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The two-state receptor model explains a drug's interaction with receptors, such as G protein-coupled receptors and ligand-gated ion channels, to induce or inhibit a biological response. When no natural ligands are present, a receptor exists in an equilibrium of inactive (Ri) and active (Ra) conformations. The inactive form does not produce a response, while the active form generates a basal effect known as constitutive activity.
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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...
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Related Experiment Video

Updated: Jun 21, 2025

Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery
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Bivalent affinity binding-inspired PPARγ immobilization with selective conformation and improved ligand-binding

Zilong Zhang1, Jiahuan Chen1, Lixiang Chen1

  • 1Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, Shaanxi 710069, China.

Journal of Chromatography. A
|July 10, 2024
PubMed
Summary

A novel bivalent affinity method immobilizes Peroxisome proliferator-activated receptor gamma (PPARγ) using DNA aptamers and Ni2+-NTA chelation. This technique enables selective bio-detection and characterization of PPARγ ligands.

Keywords:
Affinity chromatographyBivalent affinity bindingLigand-binding activityPPARγ immobilizationSelective conformation

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Protein immobilization is crucial for bio-detection but faces challenges in protein stability and device regeneration.
  • Existing methods struggle to prevent protein movement and allow for convenient regeneration.

Purpose of the Study:

  • To develop a robust and regenerable method for immobilizing Peroxisome proliferator-activated receptor gamma (PPARγ).
  • To create a bio-detection system capable of discriminating between PPARγ agonists and antagonists.

Main Methods:

  • A bivalent affinity binding strategy was employed, utilizing a DNA aptamer (Apt 2) selected via SELEX and nickel-nitrilotriacetic acid (Ni2+-NTA) chelation.
  • Both Apt 2 and Ni2+-NTA were immobilized on macroporous silica gels using L-α-allylglycine as a linker.
  • The immobilized system interacted with PPARγ and its 6×His tag for receptor immobilization.

Main Results:

  • Successful immobilization of PPARγ was confirmed through surface characterization.
  • Chromatographic studies demonstrated that immobilized PPARγ exhibited conformational selectivity, distinguishing between agonists and antagonists.
  • Accurate determination of ligand-binding parameters (affinity and rate constants) for four PPARγ agonists was achieved, showing high consistency with solution-based measurements.

Conclusions:

  • The developed bivalent affinity method provides a reliable platform for PPARγ bio-detection and ligand screening.
  • This approach offers conformational selectivity and enhanced ligand-binding activity for immobilized receptors.
  • The method's general applicability suggests potential for immobilizing other nuclear receptors, advancing bio-device development.