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Hydrogen/deuterium-exchange (H/D-Ex) of PPAR gamma LBD in the presence of various modulators

Academic Article
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Overview

authors

  • Hamuro, Y.
  • Coales, S. J.
  • Morrow, J. A.
  • Molnar, K. S.
  • Tuske, S. J.
  • Southern, M. R.
  • Griffin, Patrick

publication date

  • August 2006

journal

  • Protein Science  Journal

abstract

  • A nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), is a ligand-dependent transcription factor involved in glucose homeostasis and adipocyte differentiation. PPARgamma is the molecular target of various natural and synthetic molecules, including anti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry, was applied to study the dynamics of the PPARgamma ligand binding domain (LBD) with or without molecules that modulate PPARgamma activity. The H/D-Ex patterns of ligand-free PPARgamma LBD show that the ligand binding pocket of LBD is significantly more dynamic than the rest of the LBD. Presumably, the binding pocket is intrinsically disordered in order to accommodate different ligands. The presence of two full agonists (rosiglitazone and GW1929), a partial agonist (nTZDpa), and a covalent antagonist (GW9662), changed the dynamics/conformation of PPARgamma LBD and slowed the H/D exchange rate in various regions of the protein. The full agonists slowed the H/D exchange more globally and to a greater extent than the partial agonist or the antagonist, indicating that the full agonist stabilizes the PPARgamma LBD more than the partial agonist or the antagonist. One interesting observation is that the two full agonists significantly stabilized helix 12 while the partial agonist and the antagonist did not perturb the H/D exchange of this region. The results showed that the change in protein dynamics induced by ligand binding may be an important factor for the activation of genes and that H/D-Ex is a useful method for analyzing the biological activity of drug leads.

subject areas

  • Amides
  • Amino Acid Sequence
  • Anilides
  • Benzophenones
  • Binding Sites
  • Deuterium Exchange Measurement
  • Indoles
  • Ligands
  • Mass Spectrometry
  • Models, Molecular
  • PPAR gamma
  • Pepsin A
  • Peptide Fragments
  • Protein Conformation
  • Protein Structure, Tertiary
  • Sulfides
  • Thiazolidinediones
  • Tyrosine
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Research

keywords

  • PPAR gamma
  • hydrogen/deuterium exchange
  • mass spectrometry
  • nuclear receptor
  • protein dynamics
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Identity

PubMed Central ID

  • PMC2242592

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1110/ps.062103006

PubMed ID

  • 16823031
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Additional Document Info

start page

  • 1883

end page

  • 1892

volume

  • 15

issue

  • 8

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