scholarly journals Measuring RNA–Ligand Interactions with Microscale Thermophoresis

Biochemistry ◽  
2018 ◽  
Vol 57 (31) ◽  
pp. 4638-4643 ◽  
Author(s):  
Michelle H. Moon ◽  
Thomas A. Hilimire ◽  
Allix M. Sanders ◽  
John S. Schneekloth
Keyword(s):  
Microscale Thermophoresis ◽  
Ligand Interactions

Investigation of lipase-ligand interactions in porcine pancreatic extracts by microscale thermophoresis

2021 ◽  
Author(s):  
Ghassan Al Hamoui Dit Banni ◽  
Rouba Nasreddine ◽  
Syntia Fayad ◽  
Cyril Colas ◽  
Axel Marchal ◽  
...  
Keyword(s):  
Microscale Thermophoresis ◽  
Ligand Interactions

scholarly journals Exploring IDP–Ligand Interactions: Tau K18 as a Test Case

2020 ◽  
Vol 21 (15) ◽  
pp. 5257 ◽  
Author(s):  
Darius Vagrys ◽  
James Davidson ◽  
Ijen Chen ◽  
Roderick E. Hubbard ◽  
Ben Davis
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Test Case ◽  
Intrinsically Disordered ◽  
Microscale Thermophoresis ◽  
The Past ◽  
Ligand Interactions ◽  
Nmr Diffusion ◽  
Nmr Methods ◽  
The Impact

Over the past decade intrinsically disordered proteins (IDPs) have emerged as a biologically important class of proteins, many of which are of therapeutic relevance. Here, we investigated the interactions between a model IDP system, tau K18, and nine literature compounds that have been reported as having an effect on tau in order to identify a robust IDP–ligand system for the optimization of a range of biophysical methods. We used NMR, surface plasmon resonance (SPR) and microscale thermophoresis (MST) methods to investigate the binding of these compounds to tau K18; only one showed unambiguous interaction with tau K18. Several near neighbors of this compound were synthesized and their interactions with tau K18 characterized using additional NMR methods, including 1D ligand-observed NMR, diffusion-ordered spectroscopy (DOSY) and 19F NMR. This study demonstrates that it is possible to detect and characterize IDP–ligand interactions using biophysical methods. However, care must be taken to account for possible artefacts, particularly the impact of compound solubility and where the protein has to be immobilized.

scholarly journals Abstract P-11: Microscale Thermophoresis of Mycobacterial Cytochrome P450 with Azole Drugs

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S15-S16
Author(s):  
Ivan Kapranov ◽  
S Bukhdruker ◽  
M Karpova ◽  
Yulia Zagryadskaya ◽  
Ivan Okhrimenko ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Unsaturated Fatty Acids ◽  
Dissociation Constants ◽  
Cytochromes P450 ◽  
Microscale Thermophoresis ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Cytochrome P450 Family ◽  
The Hill

Background: Cytochrome P450 family members are found in most organisms where they are involved in the metabolism and synthesis of steroids, bile acids, unsaturated fatty acids, phenolic metabolites as well as exogenic chemicals. Drugs targeting cytochrome P450 have been shown to inhibit the growth of Mycobacterium tuberculosis, the causative agent of one of the deadliest diseases – tuberculosis. Recently, we showed that CYP124, CYP125, and CYP142 can bind and metabolize a panel of human immunoactive oxysterols in vitro (Varaksa et al., 2021) and one of them (CYP124) can metabolize antituberculosis drugs (Bukhdruker et al., 2020). Thus, inhibition of cytochrome P450 is a promising strategy for the development of new anti-tubercular drugs. The existing methods used to assess protein-ligand interactions for cytochromes P450 (spectral titration and Surface Plasmon Resonance) have a number of limitations. In this regard, we used an alternative approach for this purposes – microscale thermophoresis (MST) which was not previously used for proteins of the cytochrome P450 superfamily Methods: Here we show that MST can be used to determine the micromolar-range dissociation constants (Kd) of membrane-associated mycobacterial cytochrome CYP124 with small-molecule azole drugs. CYP124 was fluorescently labeled with Cy3-NHS and MST curves were collected at Monolith NT.115 instrument (blue/green channel, NanoTemper Technologies) in presence of various concentrations of azole compounds: econazole, ketoconazole, itraconazole, and miconazole. The experimental results were approximated by the second-order bimolecular binding equation as well as by the Hill-Langmuir equation. Results: Therefore, MST is a valuable method for the assessment of cytochrome P450 binding to their ligands for cases when traditional approaches are not applicable. The binding regime of CYP124 with azole derivatives was characterized by the structure of the CYP124 complex with carbethoxyhexyl imidazole solved with ~1Å resolution.

New insights into heparan sulphate biosynthesis from the study of mutant mice

2002 ◽  
Vol 69 ◽  
pp. 47-57 ◽  
Author(s):  
Catherine L. R. Merry ◽  
John T. Gallagher
Keyword(s):  
Growth Factors ◽  
Biosynthetic Pathway ◽  
Heparan Sulphate ◽  
Mutant Mice ◽  
Gene Products ◽  
Multiple Gene ◽  
Adhesion Proteins ◽  
Ligand Interactions

Heparan sulphate (HS) is an essential co-receptor for a number of growth factors, morphogens and adhesion proteins. The biosynthetic modifications involved in the generation of a mature HS chain may determine the strength and outcome of HS–ligand interactions. These modifications are catalysed by a complex family of enzymes, some of which occur as multiple gene products. Various mutant mice have now been generated, which lack the function of isolated components of the HS biosynthetic pathway. In this discussion, we outline the key findings of these studies, and use them to put into context our own work concerning the structure of the HS generated by the Hs2st-/- mice.

Activated CD4+ T cells enriched from spleen or colon induce apoptosis in intestinal epithellal cells via fas/fas ligand interactions

2001 ◽  
Vol 120 (5) ◽  
pp. A192-A192
Author(s):  
H TAKAISHI ◽  
T DENNING ◽  
K ITO ◽  
R MIFFLIN ◽  
P ERNST
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Fas Ligand ◽  
Ligand Interactions

Modulation of Platelet Activation by Native DNA – Initial Description of Platelet Receptor Type, Number and Discrimination for Native DNA

1981 ◽  
Vol 45 (03) ◽  
pp. 263-266 ◽  
Author(s):  
B A Fiedel ◽  
M E Frenzke
Keyword(s):  
Platelet Aggregation ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Type Number ◽  
Single Class ◽  
Receptor Ligand ◽  
Platelet Receptor ◽  
Platelet Binding ◽  
Ligand Interactions ◽  
Initial Description

SummaryNative DNA (dsDNA) induces the aggregation of isolated human platelets. Using isotopically labeled dsDNA (125I-dsDNA) and Scatchard analysis, a single class of platelet receptor was detected with a KD = 190 pM and numbering ~275/platelet. This receptor was discriminatory in that heat denatured dsDNA, poly A, poly C, poly C · I and poly C · poly I failed to substantially inhibit either the platelet binding of, or platelet aggregation induced by, dsDNA; by themselves, these polynucleotides were ineffective as platelet agonists. However, poly G, poly I and poly G · I effectively and competitively inhibited platelet binding of the radioligand, independently activated the platelet and when used at a sub-activating concentration decreased the extent of dsDNA stimulated platelet aggregation. These data depict a receptor on human platelets for dsDNA and perhaps certain additional polynucleotides and relate receptor-ligand interactions to a physiologic platelet function.

Solvent-Ligand Interactions in Colloidal Nanocrystals

2018 ◽  
Author(s):  
Jonathan De Roo ◽  
Nuri Yazdani ◽  
Emile Drijvers ◽  
Alessandro Lauria ◽  
Jorick Maes ◽  
...  
Keyword(s):  
Direct Method ◽  
Line Broadening ◽  
H Nmr ◽  
Size Dependent ◽  
Line Shape Analysis ◽  
Wide Range ◽  
Nanocrystal Synthesis ◽  
Ligand Interactions ◽  
Indispensable Tool ◽  
Theoretical Evidence

<p>Although solvent-ligand interactions play a major role in nanocrystal synthesis, dispersion formulation and assembly, there is currently no direct method to study this. Here we examine the broadening of <sup>1</sup>H NMR resonances associated with bound ligands, and turn this poorly understood descriptor into a tool to assess solvent-ligand interactions. We show that the line broadening has both a homogeneous and a heterogeneous component. The former is nanocrystal-size dependent and the latter results from solvent-ligand interactions. Our model is supported by experimental and theoretical evidence that correlates broad NMR lines with poor ligand solvation. This correlation is found across a wide range of solvents, extending from water to hexane, for both hydrophobic and hydrophilic ligand types, and for a multitude of oxide, sulfide and selenide nanocrystals. Our findings thus put forward NMR line shape analysis as an indispensable tool to form, investigate and manipulate nanocolloids.</p>

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