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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
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.
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.
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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...
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...
02:18The 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: