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The Equilibrium Binding Constant and Binding Strength02:18

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Different monodentate and polydentate ligands are used as complexing agents in complexometric titration reactions. The formation of complexes by mono- and bidentate ligands involves two or more intermediate steps, limiting their use as complexing agents. In comparison, polydentate ligands can form complexes with metal ions in a single-step process, facilitating sharper end points. This means polydentate ligands, such as amino carboxylic acid derivatives, are most commonly employed in...
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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
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Determination of Nutrient Ligand-Sensor Binding Affinity.

Xin Gu1,2,3

  • 1Department of Neurobiology, Harvard Medical School, Boston, MA, USA. xin_gu@hms.harvard.edu.

Methods in Molecular Biology (Clifton, N.J.)
|February 24, 2025
PubMed
Summary
This summary is machine-generated.

A new protocol quantifies nutrient sensor binding affinity, crucial for understanding cellular growth regulation. This method, using S-adenosylmethionine and SAMTOR, determines if nutrient sensing mechanisms are physiologically relevant.

Keywords:
Affinity beadsCompetitive binding assaysNutrient sensorsNutrientsProtein purificationRadioactive ligandsScintillationmTORC1

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Area of Science:

  • Cellular Biology
  • Biochemistry
  • Metabolism

Background:

  • Cells possess mechanisms to sense environmental nutrients for growth regulation.
  • The mechanistic Target of Rapamycin Complex 1 (mTORC1) integrates growth factors and nutrients like amino acids.
  • Nutrient sensors bind ligands to signal nutrient availability and regulate mTORC1 signaling.

Purpose of the Study:

  • To develop a reliable method for quantitatively determining nutrient sensor-ligand binding affinity (Kd).
  • To assess the physiological relevance of nutrient sensing mechanisms by comparing binding affinity to physiological metabolite ranges.
  • To provide a generalizable protocol for reproducible determination of nutrient ligand-nutrient sensor binding affinity.

Main Methods:

  • Purification of the nutrient sensor (SAMTOR).
  • Incubation with a radioactive nutrient ligand (S-adenosylmethionine).
  • Scintillation counting and mathematical calculation of binding affinity (Kd).

Main Results:

  • The protocol allows for reproducible determination of nutrient sensor binding affinity.
  • Quantitative metabolomic analysis provides the physiological range of metabolites.
  • Comparison of binding affinity to physiological ranges indicates the relevance of the sensing mechanism.

Conclusions:

  • The described protocol enables quantitative assessment of nutrient sensor-ligand binding affinity.
  • This method is essential for understanding how cells regulate growth in response to nutrient availability.
  • The S-adenosylmethionine-SAMTOR pair serves as a model for this generalizable technique.