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MicroScale Thermophoresis

Zhimei Li, Yiyuan Zhang, Lixia Chen, Hua Li
Overexpression of the prosurvival protein BCL-2 contributes to malignant cell initiation, progression and resistance to treatment. Agents that function as its natural antagonists targeting BCL-2 must provide therapeutic benefit. In SW1990 pancreatic cancer cells, amplified BCL-2 was observed, which was believed to offer advantages for malignant cell survival and lead to poor patient outcome. Using structure-based virtual ligand screening, luteolin was found to be a natural small-molecule inhibitor of BCL-2, which exhibited dose-response proapoptosis activity in a BCL-2 dependent manner in vitro...
May 17, 2018: Food & Function
Jiří Zahradník, Lucie Kolářová, Hana Pařízková, Petr Kolenko, Bohdan Schneider
Interferon gamma (IFN-γ) is one of the key players in the immune system of vertebrates. The evolution and properties of IFN-γ and its receptors in fish species are of special interest as they point to the origin of innate immunity in vertebrates. We studied the phylogeny, biophysical and structural properties of IFN-γ and its receptors. Our phylogeny analysis suggests the existence of two groups of IFN-γ related proteins, one specific for Acanthomorpha, the other for Cypriniformes, Characiformes and Siluriformes...
May 6, 2018: Fish & Shellfish Immunology
Floriane Gibault, Mathilde Coevoet, Manon Sturbaut, Amaury Farce, Nicolas Renault, Frédéric Allemand, Jean-François Guichou, Anne-Sophie Drucbert, Catherine Foulon, Romain Magnez, Xavier Thuru, Matthieu Corvaisier, Guillemette Huet, Philippe Chavatte, Patricia Melnyk, Fabrice Bailly, Philippe Cotelle
Intrinsically disordered protein YAP (yes-associated protein) interacts with TEADs transcriptional factors family (transcriptional enhancer associated domain) creating three interfaces. Interface 3, between the Ω-loop of YAP and a shallow pocket of TEAD was identified as the most important TEAD zone for YAP-TEAD interaction. Using the first X-ray structure of the hYAP50⁻71 -hTEAD1209⁻426 complex (PDB 3KYS) published in 2010, a protein-protein interaction inhibitors-enriched library (175,000 chemical compounds) was screened against this hydrophobic pocket of TEAD...
May 8, 2018: Cancers
Wenxiu Ma, Lifang Zou, Zhiyuan Ji, Xiameng Xu, Zhengyin Xu, Yangyang Yang, James R Alfano, Gongyou Chen
Xanthomonas oryzae pv. oryzae (Xoo), causal agent of bacterial blight (BB) of rice, uses transcription activator-like effectors (TALEs) to interact with the basal transcription factor gama subunit OsTFIIAγ5 (Xa5) and activates transcription of host genes. However, how OsTFIIAγ1, the other OsTFIIAγ protein, functions in the presence of TALEs remains unclear. In this study, we show that OsTFIIAγ1 plays a compensatory role in the absence of Xa5. The expression of OsTFIIAγ1, which is activated by TALE PthXo7, increased the expression of host genes targeted by avirulent and virulent TALEs...
April 28, 2018: Molecular Plant Pathology
Oscar B Torres, Alexander J Duval, Agnieszka Sulima, Joshua F G Antoline, Arthur E Jacobson, Kenner C Rice, Carl R Alving, Gary R Matyas
We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine...
April 19, 2018: Analytical and Bioanalytical Chemistry
Monika Kosmacz, Marcin Luzarowski, Olga Kerber, Ewa Leniak, Emilio Gutierrez-Beltran, Juan Camilo Moreno Beltran, Michal Gorka, Jagoda Szlachetko, Daniel Veyel, Alexander Graf, Aleksandra Skirycz
2',3'-cyclic adenosine monophosphate (2',3'-cAMP), an intriguing small molecule, is conserved among different kingdoms. This small molecule is presumably produced during RNA degradation, with increased cellular levels especially upon stress conditions. We observed its presence in protein complexes isolated from native Arabidopsis lysate, suggesting that 2',3'-cAMP might have protein partners in plants. Moreover, affinity purification (AP) experiments revealed that 2',3'-cAMP associates with the stress granule (SG) proteome...
April 4, 2018: Plant Physiology
Xia Zhong, Shaomin Yang, Tianxiang Liu, Shundong Ji, Jinrui Hu, Hongjian Li
To develop an effective long-acting antidiabetic agent, we designed a novel Exendin-4 derivative (termed LEx4) containing an albumin-binding domain (ABD), a protease-cleavable linker and a native Exendin-4. Here, we present the LEx4 with balanced glucoregulatory activity and prolonged in vivo activity. As a first step, the LEx4 with purity more than 99% was prepared. Microscale thermophoresis (MST) results demonstrated that LEx4 associates with rat and monkey serum albumin with high-affinity (Ka  = 1.26 × 106  M-1 and 1...
March 21, 2018: European Journal of Medicinal Chemistry
Benjamin Clémençon, Benjamin P Lüscher, Matthias A Hediger
INTRODUCTION: Membrane proteins represent roughly one third of the human proteome and many of them serve as targets of therapeutic drugs. An exception is the SLC solute carrier superfamily with only a handful of approved drugs targeting SLCs. Indeed, for many of the SLCs, the natural transport substrates are still unknown. A major limitation for SLCs has been the difficulty to thoroughly characterize these multimembrane spanning proteins. The intrinsic properties of membrane proteins with alternative hydrophobic and hydrophilic domains lead to instability, making the purification tasks even more challenging compared to soluble proteins...
March 23, 2018: Journal of Pharmacological and Toxicological Methods
Tanja Bartoschik, Stefanie Galinec, Christian Kleusch, Katarzyna Walkiewicz, Dennis Breitsprecher, Sebastian Weigert, Yves A Muller, Changjiang You, Jacob Piehler, Thomas Vercruysse, Dirk Daelemans, Nuska Tschammer
MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques...
March 21, 2018: Scientific Reports
Martin Köhler, Christoph Neff, Camilo Perez, Cyrill Brunner, Els Pardon, Jan Steyaert, Gisbert Schneider, Kaspar P Locher, Renato Zenobi
The application of nanobodies as binding partners for structure stabilization in protein x-ray crystallography is taking an increasingly important role in structural biology. However, the addition of nanobodies to the crystallization matrices might complicate the optimization of the crystallization process, which is why analytical techniques to screen and characterize suitable nanobodies are useful. Here, we show how chemical cross-linking combined with high-mass matrix-assisted laser/desorption ionization mass spectrometry can be employed as a fast screening technique to determine binding specificities of intact nanobody•membrane protein complexes...
March 21, 2018: Analytical Chemistry
Milton Cordeiro, Ana Rita Otrelo-Cardoso, Dmitri I Svergun, Petr V Konarev, João Carlos Lima, Teresa Santos-Silva, Pedro Viana Baptista
Selective base pairing is the foundation of DNA recognition. Here, we elucidate the molecular and structural details of a FRET-based two-component molecular beacon relying on Steady-state Fluorescence Spectroscopy, Small-angle X-ray Scattering (SAXS), Microscale Thermophoresis (MST) and Differential Electrophoretic Mobility. This molecular beacon was designed to detect the most common fusion sequences causing chronic myeloid leukemia, e14a2 and e13a2. The emission spectra indicate that the self-assembly of the different components of the biosensor occurs sequentially, triggered by the fully complementary target...
March 21, 2018: ACS Chemical Biology
Andrew M Prantner, Catherine Yin, Kalika Kamat, Khushboo Sharma, Andrew C Lowenthal, Peter B Madrid, Nathalie Scholler
Mesothelin is an epithelial marker highly expressed at the cell surface of cancer cells from diverse origins, including ovarian and pancreatic adenocarcinomas and mesotheliomas. Previously, we identified and characterized an antimesothelin nanobody (NbG3a) for in vitro diagnostic applications. The main goal of this research was to establish the potential of NbG3a as a molecular imaging agent. Site-specific biotinylated NbG3a (bNbG3a) was bound to streptavidin-conjugated reagents for in vitro and in vivo assays...
April 2, 2018: Molecular Pharmaceutics
Julie M Rainard, George C Pandarakalam, Stuart P McElroy
High-throughput screening (HTS) is a proven method for discovering new lead matter for drug discovery and chemical biology. To maximize the likelihood of identifying genuine binders to a molecular target, and avoid wasting resources following up compounds with unproductive/nonspecific mechanisms of action, it is important to employ a range of assays during an HTS campaign that build confidence of target engagement for hit compounds. Biophysical methods that measure direct target/compound engagement have established themselves as key techniques in generating this confidence, and they are now integral to the latter stages of HTS triage at the European Lead Factory (ELF)...
March 2018: SLAS Discovery
Yiwen Zhang, Zhimei Li, Qiuxia Min, Abulizi Palida, Yiyuan Zhang, Ruotian Tang, Lixia Chen, Hua Li
8-Chrysoeriol, a bioactive flavanoid, was firstly identified to bind directly to BCL-2 as BH3 mimetics by structure-based virtual ligand screening. And 3D docking model revealed the molecular basis of 8-Chrysoeriol targeting to BCL-2. The interaction between 8-Chrysoeriol and BCL-2 was further confirmed using Microscale Thermophoresis (MST) technique. Meanwhile, high expression level of antiapoptotic protein BCL-2 was detected in SW1990 pancreatic cancer cells and 8-Chrysoeriol showed obvious proapoptosis effect against SW1990 in vitro...
April 2018: Bioorganic Chemistry
Mufarreh Asmari, Ratih Ratih, Hassan A Alhazmi, Sami El Deeb
The study of biomolecular interactions is crucial to get more insight into the biological system. The interactions of protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and protein-small molecules are supporting therapeutics and technological developments. Recently, the development in a large number of analytical techniques for characterizing biomolecular interactions reflect the promising research investments in this field. In this review, microscale thermophoresis technology (MST) is presented as an analytical technique for characterizing biomolecular interactions...
February 10, 2018: Methods: a Companion to Methods in Enzymology
Marta Narczyk, Branimir Bertoša, Lucija Papa, Vedran Vuković, Ivana Leščić Ašler, Beata Wielgus-Kutrowska, Agnieszka Bzowska, Marija Luić, Zoran Štefanić
Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (Pi ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies...
April 2018: FEBS Journal
Chuannan Long, Mengmeng Liu, Xia Chen, Xiaofang Wang, Mingqiang Ai, Jingjing Cui, Bin Zeng
The present study verified whether acyl-coenzyme A (acyl-CoA)-binding protein (ACBP) affected the production of Monascus pigments (MPs) in Monascus ruber CICC41233 (MrACBP). Phylogenetic analysis revealed that the cloned Mracbp gene, which encoded the MrACBP protein, exhibited the closest match (99% confidence level) to the gene from Penicilliopsis zonata . The MrACBP and maltose-binding protein (MBP) were simultaneously expressed in Escherichia coli Rosetta DE3 in the form of a fusion protein. The microscale thermophoresis binding assay revealed that the purified MBP-MrACBP exhibited a higher affinity for myristoyl-CoA (Kd = 88...
February 2018: 3 Biotech
Michael Zepp, Marineta Kovacheva, Munkhtsetseg Altankhuyag, Gabriela Westphal, Irina Berger, Katharina S Gather, Heidegard Hilbig, Jochen Neuhaus, Gertrud M Hänsch, Franz P Armbruster, Martin R Berger
Changes in glycosylation are salient features of cancer cells. Here, we report on the diagnostic and therapeutic properties of IDK1, an antibody against tumour associated, hypoglycosylated bone sialoprotein (hypo-BSP). The affinity of the rat monoclonal antibody IDK1 for hypo-BSP, as determined by microscale thermophoresis, was three orders of magnitude higher than for mature BSP, whereas the mouse monoclonal antibody used had similar affinity for both BSP forms. IDK1 showed no activity against the proliferation or migration of normal or cancer cells growing in vitro ...
January 2018: Journal of Pathology. Clinical Research
Anmol S Adhav, Surabhi R Kokane, Rakesh S Joshi
Trehalase catalyzes hydrolysis of trehalose and plays a crucial role in insect metabolism. In the present study, phylogenetic analysis and multiple sequence alignment suggested that H. armigera trehalase-1 (HaTre-1) is closely related to other soluble trehalases with conserved signature features and functional sites. We have expressed and purified recombinant HaTre-1 having Vmax ~0.16mM/min and KM ~1.34mM. Inhibition kinetics and Microscale thermophoresis illustrated competitive inhibition of HaTre-1 by Validamycin A having Ki ~3nM and KD ~542nM, respectively...
June 2018: International Journal of Biological Macromolecules
Xiaoyu Shi, Yue Sun, Ping Wang, Lingling Gu, Lu Wang, Huan Yang, Qun Wei, Zhimei Li, Jing Luo
Calcineurin (CN) is a protein phosphatase and widely distributed in eukaryotes, with an extremely high level of expression in mammalian brain. Alpha-synuclein (α-syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. In our present study, we explored the interactions between CN and α-syn in vitro. Based on the data from microscale thermophoresis, GST pull-down assays, and co-immunoprecipitation, we found that CN binds α-syn. Furthermore, this interaction is mediated by calcium/calmodulin (Ca2+ /CaM) signaling...
February 19, 2018: Biochemical and Biophysical Research Communications
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