Equilibrium unfolding of the retinoid X receptor ligand binding domain and characterization of an unfolding intermediate.
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.
Harder
Mark E
ME
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, 97331-7305, United States. harderm@onid.orst.edu
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
D020106:Acrylamide / Q000494:pharmacology; D000077556:Alitretinoin; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D019793:Fluorescein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009566:Nitrates / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D047488:Retinoid X Receptors / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014212:Tretinoin / Q000378:metabolism; D014364:Tryptophan; D014461:Ultracentrifugation
D005456:Fluorescent Dyes; D008024:Ligands; D009566:Nitrates; D047488:Retinoid X Receptors; D000077556:Alitretinoin; D020106:Acrylamide; D014212:Tretinoin; D014364:Tryptophan; C031618:sodium nitrate; D019793:Fluorescein