| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
and ß as revealed by intrinsic tryptophan fluorescence and circular dichroism
1 Departments of Environmental & Occupational Medicine,
2 Medicine,
3 Neuroscience and Cell Biology,
4 Environmental and Occupational Health Sciences Institute,
5 The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA
6 Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA
(Requests for offprints should be addressed to T Thomas, Department of Environmental and Occupational Medicine, 125 Paterson Street, Clinical Academic Building, Room 7092, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA; Email: thomasth{at}umdnj.edu)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
and ERß) are ligand-activated nuclear receptors that mediate the action of estrogens. These receptors activate transcription by similar mechanism(s), although the overall amino acid sequence identity is only 47%. In order to compare the structural and conformational features of ER and ERß, we monitored their intrinsic tryptophan fluorescence during thermal unfolding. The 50% unfolding temperatures (TM) of ER and ERß were 39±1 and 40±2°C, respectively. Estradiol had no significant effect on the TM of ER or ERß. In contrast, binding of the estrogen-response element increased the TM of ER and ERß by 10 °C. Thermal unfolding of estradiol-bound ER and ligand-free ERß showed two-step transitions, with the formation of intermediates that were stable between 36–48 and 34–42°C, respectively. We confirmed the presence of intermediate states during thermal unfolding by circular dichroism spectroscopy. Atomic force microscopy showed that the ERß intermediate consisted of discrete globular particles, whereas the ER intermediate showed a speckled appearance, with sparse well-defined particles. Fluorescence-quenching studies showed the presence of two classes of tryptophan in unliganded ER and ERß. Binding of estradiol to ERß exposed its tryptophans, whereas estradiol reduced the accessibility of the tryptophans of ER. Our results illustrate the differential effects of ligands on the unfolding of ER and ERß, and identify partially unfolded intermediates. Differences in the conformational flexibility and stability of ER and ERß may represent functional differences of ligand-bound ERs in recruiting coactivator proteins and initiating transcription.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
and ERß) are ligand-dependent transcriptional activators belonging to the superfamily of nuclear receptors (Gustafsson 1999, McKenna et al. 2002, Katzenellenbogen & Frasor 2004, Thomas et al. 2004, McDonnell 2005). ERs mediate the function of the steroid hormone, estradiol (E2), in both males and females. ERs and other nuclear receptors share a common modular structure consisting of five domains, named A/B, C, D, E and F, and some key functions have been assigned to each domain (Metzger et al. 1995, Tsai et al. 2004). The N-terminal A/B domain contains the ligand-independent transcription activation function 1 (AF-1; Metzger et al. 1995, Tsai et al. 2004). The C domain has a characteristic zinc-finger structure responsible for the binding of estrogen-response elements (EREs; Danielian et al. 1992, Krieg et al. 2004). The D domain appears to be a hinge region that can modulate the DNA-binding ability of the receptor (Kumar et al. 1986). The E and F domains are involved in the ligand-binding function and exhibit strong ligand-dependent activation function (AF-2; Kumar et al. 1986, Brooks & Skafer 2004). ER and ERß share a modest overall sequence identity (47%), although in the DNA-binding domain (DBD) and ligand-binding domain (LBD) the amino acid identity is 97 and 59%, respectively (Kuiper et al. 1996, Mosselman et al. 1996, Tremblay et al. 1997). In addition, the A/B domain is shorter in ERß than in ER (Kuiper et al. 1996). Despite these differences, ER and ERß have similar functions as E2-induced transcriptional activators, although cellular factors and promoter context determine the degree of transactivation (Couse et al. 1997, Strom et al. 2004, Ramsey et al. 2004).
E2 binds to ER and ERß with similar binding affinities, leading to conformational changes that result in the dissociation of heat-shock proteins and dimerization (Kuiper et al. 1996). ER and ERß may form homo- or heterodimers and bind to the same ERE sequence present in estrogen-responsive genes. However, ER homodimers are more potent transcriptional activators than the heterodimers or ERß homodimers (Matthews & Gustafsson 2003). Thermodynamic and structural studies show that specific contacts between transcriptional activator proteins and DNA are associated with conformational changes in protein, DNA, or both (Pabo and Sauer 1992, Spolar and Record 1994, Greenfield et al. 2001, Margeat et al. 2003). Although the crystal-structure studies of LBD and DBDs of ER and ERß are available (Brzozowski et al. 1997, Pike et al. 1999, Manas et al. 2004) and genetic studies have defined amino acid requirements and function (Wood et al. 2001, Metivier et al. 2002, Nettles et al. 2004), physicochemical studies on the conformation of full-length ER and ERß are limited (Greenfield et al. 2001, Margeat et al. 2003, Bouter et al. 2005).
To gain insight into the role of E2 and ERE in regulating gene expression, we examined the structural and conformational changes involved in E2·ER and ER·ERE recognition. Since ER and ERß are targets for drug development for breast cancer and other diseases (Thomas et al. 2004, Turgeon et al. 2004, McDonnell 2005), detailed information on the structural differences and similarities of these two proteins might help to determine the mechanisms by which these receptors exert their biological action and facilitate drug discovery. In addition, the folding/unfolding profile of ER/ERßis of interest as protein misfolding and functional abnormalities are linked to human diseases (Yon 2002, Castro-Fernandez et al. 2005, Kamagata et al. 2004). Folding/unfolding studies also give insight into the relationship between amino acid sequence, three-dimensional structure, and function. We examined the unfolding of ER and ERß using temperature as the perturbant. We used the intrinsic tryptophan fluorescence and circular dichroism (CD) spectroscopy to detect the thermal stability and unfolding profile of the proteins. We found that the ERE significantly stabilized both ER and ERß. The ligand-free ERß and the ER·E2 complex exhibited the most stable partially unfolded state during thermal unfolding. Quenching studies using acrylamide revealed dissimilar properties of ER and ERß. Atomic force microscopy (AFM) of the partially unfolded state of ERß showed well-defined spherical particles.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Full-length recombinant ER and ERß were purchased from Invitrogen (Carlsbad, CA, USA). The specific ligand-binding activities of ER and ERß were 2800 and 3500 pmol/ml, respectively, as determined by hydroxylapatite assay (Maaroufi & Leclercq 1994). The purity of ER and ERß was tested by SDS/PAGE, followed by Coomassie Brilliant Blue staining. ER and ERß exhibited bands at 66 and 53 kDa, respectively. E2 was purchased from Sigma Chemical Co. (St Louis, MO, USA).
HPLC-/PAGE-purified oligodeoxyribonucleotides (ODNs) were purchased from Oligos, Etc. (Wilsonville, OR, USA). ODNs were dissolved in a buffer containing 10 mM Tris/HCl (pH 7.5) and 50 mM NaCl, and dialyzed three times against the same buffer before use in our experiments. An ODN having a similar percentage of GC content, arranged in a scrambled sequence, was used as a control ODN (Thomas et al. 1997). A mutant ERE with a 2 bp difference and an ODN with an ERE half-site were also used. Base sequence (upper strand) of the ERE, mutant, and control ODNs is listed below, with the palindromic ERE and mutant sequences underlined: ERE, 5'-GATCCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCAG-3'; mutant ERE, 5'-GATCCAGGTCAGAGTGCACTGAGCTAAAATAACACATTCAG-3'; control ODN, 5'-AAAGCTCGCTTCCTGAAGACGTTCTCGAAGAGAAATCTCTT-3'; ERE half-site ODN, 5'-GATCCAGGTCAGAGCATGGTGAGCTAAAATAACACATTCAG-3'.
Fluorescence measurements
The stock solution of ER/ERß was diluted to 50 nM, with a final buffer concentration of 50 mM Tris/HCl (pH 8), 150 mM KCl, 2 mM dithiothreitol (DTT), 1 mM EDTA, and 10% glycerol. The ERE solution used in our experiments was freshly made before each experiment. Complementary single strands were prepared in annealing buffer (10 mM Tris/HCl and 50 mM NaCl, pH 7.5), and equimolar concentrations mixed in the same buffer and heated in a water bath for ~10 min. The solution was allowed to cool to 22 °C and incubated at this temperature for 2 h. E2 and ERE concentrations used in our experiments were 1 µM. Before measuring the fluorescence intensity, the E2/ERE/ER reaction mixture (total volume 300 µl) was incubated for 1 h on ice.
Fluorescence experiments were carried out using a FluoroMax-2 spectrofluorometer. All measurements were done in a 10 mm quartz cuvette. The excitation and emission slits were set at 5 nm. The intrinsic tryptophan fluorescence was measured using an excitation wavelength of 295 nm to avoid tyrosine emission. Emission spectra were recorded from 310–380 nm, using 5 nm band pass for both excitation and emission. The sample temperature was controlled by a Neslab circulating-water bath. For temperature studies of ER, samples were held at each temperature for 6 min, and then the fluorescence emission was recorded. The thermal unfolding of ER and ERß was studied by recording the tryptophan fluorescence emission at 310–380 nm as a function of temperature. All the spectra were corrected for baseline and for the presence of E2 or ERE; however, E2 and ERE did not show any fluorescence at the range of study. The fraction of unfolded ER/ERß at each temperature was calculated using the following the equation (Pace 1986):
 | (1) |
where fu is the amount of unfolded fraction at a particular temperature, Fobs is the fluorescence intensity at the temperature, Fn is the fluorescence intensity of native protein, and Fd is the fluorescence intensity of the denatured (totally unfolded) protein. TM is the temperature at which 50% protein is unfolded, and calculated from a plot of fu against temperature.
CD spectroscopy
The stock solution of ER and ERß was diluted 1:3 for CD measurements. Final buffer concentration for ER and ERß was 50 mM Tris/HCl (pH 8), 150 mM KCl, 2 mM DTT, 0.3 mM EDTA, 0.3 mM sodium orthovanadate, and 10% glycerol. Data were collected on an Aviv model 215 CD spectrometer, fitted with a five-compartment thermal equilibration chamber. Spectra were collected from 260 to 200 nm at 0.5 nm intervals, collecting data for 2 s at each point for temperatures from 20 to 70 °C with 5 °C intervals, except for the intermediate temperature range (30–40 °C) where the spectra were collected at 2 °C intervals. Data were smoothed using the method of Savitsky and Golay (1964) and corrected for the contribution of the cells. Spectra were also corrected for the contribution of the ERE in samples containing ER and the ERE. The CD data were analyzed using the convex constraint algorithm (CCA) analysis program (Perczel et al. 1991) to determine the minimum number of basis spectra needed to reconstruct the data set obtained as a function of temperature.
AFM
The AFM images were obtained using a Nanoscope IIIA equipment (Digital Instruments, Santa Barbara, CA, USA) in tapping mode, operating in ambient air. A 125 µm long rectangular silicon-tip assembly was used with a spring constant of 40 Nm. The images were generated by the change in amplitude of the oscillation of the tip, as it interacted with the sample. The height differences on the surface of the sample are indicated by the color code, as shown in the AFM images. ER/ERß protein concentration of 50 µg/ml was used for the AFM measurements. AFM measurements were done for three different temperatures (22, 37, and 65 °C). Before depositing on the mica surface, samples were kept at the required temperatures for 15 min. Aliquots (5 µl) of these samples were deposited on a freshly cleaved mica surface. Before AFM measurements, the mica surface was dried at room temperature for 5 min, rinsed with two or three drops of nanopure water (Barnstead), and dried under a flow of nitrogen.
Fluorescence quenching with acrylamide
Quenching studies were conducted using acrylamide or KI as the quencher for both ER and ERß in the presence or absence of ligands (E2, ERE, or E2+ERE). The stock ER or ERß solution was diluted to 50 nM using Tris/HCl buffer (pH 7.5) containing 150 mM KCl, 2 mM DTT, 1 mM EDTA, and 10% glycerol. For acrylamide quenching, aliquots of a freshly prepared 5 M acrylamide stock solution were added to achieve the required concentration. The quenching was monitored as a decrease in the intensity of intrinsic tryptophan fluorescence emission with increasing concentration of quenchers. Prior to recording spectra, the sample was mixed gently and incubated for 5 min at 22 °C. The fluorescence spectra presented herein are averages from three scans. The spectra were corrected for background and dilution. For KI quenching experiments, aliquots of a freshly prepared KI stock solution were added to samples containing 50 nM ER or ERß to achieve the indicated KI concentration. KCl, which does not quench fluorescence, was added to maintain a constant salt concentration. Fluorescence-quenching data were fitted to the classical Stern–Volmer equation. Effective Stern–Volmer constants (KSV) were obtained from the fluorescence data according to the Stern–Volmer equation for dynamic quenching (Eftink & Ghiron 1976):
 | (2) |
where F0 and F are the fluorescence intensities in the absence and presence, respectively, of a given concentration of quencher [Q]. KSV is the quenching constant and is obtained from the slope of the linear Stern–Volmer plot. The value for KSV can be considered as a reliable reflection of the bimolecular collisional constant for collisional quenching of the tryptophan since KSV= kq
0, where kq is the bimolecular collisional constant and 0 is the lifetime constant in the absence of the quencher. However, if all tryptophan residues are not equally accessible to the quencher, a modification of the Stern–Volmer plot can be described by the Lehrer equation (Lehrer 1971):
 | (3) |
where (F0 – F) refers to the change in fluorescence intensity on addition of the quencher and fa refers to the fraction of tryptophans accessible to the quencher.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
and ERß monitored by tryptophan fluorescence
Figure 1
shows the tryptophan emission fluorescence spectra of ER and ERß with increasing temperature, from 20 to 90 °C. ER and ERß showed similar emission spectra (Fig. 1A to 1F). The highest intensity was observed at 20 °C. The intensity at 337–340 nm (
max) decreased as the temperature increased, indicating the exposure of tryptophans and quenching of fluorescence emission by the solvent. Significant red shifts (
= ~5 nm) were observed at higher temperatures, during the unfolding of ERß under all conditions and ER in the presence of E2. ER unfolded via an intermediate state in the presence of E2 (Fig. 1B); the temperature-dependent decrease in intensity of emission halted at this stage and then gradually decreased at higher temperatures. Unfolding in the absence of E2 (Fig. 1A) showed a less distinct intermediate. However, an intermediate state of ER was not detectable in the presence of the ERE (Fig. 1C) or E2+ERE (not shown),
|
shows the tryptophan emission fluorescence spectra of ERß in the absence of any ligand. ERß unfolded via a distinct intermediate state. However, unfolding of ERß did not show an intermediate in the presence of E2 (Fig. 1E). There were also no distinct intermediates for ERß in the presence of the ERE (Fig. 1F) or E2+ERE (not shown). These results indicate that specific binding of E2 and ERE converts ERß to a conformational state where an increase in temperature leads to a single-step unfolding and loss of tryptophan fluorescence.
The unfolded fractions of ER and ERß were calculated using eqn 1, and plotted against temperature. A two-step unfolding was observed for ER in the presence of E2 (36–48 °C; Fig. 2B) and ERß in the absence of E2 (34–42 °C; Fig. 2E), indicating that intermediate conformational states are stable over a range including physiological temperature. Although the thermal unfolding profile of ER·E2 (Fig. 2B) showed a two-step process, that of ERß·E2 (Fig. 2F) showed a single-step unfolding. In the presence of the ERE, ER3 unfolding showed a single-phase transition (Fig. 2G), from the native to unfolded state. We also studied the unfolding of ERs in the presence of a mutant ERE (Fig. 2D and H) or a control ODN (results not shown). ERß exhibited unfolding profiles similar to their unliganded state, indicating that mutant ERE/control ODN did not affect the thermal unfolding process. In the case of ER, the intermediate was not detectable in the presence of mutant/control ODN and barely detectable in the presence of the ERE (Fig. 2C and D). These results show that ER conformational intermediate was stabilized by E2 but not by the ERE or mutant ERE, whereas the intermediate state of ERß was lost in the presence of E2 or ERE. Thus, ER and ERß show distinct differences in the conformational flexibility and stability of the native and ligand-bound states.
|
presents the midpoint (50%) of unfolding temperature (TM) values of ER and ERß determined from the change in fluorescence as a function of temperature. Although the unfolding profiles showed differences in the stability of intermediates, overall stabilities of ER and ERß (as represented by TM values) were generally similar. In the absence of ligands, the TM values of ER and ERß were 39 ± 1 and 40 ± 2 °C, respectively. There was no significant change in the TM values of ER and ERß in the presence of E2. In contrast, the presence of the ERE increased the TM by 10 °C. These results suggested that the ERE binding provoked a thermally stable conformation in both ER and ERß. We also observed a similar stabilization of ER and ERß in the presence of E2+ERE. However, the control ODN and mutant ERE did not increase the TM of ER or ERß, demonstrating the specificity of binding of ER and ERß to the ERE.
|
and ERß. However, native EREs include functional half-site EREs, direct repeats, and other variations (Klinge 2001). To see whether a half-site ERE is sufficient to induce conformational changes in ER/ERß, we examined the effect of an ODN with the ERE half-site on the stability of ER and ERß by tryptophan fluorescence measurements. Figure 3 shows the results of these studies. We found that ER was stabilized by ODN containing the ERE half-site, yielding a TM of 49.7 ± 1.5 °C. This result indicates that a dimeric binding site is not required for the stabilization of ER by ERE. In contrast, ERß was not stabilized by the presence of ODN containing half-site ERE (TM=39 ± 1.5 °C), indicating that dimeric binding site is important for the stabilization imparted by ERE.
|
and ERß
We further examined the thermal unfolding of ER and ERß by CD spectroscopy. Figure 4 shows the temperature-dependent CD spectra for ER and ERß from 200 to 260 nm, between 20 and 70 °C. The CD spectra of ligand-free ER and E2-bound ER are shown in Fig. 4A and B, respectively. The CD spectrum of ER showed negative-ellipticity bands at 225 and 208 nm, characteristic of a protein with a high -helical content and some turns. The bands decreased sharply as the temperature increased from 20 to 30 °C and then gradually from 30 to 70 °C. Analysis of the unfolding data using the CCA algorithm showed that at least three component curves were needed to characterize the unfolding (Fig. 5A–D). As the temperature increased, the initial component curves rapidly disappeared and were replaced by a curve that lost about 50% of the total ellipticity but retained a large helical component, as manifested by its negative ellipticity at 222 and 208 nm. In the absence of E2, the intermediate component of ER showed peak levels at 30 °C (Figs. 5A and B). In the presence of E2, the intermediate component showed a broad range of stability between 30 and 45 °C (Fig 5C and D), suggesting a more stable intermediate. At temperatures higher than 50 °C the unfolded protein showed negative single-band ellipticity near 220 nm, indicating a high content of ß-pleated sheet structure, characteristic of aggregation as the protein unfolded further.
|
|
show the spectra of ERß and E2-bound ERß. The spectra of ERß showed only minor changes between 20 and 30 °C, indicating relatively higher stability of ERß at this temperature compared with ER. As in the case of ER, CCA deconvolution analysis of the temperature-dependent changes in the spectra (Fig. 5E–H) showed that at least three curves were needed to reconstruct the data set. The native state was lost above 34 °C, and was replaced by a partially folded structure that retained some helical content. This state is present at temperatures where the intermediate state was observed by tryptophan fluorescence. The intermediate state appeared to be stable between 34 and 42 °C (Fig. 5F). At higher temperatures, the protein began to aggregate. The CD spectrum of the third component is characteristic of an unfolded protein that aggregates to give some ß-pleated sheet. In the presence of E2, the intermediate component began to disintegrate at 36 °C (Fig. 5 G and H), indicating a lower stability of this component, compared with the structure in the absence of E2. AFM
We next conducted AFM experiments to determine the morphology of the intermediate states of ER/ERß. The AFM experiments were done at room temperature (22 °C) and a temperature at which the intermediates were observed (37 °C). Figure 6 shows representative AFM images obtained for ER and ERß. Table 2 presents the dimensions of the particles, measured using Nanoscope IIIA software. Figures 6A and B show AFM images of ER·E2 complexes incubated at 22 and 37 °C, respectively. At 22 °C, the ER·E2 sample showed an amorphous protein structure, with < 10% of particles of 74 ± 34 nm diameter and 2.8 ± 1.7 nm height. After incubation at 37 °C, ER·E2 samples showed molecules with a speckled appearance and particles with 37 ± 21 nm diameter and 1.5 ± 1 nm height. Figure 6C shows AFM image of a sample of ER3 without E2. In this case, images showed mostly amorphous aggregates. These results indicate that ER·E2 complexes formed compact particles, although these particles consisted of only about 20% of the aggregate images observed.
|
|
) showed that the majority of molecules were in an amorphous state, with ~25% particles of 69 ± 28 nm diameter and 2.0 ± 1.4 nm height. Samples of ERß incubated at 37 °C (Fig. 6E), however, showed compact globular nanoparticles and 90% of them had a diameter of 30 ± 7 nm and 1.1 ± 0.5 nm height. In the presence of E2, however, the majority of ERß molecules were present as aggregates (Fig. 6F). These results indicate that compact structures exist during the unfolding of ER and ERß. In the absence of E2, ERß intermediate structures appeared to be more stable than that of ER at 37 °C.
Quenching of tryptophan fluorescence of ER and ERß with acrylamide
Acrylamide is a neutral water-soluble quencher that can permeate into hydrophobic environments of proteins. As a nonionic quencher, it gives information about the exposed hydrophobic surfaces, as it penetrates the hydrophobic interiors. The fluorescence spectra of ER/ERß were recorded with increasing concentrations of acrylamide. The resulting F0/F values were plotted against the concentration of acrylamide. The Stern–Volmer plots of ER and ERß quenching, obtained in the presence and absence of ligands, are given in Fig. 7. Table 3 shows the Stern–Volmer constants (KSV) and fa values of ER and ERß in the presence/absence of ligands. The KSV values were obtained by fitting the linear part of the curve. The slope of the curve gives the KSV values according to eqn 2. The fa values (amount of exposed tryptophans) are obtained from the modified Stern–Volmer plot (not shown) according to eqn 3. Table 3 shows KSV and fa values under different conditions. Unliganded ER and ERß have KSV values of 4.22 ± 0.5 and 5.6 ± 0.15 M–1, respectively, with a slightly higher KSV value for ERß. Fully exposed tryptophan residues have KSV values of 8–9 M–1, whereas KSV values for buried or inaccessible tryptophan residues are lower and can be close to zero (Eftink & Ghiron 1981). The Stern–Volmer plots of unliganded ER and ERß are similar, non-linear with a downward curvature at higher concentrations of acrylamide, suggesting that two classes of tryptophans exist in ER and ERß, one of which is less accessible to the quencher.
|
|
had similar quenching patterns in the ligand-free state and for ER·E2·ERE complex, with KSV values of 4.22 ± 0.5 and 4.52 ± 0.9 M–1, respectively. However, the KSV value was considerably lower in the presence of E2 (2.5 ± 0.3 M–1). The KSV value of the ER·ERE complex was higher than that of native ER. These results indicate that E2 binding reduced the accessibility of tryptophans in ER, whereas ERE binding increased their accessibility. The KSV value of the ER·E2·ERE complex was in between that of the ER·E2 and ER·ERE complexes. The fa values for ER under all the conditions were ~86% or less, except for ER·ERE, for which the value was 1 (100%).
In the presence of E2, KSV values were greater than 9 M–1 for ERß, regardless of the presence of the ERE, and showed an increase compared with the KSV value in the absence of the ligand. In these cases (ERß·E2 and ERß·E2·ERE) Stern–Volmer plots were similar with an upward curvature; that is, towards the y axis, suggesting that the tryptophans were easily accessible to the quencher. The inclination toward the y axis indicates the presence of two types of quenching: static and dynamic (Eftink & Ghiron 1976). Therefore, we plotted apparent quenching constant (Kapp) against concentration of quencher (not shown) to separate the static and dynamic components. Both ERß·E2·ERE and ERß·E2 had positive slopes, indicating the close proximity of the quencher and the tryptophans. The Stern–Volmer plot of ERß·ERE showed a similar quenching pattern to that of native ERß. KSV values were similar in the presence and absence of the ERE. Results of KI quenching (data not presented) of ER and ERß in the presence of E2 and ERE were similar to those of acrylamide quenching, indicating similar patterns for ionic and nonionic quenchers.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
and ERß in the presence and absence of E2 and ERE shows common features and differences in the conformational states of ER and ERß. Unliganded ER and ERß were dissimilar in their thermal unfolding pattern, showing a relatively stable intermediate structure for ERß. However, the overall stability of ER and ERß in the presence of E2 or ERE was similar. Complex formation with the ERE increased the TM of both ER and ERß from ~40 to ~50 °C. A control ODN or mutant ERE with a 2 bp difference was unable to stabilize ER or ERß, indicating the nucleotide specificity of the interaction. Tryptophan fluorescence studies showed that native ERß unfolded with a stable intermediate state at physiological temperature, in the absence of ligands or in the presence of nonspecific DNA sequence. However, native ER was extremely labile and binding of E2 provided some stability to the partially unfolded structure. Binding of the ERE provoked ER and ERß into a conformational state that could unfold by a single thermal transition. Quenching studies showed that the tryptophan environment of ERß underwent changes due to E2 or E2+ERE binding, leaving highly exposed tryptophans in the ligand-bound ERß. In contrast, binding of E2 to ER induced a conformational state with less accessible tryptophans. AFM studies showed that incubation at physiological temperature allowed ERß to form compact, globular structures in abundance, whereas ER structures disintegrated rapidly. These results indicate distinct differences in the structure and conformation of ER and ERß in their ligand-free states, in the partially unfolded intermediates, as well as in E2-induced conformational transitions.
ER and ERß proteins have five and seven tryptophans, respectively, of which three tryptophans are located in the LBD at equivalent positions of ER and ERß, as aligned by amino acid homology (Kuiper et al. 1996, Mosselman et al. 1996). Another tryptophan is located in the LBD of ERß at position 386, and the other three are located in the A/B region (positions 27, 54, and 111). In ER, one tryptophan is located in the DBD (position 200) and one in the hinge region (position 291) between DBD and LBD. Temperature-dependent changes in tryptophan fluorescence appear to indicate the mean of the fluorescence quenching initiated by solvation. The presence of E2 did not stabilize ERs against temperature-induced fluorescence quenching, but the presence of the ERE stabilized ER and ERß. Since these effects are similar in ER and ERß, regardless of the different positions of the tryptophans, temperature-dependent effects can be considered to be due to overall changes in the conformation, rather than changes in individual tryptophans. However, presence and stability of intermediate structures of ER/ERß were different in the presence and absence of E2 and these differences were largely confirmed by independent techniques.
Tryptophan fluorescence studies using an ERE half-site ODN also indicate differences between ER and ERß. ER was stabilized by both the palindromic ERE and the half-site ERE, while ERß was stabilized by the palindromic ERE, but not the half-site ERE (Table 1). Previous studies using electrophoretic mobility-shift assays showed that both ER and ERß are able to bind to half-site ERE as dimers (Lopez et al. 2002, van de Stolpe et al. 2004). Thus differences in stability are unlikely to be due to increased stabilization of homo-dimers in the presence of the palindromic ERE compared with half-site ERE. Therefore, our results suggest higher conformational flexibility of ER in the presence of ERE or ERE-like sequences, compared with ERß.
Acrylamide-quenching studies show that unliganded ER and ERß have similar quenching behavior with KSV values of 4.2 and 5.6 M–1, respectively. The down-ward curvature of the Stern–Volmer plot of acrylamide quenching indicates two classes of tryptophans, with different accessibility to the quencher. The fa values of ER/ERß indicate that about 14–15% of total tryptophans are inaccessible to the quencher. Quenching results show that the tryptophan environment of ER and ERß undergoes contrasting changes due to the binding of E2. E2 binding reduces exposed tryptophans compared with the ligand-free ER, whereas it increases the accessibility of tryptophans in ERß (fa=1.0). These results suggest that a relatively closed conformation of the ligand-binding pocket of ER LBD is stabilized in the presence of E2, but that such a stabilization does not occur for ERß. This idea is also supported by the lack of stable intermediate structures of ERß in the presence of E2.
KSV values were higher (>9 M–1) for E2- or E2+ERE-bound ERß and the Stern–Volmer plot has an upward curvature, indicating that the tryptophans in this case are fully accessible to the quencher. The fa value (1.0) also indicates that all the tryptophans are fully exposed in these cases. However, the quenching data show that tryptophans are not fully accessible in ERß·ERE complex in the absence of E2. Although binding of the ERE stabilizes ERß in terms of TM, tryptophans remain buried in its presence, indicating that ERE binding alone is not sufficient to change the tryptophan environment. The absence of tryptophans in the DBD of ERß may also explain the lack of changes in the tryptophan environment due to ERE in the absence of E2. In contrast, binding of ERE to ER exposes tryptophans fully. Previous studies using protease sensitivity have also demonstrated independent confor-and mational changes induced by E2 or the ERE in ER ERß (Loven et al. 2001, Yi et al. 2002a).
CD studies are consistent with AFM and tryptophan fluorescence studies on the relatively high stability of intermediates formed during the unfolding of the ER·E2 complex and the ligand-free ER3. CD studies also showed the presence of intermediates during unfolding of both ERs under other conditions as well, although the intermediate states were less stable and not well defined. Proteins with > 100 amino acid residues tend to unfold through an intermediate form, known as a molten globule or compact globule, with fluctuating tertiary structure (Ptitsyn 1998, Kamagata et al. 2004). Although the shape of the unfolding curve with an inflection indicates a partially unfolded intermediate state, lack of an inflection does not necessarily exclude the existence of partially unfolded states. In the latter case, our results suggest that the intermediates are less stable. Differences in the stability of intermediate structures and the accessibility of tryptophans of ER·E2 and ERß·E2complexes may represent availability of specific domains for interaction with accessory proteins and the potential for transcriptional activation of estrogen-responsive genes by ER3 and ER3.
A partially unfolded conformational state was not detectable by tryptophan fluorescence during the thermal unfolding of ERß bound to high-affinity ligands such as E2 or ERE. The single-step transition indicates higher thermodynamic stability of the complexes compared with ligand-free protein. Although E2 does not change the TM of ERß, the labile nature of the intermediate state in the presence of E2 shows significant conformational changes in its presence. Conversely, a stable intermediate structure in E2-bound ER represents a distinct conformational change compared with that of ER. AFM revealed that the existence of compact ERß particles after heating the samples to 37 °C, whereas ER and ERß showed undefined shapes and larger particles at 22 °C.
Previous studies of the unfolding of the LBD of ER and ERß (Gee & Katzenellenbogen 2001) using intrinsic tryptophan fluorescence in the presence of guanidine hydrochloride suggested the presence of a folding intermediate. The authors suggested that partial unfolding of the LBD provided an accessible hydrophobic interior, allowing ligand binding, dissociation, or exchange. Open and closed ligand-pocket conformations of ER have also been described by studies of mutant ERs (Carlson et al. 1997). Our studies demonstrate conformational transitions and stabilization of intermediate structures in full-length ER and ERß in the presence and absence of E2. The N-terminal (A/B) regions of ER and ERß are unstructured and this region is 80 amino acids shorter in ERß than in ER (Warnmark et al. 2001b, Kumar & Thompson 2003). Ligand binding and/or DNA binding may facilitate folded structures in the N-terminal region to different extent. The A/B region in turn may make contacts with LBD or DBD in functionally organizing the conformational states of ER/ERß. The differential effects of E2 and ERE on ER/ERß may have contributions from the differences in the A/B region.
Studies on the ligand specificity of ER and ERß indicate that the overall difference in amino acid sequence of these proteins has resulted in LBD structures with unique ligand-binding affinities (Manas et al. 2004, Nettles et al. 2004). However, the functional similarities of ER and ERß do not dictate similar ligand-induced conformational changes in these proteins. Indeed the coactivator recruitment and binding affinities of ER and ERß are different (Hall et al. 2000, Warnmark et al. 2001a, Margeat et al. 2003). In addition, recent studies show that ERß binds to the corepressor proteins in the presence of ER agonistic ligands such as E2 (Webb et al. 2003). This is unusual among nuclear receptors as they generally bind corepressors in either the absence of ligands or the presence of antagonistic ligands (McKenna & O’Malley 2002).
The ratio and the level of ER and ERß vary in estrogen-responsive cells and tissues enriching the texture of their estrogenic responses. ERß is the more expressed receptor in central nervous and cardiovascular systems, and prostate gland, whereas ER plays a dominant role in mammary gland and uterus (Couse et al. 1997, Forster et al. 2004). ER is believed to play a central role in the origin and progression of breast cancer; however, recent studies suggest that ERß can suppress breast cancer progression (Paruthiyil et al. 2004, Strom et al. 2004). Thus ER and ERß appear to have different or even opposite biological actions on some tissues, although the mechanisms involved in these processes are not yet known.
Previous CD studies on the ligand-induced stabilization of ER indicated that the presence of E2 increased the TM of ER by 5 °C (Greenfield et al. 2001). Addition of ERE also increased the TM of unfolding and the presence of E2+ERE showed an additive effect. However, current studies using tryptophan fluorescence did not show a significant change in TM due to the presence of E2. However, the presence of ERE alone or E2+ERE increased the TM by 10 °C, as measured by tryptophan fluorescence. Although temperature-dependent quenching of tryptophan fluorescene as well as the CD signal at 222 nm represent unfolding of the protein, the sensitivity of these techniques to unfolding of ER/ERß might be different. Both techniques, however, detected large changes in the stability of ER/ERß induced by the presence of ERE.
AFM studies are often used to characterize intermediates formed during protein folding, unfolding, and aggregation. AFM imaging of 
D crystallin during folding showed globular structures within the first minute of re-folding, aggregation and fibril formation by 24 min, and thick bundles by 1 h (Kosinski-Collins & King 2003). Spherical particles were also reported in the case of a mutant huntington fragment which was then converted to annular structures and fibrils in a time-dependent manner (Wacker et al. 2004). Our AFM studies provide confirmatory evidence of intermediate species observed by intrinsic tryptophan fluorescene and CD studies. In addition, AFM images shows that the ER intermediate is more prone to aggregate formation, whereas the ERß intermediate exists as well-dispersed stable particles. Formation of compact intermediates, such as the molten globules and pre-molten globules, is an important step in amyloidogenesis and fibril formation (Yon 2002, Uversky & Fink 2004). Although fibrils were not observed with ER or ERß, our studies demonstrate for the first time that full-length ER and ERß can form unfolding intermediates which have different tendencies to form aggregates.
In summary, our studies show unique conformational transitions in full length ER and ERß after their binding to E2 and the ERE. The ERE stabilizes both ER and ERß by forming a specific complex with increased TM (TM ~10 °C) compared with ligand-free ERs; however, control/mutant ODNs are unable to form such a stable complex. Ligand-free ERß and E2-bound ER formed stable intermediate states during temperature-dependent unfolding. AFM studies show the formation of highly compact particles of ERß and a mixture of compact particles as well as less compact speckled structures for ER. Fluorescence-quenching studies indicate that ER and ERß have two classes of tryptophans, one class being more buried than the other. The differences in the unfolding and quenching behavior of ER·E2 and ERß·E2 complexes reveal differences in their conformational states. The ER·E2·ERE and ERß·E2·ERE complexes also have different conformations as seen from their quenching behavior. Differences in the stability and structure of unfolding intermediates and the differential effects on quenching behavior in response to ligand binding might represent the functional differences between ER and ERß in coactivator recruitment and activation of transcription.
|
Acknowledgements |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Brooks SC & Skafar DF 2004 From ligand structure to biological activity: modified estratrienes and their estrogenic and antiestrogenic effects in MCF-7 cells. Steroids 69 401–418.[CrossRef][Web of Science][Medline]
Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA & Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389 753–758.[CrossRef][Medline]
Carlson KE, Choi I, Gee A, Katzenellenbogen BS & Katzenellenbogen JA 1997 Altered ligand binding properties and enhanced stability of a constitutively active estrogen receptor: evidence that an open pocket conformation is required for ligand interaction. Biochemistry 36 14897–14905.[CrossRef][Medline]
Castro-Fernandez C, Maya-Nunez G & Conn PM 2005 Beyond the signal sequence: protein routing in health and disease. Endocrine Reviews 26 479–503.
Couse JF, Lindzey J, Grandien K, Gustafsson J-Å & Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology 138 4613–4621.
Danielian PS, White R, Lees JA & Parker MG 1992 Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO Journal 11 1025–1033.[Web of Science][Medline]
Eftink MR & Ghiron CA 1976 Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry 15 672–680.[CrossRef][Medline]
Eftink MR & Ghiron CA 1981 Fluorescence quenching studies with proteins. Analytical Biochemistry 114 199–227.[CrossRef][Web of Science][Medline]
Forster C, Kietz S, Hultenby K, Warner M & Gustafsson JA 2004 Characterization of the ERbeta–/– mouse heart. PNAS 101 14234–14239.
Gee AC & Katzenellenbogen JA 2001 Probing conformational changes in the estrogen receptor: evidence for a partially unfolded intermediate facilitating ligand binding and release. Molecular Endocrinology 15 421–428.
Greenfield N, Vijayanathan V, Thomas TJ, Gallo MA & Thomas T 2001 Increase in the stability and helical content of estrogen receptor alpha in the presence of the estrogen response element: analysis by circular dichroism spectroscopy. Biochemistry 40 6646–6652.[CrossRef][Medline]
Gustafsson J 1999 Estrogen receptor beta–a new dimension in estrogen mechanism of action. Journal of Endocrinology 163 379–383.[CrossRef][Web of Science][Medline]
Hall JM, Chang CY & McDonnell DP 2000 Development of peptide antagonists that target estrogen receptor beta-coactivator interactions. Molecular Endocrinology 14 2010–2023.
Kamagata K, Arai M & Kuwajima K 2004 Unification of the folding mechanisms of non-two-state and two-state proteins. Journal of Molecular Biology 339 951–965.[CrossRef][Web of Science][Medline]
Katzenellenbogen BS & Frasor J 2004 Therapeutic targeting in the estrogen receptor hormonal pathway. Seminars in Oncology 31 28–38.
Klinge CM 2001 Estrogen receptor interaction with estrogen response elements. Nucleic Acids Research 29 2905–2919.
Kosinski-Collins MS & King J 2003 In vitro unfolding, refolding, and polymerization of human gammaD crystallin, a protein involved in cataract formation. Protein Science 12 480–490.[CrossRef][Web of Science][Medline]
Krieg AJ, Krieg SA, Ahn BS & Shapiro DJ 2004 Interplay between estrogen response element sequence and ligands controls in vivo binding of estrogen receptor to regulated genes. Journal of Biological Chemistry 279 5025–5034.
Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S & Gustafsson J-Å 1996 Cloning of a novel receptor expressed in rat prostate and ovary. PNAS 93 5925–5930.
Kumar R & Thompson EB 2003 Transactivation functions of the N-terminal domains of nuclear hormone receptors: protein folding and coactivator interactions. Molecular Endocrinology 17 1–10.
Kumar V, Green S, Staub A & Chambon P 1986 Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO Journal 5 2231–2236.[Web of Science][Medline]
Lehrer SS 1971 Solute perturbation of protein fluorescence: the quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10 3254–3263.[CrossRef][Medline]
Lopez D, Sanchez MD, Shea-Eaton W & McLean MP 2002 Estrogen activates the high-density lipoprotein receptor gene via binding to estrogen response elements and interaction with sterol regulatory element binding protein-1A. Endocrinology 143 2155–2168.
Loven MA, Likhite VS, Choi I & Nardulli AM 2001 Estrogen response elements alter coactivator recruitment through allosteric modulation of estrogen receptor beta conformation. Journal of Biological Chemistry 276 45282–45288.
Maaroufi Y & Leclercq G 1994 Importance of A/B and C domains of the estrogen receptor for its adsorption to hydroxylapatite. Steroid Biochemistry and Molecular Biology 48 155–163.[CrossRef]
Manas ES, Xu ZB, Unwalla RJ & Somers WS 2004 Understanding the selectivity of genistein for human estrogen receptor-beta using X-ray crystallography and computational methods. Structure (Cambridge) 12 2197–2207.
Margeat E, Bourdoncle A, Margueron R, Poujol N, Cavaillès V & Royer C 2003 Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta. Journal of Molecular Biology 326 77–92.[CrossRef][Web of Science][Medline]
Matthews J & Gustafsson JA 2003 Estrogen signaling: a subtle balance between ER alpha and ER beta. Molecular Intervention 3 281–292.
McDonnell DP (2005) The molecular pharmacology of estrogen receptor modulators: implications for the treatment of breast cancer. Clinical Cancer Research 11 871s–877s.
McKenna NJ & O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108 465–474.[CrossRef][Web of Science][Medline]
Metivier R, Stark A, Flouriot G, Hubner MR, Brand H, Penot G, Manu D, Denger S, Reid G, Kos M et al. 2002 A dynamic structural model for estrogen receptor-alpha activation by ligands, emphasizing the role of interactions between distant A and E domains. Molecular Cell 10 1019–1032.[CrossRef][Web of Science][Medline]
Metzger D, Ali S, Bornert JM & Chambon P 1995 Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cells. Journal of Biological Chemistry 270 9535–9542.
Mosselman S, Polman J & Dijkema R 1996 ER beta: identification and characterization of a novel human estrogen receptor. FEBS Letters 392 49–53.[CrossRef][Web of Science][Medline]
Nettles KW, Sun J, Radek JT, Sheng S, Rodriguez AL, Katzenellenbogen JA, Katzenellenbogen BS & Greene GL 2004 Allosteric control of ligand selectivity between estrogen receptors alpha and beta: implications for other nuclear receptors. Molecular Cell 13 317–327.[CrossRef][Web of Science][Medline]
Pabo CO & Sauer RT 1992 Transcription factors: structural families and principles of DNA recognition. Annual Review of Biochemistry 61 1053–1095.[CrossRef][Web of Science][Medline]
Pace CN 1986 Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods in Enzymology 131 266–280.[Medline]
Paruthiyil S, Parmar H, Kerekatte V, Cunha GR, Firestone GL & Leitman DC 2004 Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest. Cancer Research 64 423–428.
Perczel A, Hollosi M, Tusnady G & Fasman GD 1991 Convex constraint analysis: a natural deconvolution of circular dichroism curves of proteins. Protein Engineering 4 669–679.
Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O, Ljunggren J, Gustafsson JA & Carlquist M 1999 Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO Journal 18 4608–4618.[CrossRef][Web of Science][Medline]
Ptitsyn OB 1998 Protein folding: nucleation and compact intermediates. Biochemistry (Moscow) 63 367–373.[Medline]
Ramsey TL, Risinger KE, Jernigan SC, Mattingly KA & Klinge CM 2004 Estrogen receptor beta isoforms exhibit differences in ligand-activated transcriptional activity in an estrogen response element sequence-dependent manner. Endocrinology 145 149–160.
Savitsky A & Golay MJE 1964 Smoothing and differentiation of data by simplified least square procedures. Analytical Chemistry 36 1627–1639.[CrossRef]
Spolar RS & Record MT 1994 Coupling of local folding to site-specific binding of proteins to DNA. Science 263 777–784.
Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J & Gustafsson, JA 2004 Estrogen receptor beta inhibits 17 beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. PNAS 101 1566–1571.
Thomas T, Kulkarni GD, Gallo MA, Greenfield N, Lewis JS, Shirahata A & Thomas TJ 1997 Effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide with the estrogen response element. Nucleic Acids Research 25 2396–2402.
Thomas T, Gallo MA & Thomas TJ 2004 Estrogen receptors as targets for drug development for breast cancer, osteoporosis and cardiovascular diseases Current Cancer Drug Targets 4 483–499.[CrossRef][Web of Science][Medline]
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F & Giguere V 1997 Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Molecular Endocrinology 11 353–365.
Tsai HW, Katzenellenbogen JA, Katzenellenbogen BS & Shupnik MA 2004 Protein kinase A activation of estrogen receptor alpha transcription does not require proteasome activity and protects the receptor from ligand-mediated degradation. Endocrinology 145 2730–2738.
Turgeon JL, McDonnell DP, Martin KA & Wise PM 2004 Hormone therapy: physiological complexity belies therapeutic simplicity. Science 304 1269–1273.
Uversky VN & Fink AL 2004 Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochimica et Biophysica Acta 1698 131–153.[Medline]
van de Stolpe A, Slycke AJ, Reinders MO, Zomer AW, Goodenough S, Behl C, Seasholtz AF & van der Saag PT 2004 Estrogen receptor (ER)-mediated transcriptional regulation of the human corticotropin-releasing hormone-binding protein promoter: differential effects of ERalpha and ERbeta. Molecular Endocrinology 18 2908–2923.
Wacker JL, Zareie MH, Fong H, Sarikaya M & Muchowski PJ 2004 Hsp70 and Hsp40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer. Nature Structural & Molecular Biology 11 1215–1222.
Warnmark A, Almlof T, Leers J, Gustafsson JA & Treuter E 2001a Differential recruitment of the mammalian mediator subunit TRAP220 by estrogen receptors ERalpha and ERbeta. Journal of Biological Chemistry 276 23397–23404.
Warnmark A, Wikstrom A, Wright AP, Gustafsson JA & Hard T 2001b The N-terminal regions of estrogen receptor alpha and beta are unstructured in vitro and show different TBP binding properties. Journal of Biological Chemistry 276 45939–45944.
Webb P, Valentine C, Nguyen P, Price Jr RH, Marimuthu A, West BL, Baxter JD & Kushner PJ. 2003 ERbeta Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus. Nuclear Receptor 1 1336–1378.
Wood JR, Likhite VS, Loven MA & Nardulli AM 2001 Allosteric modulation of estrogen receptor conformation by different estrogen response elements. Molecular Endocrinology 15 1114–1126.
Yi P, Driscoll MD, Huang J, Bhagat S, Hilf R, Bambara RA & Muyan M 2002 The effects of estrogen-responsive element- and ligand-induced structural changes on the recruitment of cofactors and transcriptional responses by ER alpha and ER beta. Molecular Endocrinology 16 674–693.
Yon JM 2002 Protein folding in the post-genomic era. Journal of Cellular and Molecular Medicine 6 307–327.[CrossRef][Web of Science]
Received in final form 16 July 2005
Accepted 27 July 2005
This article has been cited by other articles:
|
|
 |
|
  V. Vijayanathan, S. Venkiteswaran, S. K. Nair, A. Verma, T.J. Thomas, B. T. Zhu, and T. Thomas Physiologic levels of 2-methoxyestradiol interfere with nongenomic signaling of 17beta-estradiol in human breast cancer cells. Clin. Cancer Res., April 1, 2006; 12(7 Pt 1): 2038 - 2048. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), intermediate (I,
), and unfolded (U, •) species. Spectral features are given on the left-hand panels, and the relative amounts of native, intermediate, and unfolded forms are presented as a function of temperature on the right-hand panels.
) and ERß (•) for quenching with acrylamide. (A) Unliganded ER