Probing Conformational Changes in the Estrogen Receptor: Evidence for a Partially Unfolded Intermediate Facilitating Ligand Binding and Release

Arvin C. Gee and John A. Katzenellenbogen

Department of Chemistry University of Illinois Urbana, Illinois 61801


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Because the ligand bound to the ligand-binding domain (LBD) of nuclear hormone receptors is completely enveloped by protein, it is thought that the process of ligand binding or unbinding must involve a significant conformational change of this domain. We have used the intrinsic tryptophan fluorescence of the estrogen receptor-{alpha} (ER{alpha}) or estrogen receptor-ß (ERß) LBD, as well as bis-anilinonaphthalenesulfonate (bis-ANS), a probe for accessible interior regions of protein, to follow the guanidine-hydrochloride (Gua-HCl)-induced unfolding of this domain. In both cases, we find that the ER-LBD unfolding follows a two-phase process. At low Gua-HCl, the ER-LBD undergoes partial unfolding, whereas at high Gua-HCl, this domain undergoes a global unfolding, with bis-ANS binding preferentially to the partially unfolded state. The partially unfolded state of the ER{alpha}-LBD induced by denaturant does not bind ligand stably, but it may resemble an intermediate that this domain accesses transiently under native conditions that allow ligands to enter or exit the ligand-binding pocket.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear hormone receptors are ligand-regulated transcription factors that have a distinct domain structure. Two conserved domains, a short domain C involved in DNA binding and a large domain E responsible for ligand binding and hormone-dependent transcription activation (activation function 2, AF-2), are linked by a variable length hinge domain D. They are flanked at the N terminus by a poorly conserved A/B domain that is also involved in transcription activation (activation function 1, AF-1) and at the C terminus by a short F domain (1). The means by which the activity of these receptors is regulated by ligand is thought to involve a conformational stabilization of a portion of the ligand-binding domain (LBD) that occurs upon binding of the ligand. Aspects of this stabilization have been revealed in a number of recent crystallographic structures of nuclear hormone receptor LBDs.

X-ray structures of nuclear hormone receptor LBD complexes with various ligands show that the ligand is fully enveloped by several elements of secondary structure in the lower portion of this domain (2, 3, 4, 5). Notable as well is the fact that ligands with agonist vs. antagonist activity stabilize different conformations, particularly the orientation of the C-terminal helix 12. These alternate helix 12 positions regulate the shape and accessibility of a hydrophobic groove formed by elements of helices 3, 4, 5, and 12, which forms a docking site for the LXXLL sequence motif of the nuclear receptor interaction box (NR box) found in various coregulator proteins (6, 7, 8, 9).

What is not evident from these LBD-ligand structures is the process by which the ligand is able to enter and exit the ligand-binding pocket. The fact that the ligand is completely enveloped by the protein indicates that for ligand to exit this pocket there must be a significant conformational reorganization of a substantial region of the lower portion of the LBD. This is consistent with the very slow rate of ligand dissociation from these complexes, which can be many hours at 0–4 C and several minutes at 25 C (10, 11).

Conformational reorganization is also thought to be required for ligand to enter the LBD. For example, in a crystal structure of an LBD without a ligand (an apo-LBD), that of the retinoid X receptor-{alpha} (RXR{alpha}), the ligand-binding pocket is shown to be "collapsed" when compared with that typical for LBD-ligand complexes (12). The conformational change required to open up this pocket to allow a ligand to enter is again consistent with the very slow rate of ligand association to nuclear hormone receptor LBDs. These rates are typically several orders of magnitude slower (ca. 104 M-1sec-1) (10) than typical enzyme-inhibitor association rates, which are generally close to diffusion limited (108 M-1sec-1) (13, 14).

In this report we describe experiments in which we have examined the conformation of the LBD of the estrogen receptor {alpha} (ER{alpha}) during progressive chemically induced denaturation with guanidine hydrochloride (Gua-HCl) using fluorescence methods. Whether we follow the Gua-HCl-induced unfolding titration of this domain using intrinsic tryptophan fluorescence or bis-anilinonaphthalenesulfonate (bis-ANS), a probe for accessible interior regions of proteins, we find evidence that the ER{alpha}-LBD undergoes a two-phase unfolding process. The first phase (at low Gua-HCl) involves partial unfolding of the LBD, which opens the ligand-binding pocket, providing access to water, which quenches the tryptophan fluorescence, and allowing bis-ANS to bind. The second phase (at high Gua-HCl) corresponds to the global unfolding of the whole domain, at which point the tryptophans become fully exposed to water and experience a red shift in their fluorescence. The denaturant-induced state of the ER{alpha}-LBD that is partially unfolded may resemble a conformation that this domain accesses transiently under native conditions, allowing ligands to enter or exit the ligand-binding pocket.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tryptophan Fluorescence Monitors Protein Conformation during Gua-HCl-Induced Denaturation of the ER-LBD
Fluorescence can often be used to follow changes in protein conformation during the process of chemical denaturation, particularly when the protein contains tryptophan residues that are buried in the native structure but become exposed to water during the denaturant-induced unfolding. In such a case, two changes in tryptophan fluorescence typically occur upon protein unfolding. With progressive exposure of tryptophan residues to water, which acts as a moderately effective quencher, the emission intensity (Imax) typically decreases, although the extent of intensity change can vary considerably, depending on the local environment of the tryptophan residues in the native structure. In addition, as the tryptophan residues are shifted from the protein interior of low dielectric to an aqueous environment of high dielectric, there is a red shift in the fluorescence emission maximum ({lambda}em,max), typically from 335 nm to 356 nm (15). In general, the changes in tryptophan emission intensity and emission wavelength occur synchronously during the denaturation process (16, 17, 18, 19, 20).

The LBDs of the ERs contain three tryptophan residues (W365, 388, and 398 in ER{alpha} and W312, 335, and 345 in ERß) that are at congruent positions on helices 3 and 5 of the two ER subtypes. The location of these three tryptophan residues within the tertiary structure of the ER{alpha} LBD is illustrated in the ribbon diagram shown in Fig. 1Go. As is evident from this figure, all three of these tryptophans are in the interior of the LBD; they are also close to one another (1.31–2.00 nm), and they are positioned above the ligand-binding pocket, relatively close to the position where the ligand is bound (0.85–1.73 nm) (2). We have found that the intrinsic fluorescence of the three tryptophan residues in the ER{alpha}-LBD provides very useful information on the state of quencher access and solvation of the internal environment of the ER-LBD during the process of chemically induced denaturation.



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Figure 1. Ribbon diagram of the ER{alpha} LBD complexed with E2

The skeletal structures indicate the location of the ligand (E2) and the three tryptophan residues (W365, W388, and W398). The figure is presented in crossed stereo.

 
Intrinsic Tryptophan Fluorescence Indicates That the Denaturation of ER-LBD Is a Two-Phase Process
To follow the denaturant-induced unfolding of ER-LBD, the tryptophan fluorescence intensity (Imax) and wavelength of maximum emission ({lambda}em,max) were monitored after the protein was equilibrated with increasing concentrations of Gua-HCl. Figures 2Go and 3Go illustrate that the ER{alpha}-LBD undergoes a distinct two-phase denaturation profile in which the decrease in tryptophan emission intensity precedes the shift in wavelength of emission.



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Figure 2. Intensity and Wavelength of Maximum Emission of Intrinsic Protein Fluorescence of Unliganded or E2-Liganded ER{alpha} LBD (ER{alpha}) during Titration with Gua-HCl

Panel A, ER{alpha} without ligand (apo-ER{alpha}). Panel B, ER{alpha} liganded with E2. ER{alpha} samples (1.5 µM) were preincubated either with 4.5 µM E2 (panel B) or with buffer control (panel A) for 1 h at 4 C. A small portion was then diluted into the indicated concentration of Gua-HCl to a final concentration of 30 nM ER{alpha} and 90 nM ligand and further incubated for 1 h at 4 C. Fluorescence spectra were obtained by excitation at 285 nm, and emission was scanned from 300–420 nm. At each concentration of Gua-HCl, the relative maximum intensity (I, open circles) and the emission maximum ({lambda}max, solid squares) are plotted. For details, see text and Materials and Methods.

 


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Figure 3. Intensity of Intrinsic Protein Fluorescence of ER{alpha} LBD (ER{alpha}) without Ligand or Complexed with E2 or THC during Titration with Gua-HCl

ER{alpha} without ligand (apo-ER{alpha}, solid triangles), ER{alpha} liganded with estradiol (solid squares) or ER{alpha} liganded with THC (x) were preincubated with ligand and denatured with Gua-HCl as described in the legend to Fig. 2Go. Fluorescence spectra were obtained as described in the legend for Fig. 2Go. For details, see text and Materials and Methods.

 
When ER{alpha}-LBD is not liganded (Fig. 2AGo), the most pronounced decrease in the tryptophan Imax with little change in {lambda}em,max occurs at approximately 0.5–1 M Gua-HCl, whereas no red shift in {lambda}em,max occurs until approximately 4.5 M Gua-HCl, at which point there is little additional change in Imax. A similar two-phase denaturation profile is evident when ER{alpha}-LBD is liganded with estradiol (Fig. 2BGo); however, the major decrease in Imax does not take place until higher Gua-HCl concentrations (~2.0 M), yet there is no change in the concentration of Gua-HCl required to effect the red shift in {lambda}em,max. It is of note that when ER{alpha}-LBD is bound with a ligand of lower affinity [17{alpha}-ethynyl-4-estrene-3ß-17ß-diol (EED), relative binding affinity (RBA) = 16% vs. 100% for estradiol], the first phase of the denaturation (Imax decrease) occurs at 1.2 M Gua-HCl (not shown), intermediate between that of unliganded ER{alpha}-LBD (0.5–1 M, Fig. 2AGo) and ER{alpha}-LBD complexed with the higher affinity ligand estradiol (2.0 M, Fig. 2BGo).

The tryptophan emission spectra of the ER{alpha}-LBD in high Gua-HCl (> 4.5 M) solutions agreed well with the fluorescence spectra of an equimolar aqueous solution of N-acetyl tryptophan, which was found to have a maximum fluorescence intensity centered about 356 nm (not shown). This indicates that in high Gua-HCl, the tryptophans of ER{alpha}-LBD are fully solvated by water.

Tetrahydrochrysene methyl ketone (THC-ketone) is a fluorescent ligand we developed for ER that has been used in a variety of studies on ER ligand binding kinetics, competitive binding assays, and visualization of ER in cells by fluorescence microscopy (21, 22, 23). It has a good binding affinity for ER (RBA of 68%, vs. 100% for estradiol), and in previous work, we have shown that THC-ketone is an effective quencher of the intrinsic tryptophan fluorescence when it is bound to the receptor (11). As shown in Fig. 3Go, THC-ketone binding to ER{alpha}-LBD results in a 90% decrease in Imax relative to that of the empty receptor. This quenching of intrinsic tryptophan fluorescence by ER-bound THC-ketone is greater than that of water. In fact, the level of fluorescence from ER{alpha}-LBD with THC-ketone bound is considerably less than that of an equimolar solution of N-acetyl-tryptophan in water (corrected for the three tryptophans in ER{alpha}-LBD). As the Gua-HCl concentration is raised on the ER{alpha}-LBD-THC-ketone complex, ligand is released from the receptor ligand binding pocket at approximately 2–3 M, at which point the tryptophan Imax increases to a level equivalent to that of ER{alpha}-LBD in both the unliganded and estradiol-liganded experiments (cf. Fig 2Go, A and B). The THC-ketone has no effect on the second phase of denaturation (red shift in {lambda}em,max at 4.5 M, not shown).

We also performed analogous experiments with ERß-LBD (either unliganded or liganded with estradiol or THC-ketone) and found that this ER subtype showed two-phase denaturation profiles that were nearly identical to those with ER{alpha}-LBD (data not shown).

The Nonspecific Fluorescent Probe bis-ANS Suggests That ER-LBD Denaturation Proceeds through a Partially Folded Intermediate
bis-ANS is a compound that fluoresces strongly in hydrophobic, nonaqueous environments, but is almost nonfluorescent in water. Because of these characteristics, it has proved to be a very useful probe of accessible protein interiors (24, 25). In particular, it is considered to be a "detector" of protein molten globule states, i.e. a "liquid-like" state in which the protein has a largely condensed structure with most of the secondary and some of the tertiary structure formed, but in a much more dynamic or fluid-like state than a fully folded protein (26). Because it has essentially no binding affinity for the native ER (RBA = 0.00031% for bis-ANS), bis-ANS functions effectively as a probe of the "accessibility" of hydrophobic regions of the ER-LBD, which operationally can be considered to be its molten globule character (24, 25).

bis-ANS probing of the denaturation profile of the ER{alpha} and ERß-LBDs (both unliganded and estradiol-liganded) was conducted after equilibration with increasing concentrations of Gua-HCl, in the presence or absence of estradiol. Figure 4AGo shows that with ER{alpha}-LBD in the absence of estradiol, bis-ANS fluorescence intensity (Imax) reaches a maximum at approximately 0.5 M Gua-HCl and begins to fall at higher concentrations. In contrast, with ER{alpha}-LBD in the presence of estradiol (Fig. 4Go), bis-ANS fluorescence requires approximately 2 M Gua-HCl to reach maximum intensity. Again, with ER{alpha}-LBD liganded with the lower affinity ligand, EED, an intermediate level of Gua-HCl (ca. 1.5 M) is required for bis-ANS fluorescence to reach a maximum (not shown). Figure 4BGo shows that ERß-LBD in the absence of estradiol has a peak Imax at approximately 1 M Gua-HCl, which is shifted to approximately 2 M Gua-HCl when estradiol is present.



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Figure 4. Intensity of bis-ANS in the Presence of ER{alpha} and ERß without Ligand or Complexed with E2 during Titration with Gua-HCl

ER{alpha} (panel A) or ERß (panel B) at 2 µM were preincubated either with estradiol (6 µM, solid circles) or with buffer control (open circles) for 1 h at 4 C. Samples were then diluted into the indicated concentration of Gua-HCl to a final concentration of 30 nM ER and 90 nM ligand; 1 mM bis-ANS was added, and the samples were incubated further for 1 h at 4 C. Fluorescence spectra were obtained by excitation at 294 nm, and emission was scanned from 430–600 nm. At each concentration of Gua-HCl, the relative maximum intensity is plotted. For details, see text and Materials and Methods.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have used two types of fluorescent probes to monitor the conformation of the ER-LBD during the progress of chemical denaturation with Gua-HCl. Both by monitoring the intrinsic fluorescence of the tryptophan residues in the ER-LBD, as well as by the use of the fluorescence probe bis-ANS, we have found that the LBD unfolds by a two-phase process. The first phase (at low Gua-HCl) is characterized by an abrupt drop in tryptophan fluorescence intensity accompanied by little change in wavelength of emission, and the second phase (at high Gua-HCl) is characterized by an abrupt red shift in the wavelength of emission with little change in intensity. bis-ANS fluorescence reaches a maximum between these two phases of denaturation. Thus, as explained below, denaturant treatment appears to induce the ER-LBD to access an intermediate state that is partially unfolded and unable to bind ligand, with ligand binding stabilizing the receptor against this partial unfolding (see Fig. 5Go). One would not expect to be able to observe such a partially unfolded intermediate under native conditions, because it would have only a fleeting existence. However, it could play an important role in the process by which ligands enter and exit the binding pocket of ER-LBD.



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Figure 5. Scheme Illustrating the Four Conformational States of the ER That Are Revealed by Intrinsic Tryptophan and bis-ANS Fluorescence Studies during Gua-HCl Denaturation

The LBD of ER is illustrated by a simple shape motif that is related to the crystal structure of this domain, as illustrated at the bottom of the scheme (below the solid line). Also illustrated in this ER motif are the three tryptophan residues (W), located in the upper subdomain, and the ligand (encircled L), in the lower ligand-binding subdomain. In the unliganded state (A), the lower subdomain is likely to be collapsed (cf. Ref. 12); neither ligand nor water nor bis-ANS can enter this portion of the ER, and the tryptophans show fluorescence characteristic of protein-buried residues. At low to moderate concentrations of Gua-HCl, the domain partially unfolds (B), allowing entry of water (which lowers tryptophan fluorescence by quenching but does not result in a red shift) and entry of bis-ANS (resulting in enhanced bis-ANS fluorescence). At high Gua-HCl concentrations, global unfolding of the whole domain occurs (C) with the result that the tryptophans are now fully exposed to water and are red shifted. Ligand entry to and exit from the ligand-binding pocket occurs through the dynamic state B. When ligand-bound (D), the domain is conformationally stabilized and is more resistant to Gua-HCl-induced unfolding than is the unliganded state (A).

 
The Two-Phase Denaturation Profile of ER-LBD Is Consistent with a Partially Unfolded Intermediate of the LBD That Does Not Stably Bind Ligand
Generally, during the denaturation of a protein, the intensity of tryptophan emission and the wavelength of this emission change in a coordinated fashion process (16, 17, 18, 19, 20). Two-phase denaturation profiles are not unusual for multidomain proteins, because the unfolding of each domain can be independent of the others and can occur at a distinct concentration of denaturant (27, 28). However, multiphase denaturation profiles are usually not observed with a single protein domain. What is particularly unusual about the denaturation of the ER-LBDs is that we observe a disparity between the change in tryptophan emission intensity and the change in tryptophan emission wavelength, indicating that quencher access to these residues and their full aqueous exposure occur at different levels of denaturation.

A reasonable interpretation of our experimental results is that at low denaturant concentrations, the LBD undergoes a partial unfolding, reaching a state that allows some water molecules to enter the interior of this domain, which brings them close enough to the three interior tryptophans to quench their emission significantly (Fig. 5Go, state B). However, the tryptophans do not experience a wavelength shift because the unfolding is only partial, and thus they remain in a "protein-like" interior. The partially unfolded state also provides an accessible hydrophobic protein interior phase into which bis-ANS can enter and exhibit its characteristic enhanced emission. However, this state appears no longer able to bind ligands stably, as is evident from the loss of ligand-tryptophan resonance energy transfer quenching by the THC-ketone ligand. The further unfolding of the LBD that occurs at much higher concentration of denaturant finally exposes the tryptophans to the high dielectric of an aqueous medium, causing the expected red shift in their emission (Fig. 5Go, state C). During this final unfolding, the accessible hydrophobic protein interior character is also lost, resulting in reduced fluorescence from bis-ANS.

One nuclear receptor LBD crystal structure is very relevant to the partially unfolded conformation that we are proposing for the ER-LBD. The structure of the unliganded peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) shows that the ligand-binding pocket, which constitutes the "lower half" of this domain, is considerably more expanded than it is in a ligand-receptor complex (9, 29), whereas the structurally conserved "upper half" of this domain has the tightly folded, compact conformation that is typical for ligand-receptor complexes. The expanded conformation of the ligand binding pocket of apo-RXR{alpha} is reminiscent of the sorts of partially unfolded structures that are proposed for protein molten globules (see below), and it may be similar to the partially unfolded conformation that we are proposing is induced in ER-LBD by treatment with low concentrations of denaturant.

Other apoproteins in which cofactors, such as hemes, have been removed, are used as experimental systems to study protein molten globules. Molten globules are conformational states having most of the secondary and some of the tertiary structure of the native protein, but are more dynamic or fluid-like, and they are often proposed as intermediates in protein folding or unfolding. Good examples of these dynamic, partially unfolded states are apomyoglobin at pH 4 (30, 31) and apoflavodoxin in Gua-HCl (18, 19, 32). The finding that certain other apoproteins can be induced to adopt a molten globule state upon treatment with denaturants (or a pH shift) is reminiscent of our finding that the unliganded ER-LBD, also an apoprotein, can be induced to adopt such a partially unfolded, dynamic state upon Gua-HCl treatment.

Ligand Binding Confers Structural Stability to the ER-LBD, as Evidenced by Its Protective Effect against Guanidine Denaturation
Higher concentrations of Gua-HCl are required to effect a decrease in the tryptophan fluorescence or an increase bis-ANS fluorescence in ligand-bound ER-LBD than in unliganded receptor. This demonstrates that ligand binding does indeed provide structural stability to the receptor (Fig. 5Go, States A, B, and C). Ligand binding is protective only toward the initial phase of denaturation, i.e. partial unfolding; it does not retard the onset of the red shift in the tryptophan fluorescence, i.e. global unfolding.

Ligand-induced conformational stabilization is not unique to the ER-LBD. In the PPAR{gamma} example noted above (9, 29), ligand binding causes the open apo-PPAR{gamma} structure to collapse to a tightly folded conformation that is typical of nuclear hormone LBD-ligand complexes (12). More recently, Johnson et al. (33) have used nuclear magnetic resonance to document this ligand-induced stabilization of the ligand binding pocket in PPAR{gamma}, and related work in ER has been done by Luck et al. (34). Pissios et al. (35) has described another approach to study ligand-induced changes in the ER LBD. When the LBD is covalently cut between helix-1 and the rest of the LBD, these two elements interact weakly, if at all, in the absence of ligand, but they do interact when estradiol is added. This suggests that ligand binding stabilizes the packing of helix-1 against the remaining portions of the LBD to such a degree that it remains associated by strictly noncovalent interactions. Since helix-1 interacts only with the upper zone of the LBD, ligand binding apparently has a stabilizing effect on the conformation of this upper zone.

Our recent finding that the binding of coactivator peptides to the ER-LBD dramatically slows the rate of ligand dissociation is also supportive of the competition between those forces that stabilize the tightly folded conformation of ER and those unfolding motions that are required for ligand dissociation (11). By binding in the coactivator groove, these peptides appear to lock the ER-LBD in the folded conformation, effectively trapping the ligand in the binding pocket to an even greater degree.

These observations and others also highlight differences between in vitro experiments and the cellular context. In all of the experiments described above, only the LBDs of these receptors are being studied alone and in vitro, whereas in the cell, these receptors are full length, which may have some effect on ligand binding (10), and there are, as well, many other proteins with which these receptors can interact. The latter include chaperone proteins that bind preferentially to unliganded receptors and may stabilize them in an unfolded state (36, 37). In fact, with the glucocorticoid receptor, association of the aporeceptor with chaperone proteins appears to be essential for ligand binding (38, 39). Thus, the results from our experiments on the ER-LBD in vitro may not directly predict the behavior of full-length ER in vivo. Nevertheless, they are instructive of the conformational dynamics that are an inherent property of the ER-LBD.

The Dynamic Character of the ER-LBD Is Essential for Its Regulation of Transcription
All of our results suggest that the LBD is a dynamic protein capable of entering multiple conformational states (Fig. 5Go): it needs to be dynamic for ligand to enter and exit; in the absence of ligand, it needs to be in a less rigid or soft state that does not afford effective docking sites for coactivator proteins (state A), and in the presence of ligands, it needs to adopt stable conformations that provide the sort of rigid exterior texture that is required to promote the interaction of coactivators (state D). This general need for the ligand to induce conformational stability is inherent to the ability of the ligand to turn on or turn off the activity of the receptor, a situation that is now widely appreciated in regulatory proteins (40, 41).

Normally, ER is not exposed to denaturants that can induce the partial unfolding of this domain that would facilitate ligand binding and dissociation. Yet, both apo-ER and ligand-bound ER must be able to access such a state under native conditions, although they would need to do so only transiently (Fig. 5Go, state B). On the basis of the studies we have performed here, we propose that ER under native conditions can transiently access a partially unfolded intermediate in which the ligand-binding pocket has a more open, dynamic character, allowing ligand binding, dissociation, and exchange to occur freely. Whereas such a transient, dynamic state would not be detectable under native conditions, it appears that an intermediate that displays the character expected from this partially unfolded state can be induced in the ER-LBD at intermediate concentrations of Gua-HCl.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Radiolabeled estradiol ([3H]E2) ([6,7-3H]estra-1,3,5,(10)-triene-3,17-ß-diol), 54 Ci/mmol, was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Isopropyl-ß-D-thiogalactopyranoside, imidazole, ß-mercaptoethanol, N-acetyl tryptophan, and unlabeled E2 were obtained from Sigma (St. Louis, MO). bis-ANS was obtained from Molecular Probes, Inc. Eugene, OR). The THC-ketone was prepared as described previously. The competent BL21(DE3)pLysS Escherichia coli were obtained from Novagen (Madison, WI).

ER Preparations
The human ER{alpha}-LBD, comprising amino acids 304–554 of human ER (hER), was expressed from a pET-15b vector in BL21(DE3)pLysS E. coli and purified as described previously (10). A pET-15b ERß construct (ERß-LBD) coding for the corresponding amino acids in ERß (256–505) (42) was also prepared using the same cloning site, and the ERß-LBD was expressed and purified in the same fashion as the ER{alpha}-LBD.

Tryptophan Fluorescence-Monitored Guanidine Denaturation Assay
For the fluorescence-monitored Gua-HCl denaturation assays of wild-type hER-LBD, purified hER-LBD was diluted to 1,500 nM in Tris-glycerol buffer (50 mM Tris, pH 8.0, 10% glycerol). For assays investigating the effect of ligand binding, the receptor was saturated by adding E2 or THC to a final concentration of 4,500 nM. The resultant solution was then incubated on ice for 1 h before denaturing with the Gua-HCl stock solutions. A 20 µl portion of the hER-LBD solution was added to 980 µl of a stock Gua-HCl solution, yielding a final hER-LBD concentration of 30 nM and a final ligand concentration of 90 nM (if ligand was used for the experiment). The Gua-HCl concentration of each stock solution was determined using the method described by Pace and Scholtz (43). The hER-LBD/Gua-HCl solutions were then further incubated on ice for 1 h. An aliquot (900 µl) of the solution was placed into a 5 x 5 mm quartz fluorescence cuvette, and the cuvette was placed into the sample chamber of a Fluorolog II (model IIIc) fluorometer (Spex Industries, Inc., Edison, NJ). The sample chamber was held at a constant 4 C, and the sample was excited at 285 nm (1.25-mm slits), and the tryptophan emission was observed from 300 nm to 420 nm (1.25-mm slits). Five emission scans were averaged for each sample, and a blank spectrum (buffer alone) was subtracted and was smoothed using a 21-point Savitsky-Golay algorithm using the Datamax software (Spex Industries, Inc.). The emission spectra were then analyzed for peak emission intensity and wavelength using Excel 97 (Microsoft Corp., Redmond, WA).

A 90 nM solution of N-acetyl tryptophan in Tris-glycerol buffer was used to determine the fluorescence spectral profile of fully solvated tryptophan. This spectrum was determined in an identical fashion to that of hER-LBD, and it showed an emission maximum at 356 nm.

bis-ANS Probing of Receptor Conformational Flexibility
For these experiments, hER-LBD was denatured in Gua-HCl solutions that had been prepared as described above. All samples were prepared as in the tryptophan fluorescence-monitored denaturation assay except that purified hER-LBD was initially diluted to 2,000 nM and the ligand concentrations were adjusted accordingly. To denature the protein, 15 µl of the 2,000 nM hER-LBD solutions were diluted into 980 µl of the stock Gua-HCl solutions to achieve a final hER-LBD concentration of 30 nM. A 5 µl portion of 0.2 M bis-ANS in EtOH was then added to each sample to yield a final bis-ANS concentration of 1 mM, and the samples were further incubated on ice for 1 h. The fluorescence of bis-ANS was measured exactly as for the tryptophan fluorescence except that the samples were excited at 294 nm and the emission spectra were scanned from 430 to 600 nm. The collected data were analyzed using the same methods as described above, but the wavelength of maximum emission intensity was not determined.


    FOOTNOTES
 
Address requests for reprints to: John A. Katzenellenbogen, Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana Illinois 61801. E-mail: jkatzene{at}uiuc.edu

This research was supported by NIH Grant PHS 1R37 DK-15556.

Received for publication July 28, 2000. Revision received September 25, 2000. Accepted for publication October 13, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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