A review on the ligand binding studies by isothermal titration calorimetry

2006 ◽  
Vol 3 (1) ◽  
pp. 1-21 ◽  
Author(s):  
A. A. Saboury
Keyword(s):  
Ligand Binding ◽  
Isothermal Titration Calorimetry ◽  
Titration Calorimetry ◽  
Binding Studies

scholarly journals Interbase-FRET binding assay for pre-microRNAs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mattias Bood ◽  
Anna Wypijewska del Nogal ◽  
Jesper R. Nilsson ◽  
Fredrik Edfeldt ◽  
Anders Dahlén ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Drug Targets ◽  
Structural Changes ◽  
Small Sample Size ◽  
Resonance Energy ◽  
Small Sample ◽  
Titration Calorimetry ◽  
Drug Candidate ◽  
Binding Studies ◽  
Aberrant Expression

AbstractThe aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA–target binding studies.

scholarly journals Phosphopantetheine Adenylyltransferase from Escherichia coli: Investigation of the Kinetic Mechanism and Role in Regulation of Coenzyme A Biosynthesis

2007 ◽  
Vol 189 (22) ◽  
pp. 8196-8205 ◽  
Author(s):  
J. Richard Miller ◽  
Jeffrey Ohren ◽  
Ronald W. Sarver ◽  
W. Thomas Mueller ◽  
Piet de Dreu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Coenzyme A ◽  
Forward Direction ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Titration Calorimetry ◽  
Antibacterial Drug ◽  
Binding Modes ◽  
Binding Studies

ABSTRACT Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.

scholarly journals Resolving Three-State Ligand-Binding Mechanisms by Isothermal Titration Calorimetry: A Simulation Study

2017 ◽  
Author(s):  
Evgenii L. Kovrigin
Keyword(s):  
Ligand Binding ◽  
Isothermal Titration Calorimetry ◽  
Simulation Study ◽  
Titration Calorimetry ◽  
Induced Fit ◽  
State Process ◽  
Binding Mechanisms

ABSTRACTIn this paper, I theoretically analyzed ITC profiles for three-state equilibria involving ligand binding coupled to isomerization or dimerization transitions. Simulations demonstrate that the mechanisms where the free or ligand-bound protein undergoes dimerization (such that the ligand cannot bind to or dissociate from the dimer) produce very distinctive titration profiles. In contrast, profiles of the pre-existing equilibrium or induced-fit models cannot be distinguished from a simple two-state process, requiring data from additional techniques to positively identify these mechanisms.

Thermodynamic studies of ligand binding to the human homopentameric glycine receptor using isothermal titration calorimetry

2012 ◽  
Vol 30 (2) ◽  
pp. 169-183 ◽  
Author(s):  
Annemarie Beate Wöhri ◽  
Per Hillertz ◽  
Per-Olof Eriksson ◽  
Johan Meuller ◽  
Niek Dekker ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Isothermal Titration Calorimetry ◽  
Glycine Receptor ◽  
Titration Calorimetry ◽  
Thermodynamic Studies
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