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Related Concept Videos

Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
The Equilibrium Binding Constant and Binding Strength02:18

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:

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Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery
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Ligand binding analysis and screening by chemical denaturation shift.

Arne Schön1, Richard K Brown, Burleigh M Hutchins

  • 1Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.

Analytical Biochemistry
|September 3, 2013
PubMed
Summary

This study introduces chemical denaturation as a superior method for identifying drug ligands by accurately measuring binding affinities. This technique offers a wider dynamic range and more reliable results than traditional Tm shift assays.

Keywords:
Chemical denaturationLigand binding analysis

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Area of Science:

  • Biochemistry and Structural Biology
  • Drug Discovery and Development

Background:

  • Identifying small molecule ligands is crucial for developing drugs, particularly for proteins lacking intrinsic activity.
  • Accurate and rapid measurement of ligand binding affinities is essential for efficient drug discovery.
  • Traditional methods like Tm shift assays have limitations in determining precise binding affinities and rank ordering at physiological temperatures.

Purpose of the Study:

  • To present chemical denaturation as a robust method for quantifying ligand binding affinities.
  • To develop analytical equations for interpreting chemical denaturation data.
  • To demonstrate the application of chemical denaturation for ligand identification and characterization.

Main Methods:

  • Utilized chemical denaturation to induce protein unfolding.
  • Measured the shift in denaturation curves caused by ligand binding.
  • Developed and applied analytical equations to calculate binding affinities from denaturation shifts.

Main Results:

  • Chemical denaturation shifts provide accurate binding affinities across a wide dynamic range (high micromolar to sub nanomolar).
  • This method is effective even when ligand binding alters the cooperativity of protein unfolding.
  • Experimental examples demonstrate the utility of chemical denaturation for ligand characterization.

Conclusions:

  • Chemical denaturation offers a versatile and accurate approach for determining ligand binding affinities.
  • It overcomes limitations of Tm shift assays, providing reliable rank ordering at physiological temperatures.
  • This technique is valuable for identifying and characterizing small molecule ligands in drug development.