Increase in the stability and helical content of estrogen receptor alpha in the presence of the estrogen response element: analysis by circular dichroism spectroscopy.
Ligand-dependent stabilization of the estrogen receptor (ER) is often postulated, with limited support from experimental data. We studied the thermal unfolding of recombinant ERalpha by circular dichroism (CD) spectroscopy. The T(M) of unfolding of ERalpha was 38 +/- 2.4 degrees C, and the van't Hoff enthalpy of unfolding was 31.7 +/- 3.4 kcal/mol in the absence of ligands. Addition of estradiol (E(2)) increased the T(M) to 43.6 +/- 2.3 degrees C, while addition of E(2) and an oligonucleotide harboring the estrogen response element (ERE) increased the T(M) to 47.9 +/- 1.6 degrees C. Addition of the antiestrogen 4-hydroxytamoxifen (HT) alone did not increase the T(M); however, a combination of HT and the ERE increased the T(M) to 48.9 +/- 1.0 degrees C. The ERE alone increased the T(M) to 46.1 +/- 0.9 degrees C. Addition of E(2) alone had no effect on the apparent enthalpy of unfolding; however, the ERE alone increased the apparent enthalpy from 31.7 to 36.1 kcal/mol. ERalpha samples containing the ERE also exhibited an increase in the negative ellipticity at 208 and 222 nm, relative to that of ligand-free ERalpha, suggesting a stabilization of the alpha-helix. CD data analysis further showed that the presence of the ERE caused a large increase in alpha-helical content of ERalpha in both the presence and absence of the ligands. This increase in alpha-helical content of ERalpha was not observed in the presence of a nonspecific oligonucleotide. These results show that the ERE can increase the thermal stability of ERalpha, enhance its alpha-helical content, and facilitate the cooperativity of the folding transition.
Greenfield
N
N
Departments of Neuroscience and Cell Biology, Medicine, and Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Vijayanathan
V
V
Thomas
T J
TJ
Gallo
M A
MA
Thomas
T
T
D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000737:chemistry; D020847:Estrogen Receptor Modulators / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011960:Receptors, Estrogen / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020218:Response Elements / Q000235:genetics*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D020847:Estrogen Receptor Modulators; D047628:Estrogen Receptor alpha; D004967:Estrogens; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011960:Receptors, Estrogen; D012097:Repressor Proteins; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol