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Related Concept Videos

Cooperative Allosteric Transitions01:58

Cooperative Allosteric Transitions

Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
Cooperative Allosteric Transitions01:58

Cooperative Allosteric Transitions

Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
Cooperative Allosteric Transitions01:58

Cooperative Allosteric Transitions

Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
Allosteric Regulation01:08

Allosteric Regulation

Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
Allosteric Regulation01:08

Allosteric Regulation

Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
ATP Energy Storage and Release01:31

ATP Energy Storage and Release

ATP is a highly unstable molecule. Unless quickly used to perform work, ATP spontaneously dissociates into ADP and inorganic phosphate (Pi), and the free energy released during this process is lost as heat. The energy released by ATP hydrolysis is used to perform work inside the cell and depends on a strategy called energy coupling. Cells couple the exergonic reaction of ATP hydrolysis with endergonic reactions, allowing them to proceed.
One example of energy coupling using ATP involves a...

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Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
13:57

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Published on: February 18, 2014

Binding techniques to study the allosteric energy cycle.

James K Kranz1, José C Clemente

  • 1Biopharmaceuticals Research & Development, GlaxoSmithKline, Upper Merion, PA, USA. james.k.kranz@gsk.com

Methods in Molecular Biology (Clifton, N.J.)
|November 5, 2011
PubMed
Summary
This summary is machine-generated.

This study quantifies ligand binding energies and conformational dynamics using circular dichroism (CD) spectroscopy and thermal shift assays. The method determines pairwise coupling free energy to understand allosteric regulation in proteins like dihydrofolate reductase (DHFR) and glutamate dehydrogenase (GDH).

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Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
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Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins
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Area of Science:

  • Biophysics
  • Biochemistry
  • Structural Biology

Background:

  • Allostery and cooperativity are key to understanding ligand binding.
  • Quantifying ligand binding energies and conformational dynamics is crucial for studying allosteric effects.
  • Circular dichroism (CD) spectroscopy offers insights into protein secondary and tertiary structures.

Purpose of the Study:

  • To present a method combining CD spectroscopy with thermal shift assays for quantifying ligand binding constants.
  • To determine pairwise coupling free energy to characterize allosteric interactions.
  • To investigate allosteric regulation in model proteins like DHFR and GDH.

Main Methods:

  • Utilizing CD spectroscopy integrated with thermal shift assays to measure protein denaturation.
  • Quantifying ligand binding constants based on their stabilizing effect on protein thermal stability.
  • Calculating pairwise coupling free energy from binding constants of two ligands.

Main Results:

  • The developed method allows for the quantification of ligand binding energies and allosteric coupling.
  • Demonstrated application on dihydrofolate reductase (DHFR) showing positive cooperativity for NADP and methotrexate binding.
  • Observed negative cooperativity in mammalian glutamate dehydrogenase (GDH) with NAD/NADPH, ADP activation, and GTP inhibition.

Conclusions:

  • CD-based thermal shift assays provide a robust approach to study protein-ligand interactions and allosteric mechanisms.
  • The method is effective for characterizing cooperativity and coupling free energies in biological systems.
  • This technique offers valuable insights into the functional regulation of enzymes like DHFR and GDH.