a European Journal of Chemical Biology
Weiying Zhang, Xiangyang Li, Guoping Zhang, Yan Ding, Longlu Ran, Liangzhi Luo, Jian Wu, Deyu Hu, Baoan Song
Tobacco mosaic virus (TMV) is an important plant virus that can cause considerable crop loss. Our group synthesized a series of enantiomeric α-aminophosphonate derivatives with high anti-TMV activities. The activity of (R)-diphenyl-1-(4-methylbenzothiazole-2-amino)-1-(thiphene-2-yl)-methylphosphonate (Q-R) was found to be superior to that of (S)-diphenyl-1-(4-methyl benzothiazole-2-amino)-1-(thiphene-2-yl)-methylphosphonate (Q-S). However, the mechanism for inhibition of the R-isomer (Q-R) of infection activity is not clear...
January 2017: International Journal of Biological Macromolecules
T Dau, E V Edeleva, S A I Seidel, R A Stockley, D Braun, D E Jenne
High abundance proteins like protease inhibitors of plasma display a multitude of interactions in natural environments. Quantitative analysis of such interactions in vivo is essential to study diseases, but have not been forthcoming, as most methods cannot be directly applied in a complex biological environment. Here, we report a quantitative microscale thermophoresis assay capable of deciphering functional deviations from in vitro inhibition data by combining concentration and affinity measurements. We obtained stable measurement signals for the substrate-like interaction of the disease relevant inhibitor α-1-antitrypsin (AAT) Z-variant with catalytically inactive elastase...
October 14, 2016: Scientific Reports
Achim Löf, Jochen P Müller, Martin Benoit, Maria A Brehm
The large multimeric plasma glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis by recruiting platelets to sites of vascular injury. VWF multimers respond to elevated hydrodynamic forces by elongation, thereby increasing their adhesiveness to platelets. Thus, the activation of VWF is force-induced, as is its inactivation. Due to these attributes, VWF is a highly interesting system from a biophysical point of view, and is well suited for investigation using biophysical approaches. Here, we give an overview on recent studies that predominantly employed biophysical methods to gain novel insights into multiple aspects of VWF: Electron microscopy was used to shed light on the domain structure of VWF and the mechanism of VWF secretion...
September 28, 2016: Advances in Biological Regulation
Mindy I Davis, Rajan Pragani, Jennifer T Fox, Min Shen, Kalindi Parmar, Emily F Gaudiano, Li Liu, Cordelle Tanega, Lauren McGee, Matthew D Hall, Crystal McKnight, Paul Shinn, Henrike Nelson, Debasish Chattopadhyay, Alan D D'Andrea, Douglas S Auld, Larry J DeLucas, Zhuyin Li, Matthew B Boxer, Anton Simeonov
Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 μm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines...
November 18, 2016: Journal of Biological Chemistry
Markéta Svobodová, Vasso Skouridou, Mary Luz Botero, Miriam Jauset-Rubio, Thomas Schubert, Abdulaziz S Bashammakh, Mohammad S El-Shahawi, Abdulrahman O Alyoubi, Ciara K O'Sullivan
The rapid and sensitive detection of small molecules is garnering increasing importance, and aptamers show great promise in replacing expensive, elaborate detection platforms exploiting chromatographic separation or antibody-based assays. The characterization of aptamer interaction with small molecule targets is not facile, and there is a mature need for a rapid, high-throughput technique for the analysis of aptamer-small molecule kinetics and affinity. In this work we present methodologies for the evaluation of aptamer-small molecule interactions, using the aptamers reported against the steroid 17β-estradiol as a model system...
March 2017: Journal of Steroid Biochemistry and Molecular Biology
Petra Zadravec, Lucie Marečková, Hana Petroková, Vesna Hodnik, Milica Perišić Nanut, Gregor Anderluh, Borut Štrukelj, Petr Malý, Aleš Berlec
Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display...
2016: PloS One
Kristina Szameit, Katharina Berg, Sven Kruspe, Erica Valentini, Eileen Magbanua, Marcel Kwiatkowski, Isaure Chauvot de Beauchêne, Boris Krichel, Kira Schamoni, Charlotte Uetrecht, Dmitri I Svergun, Hartmut Schlüter, Martin Zacharias, Ulrich Hahn
G-quadruplexes have recently moved into focus of research in nucleic acids, thereby evolving in scientific significance from exceptional secondary structure motifs to complex modulators of gene regulation. Aptamers (nucleic acid based ligands with recognition properties for a specific target) that form Gquadruplexes may have particular potential for therapeutic applications as they combine the characteristics of specific targeting and Gquadruplex mediated stability and regulation. We have investigated the structure and target interaction properties of one such aptamer: AIR-3 and its truncated form AIR-3A...
October 2, 2016: RNA Biology
Tatiana P Soares da Costa, Sebastien Desbois, Con Dogovski, Michael A Gorman, Natalia E Ketaren, Jason J Paxman, Tanzeela Siddiqui, Leanne M Zammit, Belinda M Abbott, Roy M Robins-Browne, Michael W Parker, Geoffrey B Jameson, Nathan E Hall, Santosh Panjikar, Matthew A Perugini
Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not. The 1.65-Å resolution structure of the Gram-negative Legionella pneumophila DHDPS and the 1.88-Å resolution structure of the Gram-positive Streptococcus pneumoniae DHDPS bound to lysine, together with comprehensive functional analyses, show that this dogma is incorrect...
August 2, 2016: Structure
Tan Qin, Fangjin Chen, Xiaolong Zhuo, Xiao Guo, Taikangxiang Yun, Ying Liu, Chuanmao Zhang, Luhua Lai
Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations...
August 11, 2016: Journal of Medicinal Chemistry
Yuhua Zhan, Yongliang Yan, Zhiping Deng, Ming Chen, Wei Lu, Chao Lu, Liguo Shang, Zhimin Yang, Wei Zhang, Wei Wang, Yun Li, Qi Ke, Jiasi Lu, Yuquan Xu, Liwen Zhang, Zhihong Xie, Qi Cheng, Claudine Elmerich, Min Lin
Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade...
July 26, 2016: Proceedings of the National Academy of Sciences of the United States of America
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