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Related Concept Videos

Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...

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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
13:26

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Published on: September 13, 2014

Quantifying Escherichia coli glutaredoxin-3 substrate specificity using ligand-induced stability.

Tobias H Elgán1, Kurt D Berndt

  • 1Department of Biosciences and Nutrition, Karolinska Institutet, S-141 57, Huddinge, Sweden.

The Journal of Biological Chemistry
|September 2, 2008
PubMed
Summary

This study quantifies protein-ligand interactions using ligand-induced stability, revealing key binding contributions for glutaredoxin-3 and glutathione. This method offers insights into substrate specificity in thioredoxin superfamily proteins.

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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Published on: December 5, 2019

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Interactions

Background:

  • Traditional protein-ligand affinity quantification methods (kinetic, equilibrium) are limited for stable covalent complexes.
  • Ligand binding alters protein conformational stability, offering an alternative measurement basis.
  • Glutaredoxin-3 (Grx3) interacts with glutathione (GSH), a biologically relevant substrate.

Purpose of the Study:

  • To develop and apply a novel method for quantifying protein-ligand interactions, particularly for covalent binding.
  • To dissect the specific contributions of different molecular groups in glutathione to its binding with Grx3.
  • To understand substrate specificity within the thioredoxin superfamily.

Main Methods:

  • Utilized ligand-induced stability changes to quantify binding interactions.
  • Employed a series of glutathione analogs to probe individual molecular group contributions.
  • Leveraged the natural catalytic cycle of Grx3, allowing trapping in covalent mixed disulfides.
  • Interpreted conformational stability changes as interaction energies.

Main Results:

  • Ionic interactions at the N- and C-termini of glutathione are primary drivers of Grx3 binding.
  • Van der Waals interactions involving glutathione's gamma-glutamate moiety significantly contribute to specificity.
  • Quantified the additive and independent nature of these specific interactions.

Conclusions:

  • Ligand-induced stability is a versatile method for probing substrate specificity, especially for covalent interactions.
  • Elucidated the key binding determinants for the Grx3-glutathione interaction.
  • Provides a foundation for understanding substrate accommodation in the thioredoxin superfamily.