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MicroScale Thermophoresis

Yanli Xie, Xiaolu Zhu, Yuan Li, Cheng Wang
The Fe(III) chelating activity of anthocyanin extracted from black soybean coats was investigated at pH 3.0, 5.0, 6.5, 7.0 and 7.4 with fluorescence spectroscopy and microscale thermophoresis (MST). Cyanidin-3-glucoside (C3G) was determined to be 98% of the total anthocyanin by HPLC. The binding affinity (Ka) exhibited significant pH-dependent behavior; Ka was 9.7167, 1.0837, 1.4284, 5.4550 and 3.0269×104 M-1 at pH 3.0, 5.0, 6.5, 7.0 and 7.4, respectively (p<0.05). The MST data showed that ΔG<0 and ΔH<0, demonstrating that chelation is spontaneous and exothermic...
January 15, 2018: Journal of Agricultural and Food Chemistry
Michelle H Moon, Thomas Hilimire, Allix M Sanders, John S Schneekloth
In the past twenty years, there has been a dramatic increase in the study of RNA. RNA has gone from being known as an intermediate in the central dogma of molecular biology to a molecule with a large diversity of structure and function that is involved in all aspects of biology. As new functions are rapidly discovered, it has become clear that there is a need for RNA-targeting small molecule probes to investigate RNA biology and clarify the potential for therapeutics based on RNA/small molecule interactions...
January 12, 2018: Biochemistry
Torun Ekblad, Patricia Verheugd, Anders E Lindgren, Tomas Nyman, Mikael Elofsson, Herwig Schüler
Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay...
January 1, 2018: SLAS Discovery
Fengyan Bao, Kaiyin Yang, Canrong Wu, Suyu Gao, Penghe Wang, Lixia Chen, Hua Li
Hexokinase 2 (HK2), a rate-limiting enzyme in the first step of glycolysis pathway, expresses at high level in cancer cells compared with normal cells. HK2 provides a new target for cancer therapy due to its pivotal role in tumor tumourigenic and metastatic process. The structure-based virtual ligand screening in a small in-house database of natural products predicted that a new steroid, (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (2) from Ganoderma sinense has high binding affinity to HK2 with significant calculated binding free energy...
January 3, 2018: Fitoterapia
Romain Magnez, Bryan Thiroux, Solenne Taront, Zacharie Segaoula, Bruno Quesnel, Xavier Thuru
The characterization of protein interactions has become essential in many fields of life science, especially drug discovery. Microscale thermophoresis (MST) is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. In addition, one of the major advantages of this technique is that no tedious purification step is necessary to access the protein of interest. Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 couple, which is involved in tumour escape processes, without purification of the target protein from cell lysates...
December 15, 2017: Scientific Reports
Damien Morger, Franziska Zosel, Martin Bühlmann, Sara Züger, Maximilian Mittelviefhaus, Benjamin Schuler, Jeremy Luban, Markus G Grütter
Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remains largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice...
December 13, 2017: Journal of Virology
Maria-Jose Garcia-Bonete, Maja Jensen, Christian V Recktenwald, Sandra Rocha, Volker Stadler, Maria Bokarewa, Gergely Katona
A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners...
December 1, 2017: Scientific Reports
Lorenz M R Keil, Friederike M Möller, Michael Kieß, Patrick W Kudella, Christof B Mast
Proton gradients are essential for biological systems. They not only drive the synthesis of ATP, but initiate molecule degradation and recycling inside lysosomes. However, the high mobility and permeability of protons through membranes make pH gradients very hard to sustain in vitro. Here we report that heat flow across a water-filled chamber forms and sustains stable pH gradients. Charged molecules accumulate by convection and thermophoresis better than uncharged species. In a dissociation reaction, this imbalances the reaction equilibrium and creates a difference in pH...
December 1, 2017: Nature Communications
Peggy Reich, Regina Stoltenburg, Beate Strehlitz, Dieter Frense, Dieter Beckmann
In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1...
November 21, 2017: International Journal of Molecular Sciences
Yexuan Mao, Lanlan Yu, Mengfan Mao, Chuanguo Ma, Lingbo Qu
Type 2 diabetes mellitus, a kind of conformational disease, has become an epidemic disease, which seriously endangers the quality of life and health of human beings. The deposition of human islet amyloid polypeptide (hIAPP) has been considered as one of the major pathological features of type 2 diabetes mellitus. As lipopeptides have some hydrophobic groups, which are similar to the reported aggregation inhibitors, and some lipopeptides could prevent cells from depositing of amyloid fibrils, several potential lipopeptide inhibitors have been engineered and synthesized, which have been assessed for their inhibitory effect in preventing amyloid fibrils formation of hIAPP11-20 by using the conventional thioflavin-T fluorescence assay and new technique microscale thermophoresis (MST)...
November 21, 2017: Journal of Peptide Science: An Official Publication of the European Peptide Society
Syntia Fayad, Philippe Morin, Reine Nehmé
Elastase, collagenase, hyaluronidase and tyrosinase, are very interesting enzymes due to their direct implication in skin aging and as therapeutic hits. Different techniques can be used to study these enzymes and to evaluate the influence of effectors on their kinetics. Nowadays, analytical techniques have become frequently used tools for miniaturizing enzyme assays. The main intention of this article is to review chromatographic and electrophoretic tools that study the four enzymes above mentioned. More specifically, the use of high-performance liquid chromatography and capillary electrophoresis and their derivative techniques for monitoring these enzymes will be investigated...
November 9, 2017: Journal of Chromatography. A
Bernardina Scafuri, Antonio Varriale, Angelo Facchiano, Sabato D'Auria, Maria Elisabetta Raggi, Anna Marabotti
We have applied a combined computational procedure based on inverse and direct docking in order to identify putative protein targets of a panel of mycotoxins and xenobiotic compounds that can contaminate food and that are known to have several detrimental effects on human health. This procedure allowed us to identify a panel of human proteins as possible targets for aflatoxins, gliotoxin, ochratoxin A and deoxynivalenol. Steady-state fluorescence and microscale thermophoresis experiments allowed us to confirm the binding of some of these mycotoxins to acetylcholinesterase and X-linked neuroligin 4, two proteins involved in synapse activity and, particularly for the second protein, neuronal plasticity and development...
November 9, 2017: Scientific Reports
Nikola Štambuk, Paško Konjevoda, Petra Turčić, Katalin Kövér, Renata Novak Kujundžić, Zoran Manojlović, Mario Gabričević
Sense and antisense peptides, i.e. peptides specified by complementary DNA and RNA sequences, interact with increased probability. Biro, Blalock, Mekler, Root-Bernstein and Siemion investigated the recognition rules of peptide-peptide interaction based on the complementary coding of DNA and RNA sequences in 3'→5' and 5'→3' directions. After more than three decades of theoretical and experimental investigations, the efficiency of this approach to predict peptide-peptide binding has been experimentally verified for more than 50 ligand-receptor systems, and represents a promising field of research...
October 28, 2017: Bio Systems
Christian T Hjuler, Nicolai N Maolanon, Jørgen Sauer, Jens Stougaard, Mikkel B Thygesen, Knud J Jensen
Glycobiology, in particular the study of carbohydrate-protein interactions and the events that follow, has become an important research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified here by rhizobial Nod factors and exopolysaccharide fragments...
November 2017: Nature Protocols
Shih-Chia Tso, Qiuyan Chen, Sergey A Vishnivetskiy, Vsevolod V Gurevich, T M Iverson, Chad A Brautigam
The emergence of microscale thermophoresis (MST) as a technique for determining the dissociation constants for bimolecular interactions has enabled these quantities to be measured in systems that were previously difficult or impracticable. However, most models for analyses of these data featured the assumption of a simple 1:1 binding interaction. The only model widely used for multiple binding sites was the Hill equation. Here, we describe two new MST analytic models that assume a 1:2 binding scheme: the first features two microscopic binding constants (KD(1) and KD(2)), while the other assumes symmetry in the bivalent molecule, culminating in a model with a single macroscopic dissociation constant (KD,M) and a single factor (α) that accounts for apparent cooperativity in the binding...
October 17, 2017: Analytical Biochemistry
Victor Bandlow, Susanne Liese, Daniel Lauster, Kai Ludwig, Roland R Netz, Andreas Herrmann, Oliver Seitz
Attachment of the Influenza A virus onto host cells involves multivalent interactions between virus surface hemagglutinin (HA) and sialoside-containing glyco ligands. Despite the development of extremely powerful multivalent binders of the Influenza virus and other viruses, comparably little is known about the optimal spacing of HA ligands, which ought to bridge binding sites within or across the trimeric HA molecules. To explore the criteria for ligand economical high affinity binding, we systematically probed distance-affinity relationships by means of two differently behaving scaffold types based on (i) flexible polyethylene glycol (PEG) conjugates and (ii) rigid self-assembled DNA·PNA complexes...
November 15, 2017: Journal of the American Chemical Society
Andrea Topf, Peter Franz, Georgios Tsiavaliaris
Here, we present a MicroScale Thermophoresis (MST)-based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases: nucleation, elongation, and steady-state equilibration. Titration experiments allowed us to evaluate the effects of actin-binding proteins (ABPs) on polymerization, including DNase I-induced inhibition and mDia2FH1FH2 (mDia2)-assisted acceleration of nucleation...
October 1, 2017: BioTechniques
Alice Coletti, Francesca Camponeschi, Elisa Albini, Francesco Antonio Greco, Vincenzo Maione, Chiara Custodi, Federica Ianni, Ursula Grohmann, Ciriana Orabona, Francesca Cantini, Antonio Macchiarulo
Indoleamine 2,3-dioxygenase 1 (IDO1) is attracting a great deal of interest as drug target in immune-oncology being highly expressed in cancer cells and participating to the tumor immune-editing process. Although several classes of IDO1 inhibitors have been reported in literature and patent applications, only few compounds have proved optimal pharmacological profile in preclinical studies to be advanced in clinical trials. Accordingly, the quest for novel structural classes of IDO1 inhibitors is still open...
December 1, 2017: European Journal of Medicinal Chemistry
Lukas Traxler, Petr Rathner, Marc Fahrner, Michael Stadlbauer, Felix Faschinger, Tatsiana Charnavets, Norbert Müller, Christoph Romanin, Peter Hinterdorfer, Hermann J Gruber
Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3...
October 11, 2017: Angewandte Chemie
Adrian Michael Mueller, Dennis Breitsprecher, Stefan Duhr, Philipp Baaske, Thomas Schubert, Gernot Längst
Interactions between nucleic acids and proteins are driving gene expression programs and regulating the development of organisms. The binding affinities of transcription factors to their target sites are essential parameters to reveal their binding site occupancy and function in vivo. Microscale Thermophoresis (MST) is a rapid and precise method allowing for quantitative analysis of molecular interactions in solution on a microliter scale. The technique is based on the movement of molecules in temperature gradients, which is referred to as thermophoresis, and depends on molecule size, charge, and hydration shell...
2017: Methods in Molecular Biology
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