Dataset Information

Equilibrium unfolding of the retinoid X receptor ligand binding domain and characterization of an unfolding intermediate.

ABSTRACT: The retinoid X receptor (RXR) is a ligand-activated transcription factor that plays an important role in growth and development and the maintenance of cellular homeostasis. A thermodynamic ultraviolet circular dichroism, tryptophan fluorescence and ligand binding activity with guanidine as a chemical denaturant are consistent with a two step mechanism. The dimeric LBD equilibrates with a monomeric intermediate (DeltaG(0)(H(2)O) equal to 8.3 kcal/mol) that is in equilibrium with the unfolded state (DeltaG(0)(H(2)O) equal to 2.8 kcal/mol). The intermediate was characterized by analytical ultracentrifugation, spectroscopy, and collisional fluorescence quenching, which imply that the monomeric intermediate maintains a high degree, but not all, of native secondary structure. Although intrinsic fluorescence from native and intermediate suggests little change in tryptophan environments, fluorescence intensities from fluorescein reporter groups differ significantly between the two structures. Analysis of the collisional quenching results imply that the intermediate is characterized by tryptophans with increased accessibility to small solutes and less overall compactness than the native protein.

SUBMITTER: Harder ME

PROVIDER: S-EPMC2805030 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Equilibrium unfolding of the retinoid X receptor ligand binding domain and characterization of an unfolding intermediate.

Harder Mark E ME Malencik Dean A DA Yan Xuguang X Broderick David D Leid Mark M Anderson Sonia R SR Deinzer Max L ML Schimerlik Michael I MI

Biophysical chemistry 20081216 1

The retinoid X receptor (RXR) is a ligand-activated transcription factor that plays an important role in growth and development and the maintenance of cellular homeostasis. A thermodynamic ultraviolet circular dichroism, tryptophan fluorescence and ligand binding activity with guanidine as a chemical denaturant are consistent with a two step mechanism. The dimeric LBD equilibrates with a monomeric intermediate (DeltaG(0)(H(2)O) equal to 8.3 kcal/mol) that is in equilibrium with the unfolded stat ...[more]

PMID: 19167149

Similar Datasets

Project description:Ligand-induced conformational perturbations in androgen receptor (AR) are important in coactivator recruitment and transactivation. However, molecular rearrangements in AR ligand-binding domain (AR-LBD) associated with agonist binding and their kinetic and thermodynamic parameters are poorly understood. We used steady-state second-derivative absorption and emission spectroscopy, pressure and temperature perturbations, and 4,4'-bis-anilinonaphthalene 8-sulfonate (bis-ANS) partitioning to determine the kinetics and thermodynamics of the conformational changes in AR-LBD after dihydrotestosterone (DHT) binding. In presence of DHT, the second-derivative absorption spectrum showed a red shift and a change in peak-to-peak distance. Emission intensity increased upon DHT binding, and center of spectral mass was blue shifted, denoting conformational changes resulting in more hydrophobic environment for tyrosines and tryptophans within a more compact DHT-bound receptor. In pressure perturbation calorimetry, DHT-induced energetic stabilization increased the Gibbs free energy of unfolding to 8.4 +/- 1.3 kcal/mol from 3.5 +/- 1.6 kcal/mol. Bis-ANS partitioning studies revealed that upon DHT binding, AR-LBD underwent biphasic rearrangement with a high activation energy (13.4 kcal/mol). An initial, molten globule-like burst phase (k approximately 30 sec(-1)) with greater solvent accessibility was followed by rearrangement (k approximately 0.01 sec(-1)), leading to a more compact conformation than apo-AR-LBD. Molecular simulations demonstrated unique sensitivity of tyrosine and tryptophan residues during pressure unfolding with rearrangement of residues in the coactivator recruitment surfaces distant from the ligand-binding pocket. In conclusion, DHT binding leads to energetic stabilization of AR-LBD domain and substantial rearrangement of residues distant from the ligand-binding pocket. DHT binding to AR-LBD involves biphasic receptor rearrangement including population of a molten globule-like intermediate state.

Dataset Information

Equilibrium unfolding of the retinoid X receptor ligand binding domain and characterization of an unfolding intermediate.

Publications

Equilibrium unfolding of the retinoid X receptor ligand binding domain and characterization of an unfolding intermediate.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets