A biosensor for estrogenic substances using the quartz crystal microbalance

A biosensor for estrogenic substances using the quartz crystal microbalance

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 345 (2005) 277–283 www.elsevier.com/locate/yabio A biosensor for estrogenic substances using the quar...

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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 345 (2005) 277–283 www.elsevier.com/locate/yabio

A biosensor for estrogenic substances using the quartz crystal microbalance Kendra S. Carmon a, Ruth E. Baltus b, Linda A. Luck a,* a


Department of Chemistry, Clarkson University, Potsdam, NY 13699, USA Department of Chemical Engineering, Clarkson University, Potsdam, NY 13699, USA Received 11 May 2005 Available online 3 August 2005

Abstract This article describes a biosensor that detects estrogenic substances using a quartz crystal microbalance with a genetically engineered construct of the hormone-binding domain of the a-estrogen receptor. The receptor was immobilized to a piezoelectric quartz crystal via a single exposed cysteine, forming a uniform orientation on the crystal surface. Our results illustrate that this sensor responds to a variety of ligands that are known to bind to the estrogen receptor. No response was observed for nonbinding substances such as testosterone and progesterone. The sensitive response of this biosensor to estrogenic substances results from changes in the structural rigidity of the immobilized receptor that occurs with ligand binding. Agonist and antagonist show different responses.  2005 Elsevier Inc. All rights reserved. Keywords: Estrogen receptor; Biosensor; Quartz crystal microbalance; Estrogens; Xenoestrogens; Surface chemistry

A device that can monitor substances that bind to the estrogen receptor would be a very valuable tool. Xenoestrogens have become a topic of public concern because they have potential adverse effects on reproductive health. These substances alter reproductive function and development through their direct interaction with the estrogen receptor. Compounds notorious for this action include natural plant estrogens and mycoestrogens, growth-promoting pharmaceuticals, and chemicals spread in water, sewage sludge, or the atmosphere such as detergents and surfactants, plastics, pesticides, and industrial chemicals [1]. These endocrine-disrupting compounds exert their action by their ability to act as hormone agonists or antagonists or to disrupt hormone synthesis, storage, or metabolism. With such far-reaching, potentially adverse effects on human health, there has been a great need to find good methodologies to screen for such *

Corresponding author. Fax: +1 315 268 6610. E-mail address: [email protected] (L.A. Luck).

0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.07.018

chemicals [2]. Because a large number of endocrine disruptors are estrogen-like and exert their effects influence through the estrogen receptor (ER),1 our studies have focused on the development of a biosensor that uses this protein. Because of its function as an allosteric liganddependent transcription factor, the ER has effects on the cardiovascular and reproductive systems as well as on a wide variety of tissues, including breast, bone, and liver [3]. Ligand binding at the C-terminal hormone-binding domain (HBD) of ER promotes a conformational change that is the driving force for a host of events within the cell, including dimerization, DNA binding, interaction with transcriptional factors, and gene transcription [4]. It is believed that the ligand-binding event is responsible for the ultimate biologies of the estrogen-sensitive tissues. The HBD functions indepen1 Abbreviations used: ER, estrogen receptor; HBD, hormone-binding domain; H12, helix 12; RQCM, research quartz crystal microbalance; E2, 17b-estradiol; QCM, quartz crystal microbalance; DES, diethylstilbestrol.


Biosensor for estrogenic substances / K.S. Carmon et al. / Anal. Biochem. 345 (2005) 277–283

dently and is known to bind a repertoire of ligands, including tamoxifen, estradiol, diethylstilbestrol, and raloxifene, each of which in turn affects the structure of the protein in very different ways. X-ray crystallographic studies of the ER with various ligands have shown that agonists and antagonists stabilize distinctly different conformations of the protein, primarily with respect to the carboxyl-terminal helix 12 (H12) [5–9]. Our laboratory has taken advantage of the HBD and its substrate-induced changes in conformation and structural rigidity to develop a technique to screen for compounds that bind to the estrogen receptor. The development of this biosensor combines the powerful specificity of a protein for its substrate with a sensitive device to measure the response of ligand capture. The heart of this quartz crystal piezoelectric methodology combines a method for immobilizing a target receptor, the HBD, to the electrode surface of a quartz crystal and a signal transduction method to measure the response of that target at the surface when ligand binds. The principle of the electrochemical methodology is based on detecting frequency changes of the crystal resulting from changes on the surface of the crystal [10,11]. The Sauerbrey equation describes the frequency change of the crystal when a rigid mass is added to the surface [12]: 2f 2 Df ¼  pffiffiffiffiffiffiffiffiffi Dm ¼ C f Dm; qq lq


where Df is the frequency shift resulting from the additional mass per area (Dm), f is the intrinsic crystal frequency, qq is the density of the quartz, and lq is the shear modulus of the quartz film. For a 5-MHz crystal, which we use in our instrument, Cf = 56.5 Hz cm2/lg. Changes in the viscosity or stiffness of a layer on the piezoelectric surface can also result in changes in the frequency of the resonating quartz [13]. For our studies, a genetically engineered construct of the HBD was immobilized on the surface of the crystal through a sulfur–gold bond. This biomodified surface has the capability to bind ligands with estrogenic activity. The binding event is detected as a change in frequency of the crystal that can be correlated to the ligand-induced changes in the characteristics of the protein surface. This method is highly specific for estrogenic substances because the target for the signal in the device is the same as the protein target in the body.

primer sequences were designed as follows. The C381S mutant was made and sequenced, and this DNA was used to make the double mutant HBD-C381,530S: Cys381 fi Ser Wild-type sequence: 5 0 -CCACCTTCTAGAA [TGT] GCCTGGCTAGAG-3 0 Primer I: 5 0 -CCACCTTCTAGAA [TCT] GCCTGG CTAGAG-3 0 Primer II: 5 0 -CTCTAGCCAGGC [AGA] TTCTAG AAGGTGG-3 0 Cys530 fi Ser Wild-type sequence: 5 0 -CTGTACAGCATGAAG [TGC] AAGAACGTGG-3 0 Primer I: 5 0 -CTGTACAGCATGAAG [TCC] AAGA ACGTGG-3 0 Primer II: 5 0 -CCACGTTCTT [GGA] CTTCATGCT GTACAG-3 0 Mutations were confirmed by sequencing at the Molecular Core Facility of the Trudeau Institute in Saranac Lake, New York, USA. The confirmed HBDC381,530S plasmid was used to transform into Escherichia coli BL21 (DE3) competent cells. Protein growth and isolation were done according to published procedures [14]. Purity of the protein was assessed by SDS– PAGE gels. Piezoelectric sensor experimental setup The research quartz crystal microbalance (RQCM, Maxtek, Torrance, CA, USA) is composed of the sensor, oscillator unit, frequency counter, voltage supply, and PC interface connection for signal output visualization. The RQCM sensors employed were commercially available, 5 MHz, polished AT-cut quartz crystals (2.54 cm diameter) with gold electrodes on both sides. The crystal was placed in the holder and positioned by an O-ring so that only one side was exposed to the protein, ligand, and buffer solutions. A 250-ml beaker containing 200 ml of T/G buffer (50 mM Tris, 10% glycerol, and 10 mM b-mercaptoethanol, pH 8.0) served as the ‘‘measurement cell.’’ This was done to decrease fluctuations in the baseline frequency. The setup has been described in detail elsewhere [15]. Attachment of HBD-C381,530S to the gold surface and frequency detection

Materials and methods Design and construction of the mutant HBD protein as the biotarget Site-directed mutagenesis was performed by PCR using the pET-HBD of the a-ER obtained from the Katzenellenbogen laboratory [14]. The oligonucleotide

The crystal holder was immersed into the liquid environment of the measurement cell until a stable frequency resonance was achieved and recorded. Frequency measurement was then temporarily halted. The holder was removed, and then 400 ll of HBD-C381,530S (0.7– 1.0 mg/ml) was incubated on the crystal surface. An incubation time of 0.5 h was determined to be optimal

Biosensor for estrogenic substances / K.S. Carmon et al. / Anal. Biochem. 345 (2005) 277–283


for significant protein attachment. The immobilization method was based on the formation of a direct covalent bond between the thiolated group of the surface-exposed C417 and the gold electrode of the crystal. After incubation, the crystal with immobilized protein was washed and returned to the measurement cell. Frequency was monitored until a stable reading was again obtained. Detection of ligand binding The crystal was removed from the measurement cell, and then a 400-ll quantity of 20 lM 17b-estradiol (E2) was introduced to HBD-C381,530S-immobilized crystal surface. Following a 2-h incubation with ligand, the crystal was immersed in the measurement cell. The frequency was monitored until a stable baseline was again reached. Analogous experiments were conducted with all ligands. Testosterone and progesterone served as negative controls. Because the ligands are water insoluble, they were initially diluted in ethanol. However, ethanol concentration in the ligand solution did not exceed 1% during frequency measurements.

Results and discussion Genetic engineering of the estrogen receptor The wild-type ERa HBD has four cysteine residues at positions 381, 417, 447, and 530. Previous work from the Katzenellenbogen laboratory showed that mutating these cysteines to serines in a construct that includes residues 304–554 has minimal effect on the binding ability of the protein [14]. We have capitalized on this finding and produced an HBD construct that will immobilize in a site-specific orientation to the gold surface of the piezoelectric crystal via a sulfur-to-gold covalent bond with Cys417. In the native folded protein, cysteines 381, 417, and 530 are located at the protein surface and are exposed to solvent. Therefore, they have the potential to form strong covalent attachments to the gold surface of the piezoelectric crystal without perturbing the protein conformation. In contrast, Cys447 resides in the internal hydrophobic area of the protein and, therefore, cannot bind to the gold unless the protein becomes denatured. To ensure that HBD immobilization allows only one orientation on the crystal and that the gold-to-sulfur bond occurs only at the 417 site, two cysteine-to-serine mutations were created at positions 381 and 530, producing a double mutant (HBDC381,530S). The attachment of the HBD-C381,530S by amino acid 417 to the crystal surface is illustrated in Fig. 1. This cysteine is an ideal site for attachment because this site is not involved in ligand binding, dimer

Fig. 1. Illustration of the HBD on the gold surface of the piezoelectric crystal. Shown in space-filling forms are the cysteine residues, estradiol in blue, helix 12 in yellow, and the placement of the gold sulfur bonds to each of the dimer lobes [5]. Our mutant used in these experiments has C381 and C530 changed to serine residues.

formation, or any conformational changes associated with ligand binding. From crystallography measurements, it has been determined that the HBD with bound ligand estradiol is a dimer with dimensions of approximately ˚ [5]. The estrogen receptor has been 55 · 55 · 64 A reported to exist as a dimer, even in the absence of ligand with a dimer dissociation half-life of nearly 1 h [14]. Therefore, the formation of the Au–S bond from one monomer of the dimer presumably promotes formation of the second gold-to-sulfur bond from the cysteine of the other monomer component. With the orientation on the gold surface shown in Fig. 1, it is estimated that each HBD dimer covers an area of approximately ˚ . With the gold electrode having an area of 55 · 55 A 2 1.27 cm , approximately 5.1 · 1012 HBD dimers are expected to be found in a monolayer on the surface. Careful washing of the surface with buffer and urea after immobilization of the HBD ensures that there is a monolayer of protein on the surface. Immobilization of HBD-C381,530S on the piezoelectric crystal and the addition of ligands Immobilization of HBD-C381,530S to the crystal surface resulted in a decrease in crystal frequency of 54 Hz relative to the frequency measured for the clean proteinfree crystal. Using the Sauerbrey equation Eq. (1) with a frequency shift of 54 Hz yields Dm = 0.96 lg/cm2, corresponding to 2.5 · 1013 dimer molecules deposited on the surface. This value is approximately five times larger


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than the estimate of 5.1 · 1012 that was calculated from dimensions derived from crystallography measurements with the HBD. The Sauerbrey equation is derived for a rigid film added to the piezoelectric surface. The HBD receptor films are expected to be reasonably fluid and viscous prior to ligand binding. The nonrigid nature of these films should dampen the crystal oscillation. This means that it is likely that there are more than 2.5 · 1013 dimers on the surface. In our measurements, the HBD is in a liquid environment where its effective size is expected to be smaller than the size estimated from crystallography. In addition, it is likely that the HBD can immobilize on the surface in a manner such that one HBD dimer occupies an ˚ footprint estimated from area less than the 55 · 55 A crystallography. Therefore, the estimate of 5.1 · 1012 dimers on the surface can be considered as a lower limit. Given the uncertainty in the interpretation of frequency measurements in terms of added mass and in the estimation of HBD size in solution from crystallography measurements, the frequency shift observed with receptor immobilization can be considered to be in general agreement with expectations based on crystallography measurements. Exposure of immobilized receptor to E2 (natural estrogen) showed an additional frequency shift of 25 Hz. Calculations based only on mass Eq. (1) indicate that a frequency shift of less than 1 Hz is expected from the addition of E2 to the immobilized protein on the surface. We believe that this unexpectedly large frequency shift observed with ligand binding occurs due to changes in the conformation or rigidity of the receptor that alter the nanoscale environment at the sensor surface. The result is a much larger frequency response than expected from mass considerations alone. This immobilized protein surface change is the exquisite molecular recognition element of our biosensor. Exposure of the immobilized receptor to ligands that do not bind shows no change in frequency. Because there is no change in the protein, there is no change in the sensor surface. Recent studies in our laboratory have documented an unexpectedly large frequency response from a quartz crystal microbalance (QCM) study of glucose binding to a glucose/galactose receptor from E. coli that we attributed to ligand-induced conformational changes in the protein [15]. When ligand binds, the glucose/galactose-binding protein undergoes a structural change, as do many other periplasmic binding proteins. The ligand-free protein is flexible and poised to capture the ligand. On binding, the protein adopts a more rigid form that enables the transport of ligand to the transport or chemotaxis assemblies in the membrane. Calculations of the frequency shift expected for a flexible viscous film and for a considerably more rigid film, along with atomic force microscopy studies of the rigidity of this protein

with and without ligand, provide support for this explanation [15,16]. This scenario is similar to changes undergone by the estrogen receptor and other members of the steroid superfamily. The apo forms of these proteins resemble molten globules and are dynamic or fluid-like in structure. Ligand binding reduces this flexibility. For example, NMR studies have shown that the structure of the ligand-free peroxisome proliferator-activated receptor is open and expanded, whereas the receptor–ligand complex collapses to a tightly folded conformation that is considerably more rigid [17]. KatzenellenbogenÕs laboratory found that conformational reorganization was necessary for the HBD to release the ligand [18]. In the absence of ligand, the HBD is in a ‘‘soft state’’ that precludes docking of coactivator proteins. In the presence of estradiol, the HBD provides a rigid exterior texture that promotes interaction with coactivators and subsequent biological activity [18]. Our laboratory has also used 19F NMR to further document ligand-induced changes in the rigidity of the HBD [19]. Thus, the frequency responses we observe in the QCM experiments reflect changes in the protein on the quartz surface and not the added mass of the ligand. Subsequent testing of a number of ligands that are known to interact with the estrogen receptor also showed frequency shifts in the QCM signal. The results with other estrogenic compounds are summarized in Fig. 2. These results show a frequency response for estrogens, antiestrogens, and phytoestrogens, with the ratio of the frequency shift resulting from ligand binding to the change in frequency observed with receptor immobilization ranging from 0.1 to 0.5. No frequency

Fig. 2. QCM frequency response to ligand binding. This graph represents the normalized frequency shifts from the QCM for ligand binding relative to the immobilization of HBD-C530,381S for two trials with each ligand.

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shift was observed when the nonbinding ligands testosterone and progesterone were introduced to the immobilized HBD on the surface of the piezoelectric crystal. The results shown in Fig. 2 indicate a different frequency response with different ligands for HBD. A reasonable interpretation of our QCM experimental results is that the HBD adopts a unique structure or level of rigidity with each type of ligand. Three-dimensional studies of the estrogen receptor with a variety of ligands allow us to visualize the static structural change mediated by each ligand and to relate these data to the frequency differences observed in the QCM experiments. According to X-ray crystallography, the structure of the estrogen receptor structure is akin to a pot with a lid [5,6]. Other nuclear receptors have been described as having a mousetrap mechanism for ligand binding [20]. The lid is the flexible helix known as H12 that covers the binding pocket, as shown in Fig. 1. As mentioned above, the empty protein is flexible, and so no crystal structure has been solved to date. In the apo state, it is assumed that H12 in nuclear receptors is in an extremely dynamic state and is exposed to solvent [21]. When the estrogen receptor binds E2, H12 fits snugly on the binding pocket [5–7]. This lid does not have any interaction with the ligand in the pocket but rather serves as a stabilizing entity for the structure. With H12 covering the binding pocket, the AF-2 groove is available to bind coactivators [22]. The orientation of H12 is highly sensitive to the bound ligand, and the nature of the coactivator-binding groove is highly dependent on the position and flexibility of H12 [6,7]. The interaction of the HBD with diethylstilbestrol (DES) shows a ligand completely encased within the binding cavity. However, the internal interactions with DES provide different packing configurations from those of the HBD with estradiol [6]. These interactions allow DES to bind tightly to the protein and to directly affect the overall conformation of the protein in a different manner from that of the HBD with bound estradiol. In contrast, when the antagonists tamoxifen and 4-hydroxy-tamoxifen bind to the HBD, the ligand protrudes from the cavity so that steric effects redirect H12 to the groove on the side of the cleft, thereby blocking interactions with coactivators required for agonistic activity [6]. One would expect that the displacement of the lid would lead to a less rigid structure than the estradiol-bound complex, especially with the ligand extending outside of the pocket. Genistein, a partial agonist to the HBD, showed a structure of the b-HBD complex with H12 in a quasi-antagonist position in one study [8]. However, a more recent study that crystallized complexes of the HBDs with coactivator fragments showed, in contrast, an agonist-like structure for both b-HBD and a-HBD [23]. Both studies corroborated the fact that the helix is in a flexible position and does not completely block the AF-2 site. The latter study showed that the helix


can be easily displaced to accommodate coactivator binding. We hypothesize that the frequency shifts measured in this study can be explained through these structural data. The largest frequency shift is shown with the endogenous ligand E2. With this ligand in the pocket, the protein adopts a conformation that is rigid and rather stable. Estriol, which is a strong agonist with a slight change in the chemical structure around the carbon 16, exhibits the next highest frequency shift. This compound binds to the pocket with somewhat lower affinity, and the lower frequency shift may reflect minor changes in the conformation of the receptor–ligand complex. The surface topography of H12 with estriol is expected to be similar to that of the E2 complex, but the overall conformation of the complex may be less rigid. DES binds to the HBD much tighter than does E2 [24]. However, DES does not make the same contacts within the cavity as does the natural ligand. This altered binding mode may slightly change internal structural units that translate to the surface topology. The antagonists tamoxifen and 4-hydroxytamoxifen are bound to the HBD, but a portion of the ligand extends outside of the cavity and displaces H12 to an alternate position. The conformation of the H12 on the side of the binding pocket leads to a less rigid complex than that of the HBD complexed with E2. Genistein shows the lowest frequency change of the binding ligands. This can be explained by the fact that, with this ligand, the H12 is in a quasi-position between the top of the binding cavity and the AF-2 position of protein. This is an unstable position for the helix and induces dynamic or flexible character in the complex. Our results indicate that the frequency response to ligand binding can be correlated with the nature of the ligand. A host of previous structural studies of the ligandbound receptor have provided knowledge of the ligand-induced structural changes and how they relate to the regulation of the estrogen receptor and the ultimate biologies. Although these studies provide information about the overall picture of the proteins, they do not provide information about the structural integrity or rigidity of the protein, which may be a key player in its function. The sensitivity of the QCM response to ligand concentration was examined by measuring the frequency shift resulting when solutions with estrogen concentration ranging from 0.1 to 1000 lM were introduced to the HBD immobilized on a quartz crystal. Results for the normalized frequency response resulting from ligand binding as a function of ligand concentration are shown in Fig. 3. This graph illustrates the response of the biosensor to the concentration of estrogens in the micromolar range. In this study, we have demonstrated the design and creation of a biosensor for estrogenic substances by


Biosensor for estrogenic substances / K.S. Carmon et al. / Anal. Biochem. 345 (2005) 277–283

Fig. 3. Ratio of QCM frequency response to binding 17b-estradiol binding relative to QCM frequency response to immobilization of HBD-C530,381S as a function of estrogen concentration.

combining the molecular control of genetic engineering and an ultra-sensitive detection methodology, the QCM, which has been underused for the detection of surface changes. A challenge in building biosensors is the protein immobilization and the stability of the surface during experimentation. Our direct linkage to the gold surface shows a distinct advantage over many multiplayer platforms used in prior studies. A previous report of a bioaffinity sensor using an immobilized HBDa/HSP90 on a QCM showed a change in frequency when estradiol was added to the surface [25]. Those authors claimed that this may be caused by the dissociation of HSP90 from the initial complex. In the same study, the authors showed another biosensor based on ligand-induced properties of estrogen. This sensor absorbed a transcriptional activator to the surface via the same metal linkage and showed a response when fulllength estrogen receptor was added with estradiol. Both of these sensors relied on the immobilization of the complex to the surface via a Ni(II)-mediated chemisorption using the His tag on the HBD and the coactivator through a thiol-modified linker. The Ni(II) metal linkage, which is also used in the purification scheme, is notoriously unstable. Destabilization of this linkage may account for part of the response of the sensor when estradiol is added. Our direct covalent linkage has a distinct advantage over techniques often used in QCM and surface plasmon resonance experiments in that the method gave reproducible signals. We are observing changes in the surface due to the ligand-induced changes in the protein surface; we are not observing mass changes.

The results presented here illustrate that it is possible to monitor the binding of small ligands to an immobilized receptor surface. This has allowed the rapid monitoring of the presence of ligands that bind to the estrogen receptor as well as the characterization of those ligands. Receptors are uniformly bound on the surface because there is only one mode of binding, thereby minimizing surface heterogeneities. The validity of this biosensor was demonstrated by its ability to discriminate between ligands and nonligands as well as its ability to detect E2 in the micromolar range. The natural ligand E2 exhibits the greatest response, demonstrating the innate structural integrity of the complex with the endogenous ligand. Ligands that do not bind to the estrogen receptor, such as testosterone and progesterone, show no response. As a group, binding of agonists shows a larger frequency response than is observed with binding of antagonists. Our results demonstrate that it is possible to detect binding of small ligands to large proteins captured on the surface of the QCM by sensing changes in the structural rigidity of the protein that occur on ligand binding. This information may offer new insight into receptor–ligand interactions and may lead to a new screen for xenoestrogens in the environment.

Acknowledgments We gratefully acknowledge funding for this work from the National Institutes of Health (Grant R03-CA 89705) and the National Science Foundation (Grant CTS-032968). We also thank John Katzenellenbogen for the plasmid HBDa (residues 304–554) and for many helpful discussions.

References [1] D. Crews, E. Willingham, J.K. Skipper, Endocrine disruptors: present issues, future directions, Q. Rev. Biol. 75 (2000) 243–260. [2] S.O. Mueller, K.S. Korach, Mechanisms of estrogen receptormediated agonistic and antagonistic effect, in: The Handbook of Environmental Chemistry, vol. 3 (2001). [3] P.H. Driggers, J.H. Segars, Estrogen action and cytoplasmic signalling pathways, Trends Endocrinol. Metab. 13 (2002) 422– 427. [4] G.G. Kuiper, B. Carlsson, K. Grandien, E. Enmark, J. Haggblad, S. Nilsson, J.A. Gustafsson, Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta, Endocrinology 138 (1997) 863–870. [5] A.M. Brzozowski, A.C.W. Pike, Z. Dauter, R.E. Hubbard, T. Bonn, O. Engstrom, L. Ohman, G.L. Greene, J. Gustafsson, M. Carlquist, Molecular basis of agonism and antagonism in the oestrogen receptor, Nature 389 (1997) 753–758. [6] A.K. Shiau, D. Barstad, P.M. Loria, L. Cheng, P.J. Kushner, D.A. Agard, G.L. Greene, The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen, Cell 95 (1998) 927–937.

Biosensor for estrogenic substances / K.S. Carmon et al. / Anal. Biochem. 345 (2005) 277–283 [7] A.C.W. Pike, A.M. Brzozowski, R.E. Hubbard, A structural biologistÕs view of the oestrogen receptor, J. Steroid Biochem. Mol. Biol. 74 (2000) 261–268. [8] A.C.W. Pike, A.M. Brzozowski, R.E. Hubbard, T. Bonn, A. Thorsell, O. Engstrom, J. Ljunggren, J. Gustafsson, M. Carlquist, Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and full antagonist, EMBO J. 18 (1999) 4608–4618. [9] E.H. Kong, A.C.W. Pike, R.E. Hubbard, Structure and mechanism of the oestrogen receptor, Biochem. Soc. Trans. 31 (2003) 56–59. [10] A. Janshoff, H.J. Galla, C. Steinam, Piezoelectric mass-sensing devices as biosensors: an alternative to optical biosensors? Angew. Chem. 39 (2000) 4004–4032. [11] K. Marx, Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution–surface interface, Biomacromolecules 4 (2003) 1099– 1120. [12] G. Sauerbrey, Z. Phys. 155 (1959) 206–222. [13] F. Hook, B. Kasemo, Variations in coupled water, viscoelastic properties, and film thickness of a Mefp-1 protein film during adsorption and cross-linking: a quartz crystal microbalance with dissipation monitoring, ellipsometry, and surface plasmon resonance study, Anal. Chem. 73 (2001) 5796–5804. [14] A. Tamrazi, K.E. Carlson, J.R. Daniels, K.M. Hurth, J.A. Katzenellenbogen, Estrogen receptor dimerization: ligand binding regulates dimer affinity and dimer dissociation rate, Mol. Endocrinol. 16 (2002) 2706–2719. [15] K.S. Carmon, R.E. Baltus, L.A. Luck, A piezoelectric quartz crystal biosensor: the use of two single cysteine mutants of the periplasmic E. coli glucose/galactose receptor as target proteins for the detection of glucose, Biochemistry 43 (2004) 1429–1456. [16] I. Sokolov, S. Venkatesh, L.A. Luck, Change in rigidity in the activated form of the glucose/galactose receptor from E. coli: a phenomenon that will be key to the development of biosensors, Biophys. J. (under review).


[17] B.A. Johnson, E.M. Wilson, Y. Li, D.E. Moller, R.G. Smith, G. Zhou, Ligand-induced stabilization of PPARc monitored by NMR spectroscopy: implications for nuclear receptor activation, J. Mol. Biol. 298 (2000) 187–194. [18] A.C. Gee, J.A. Katzenellenbogen, Probing conformational changes in the estrogen receptor: evidence for a partially unfolded intermediate facilitating ligand binding and release, Mol. Endocrinol. 15 (2001) 421–428. [19] L.A. Luck, J.L. Barse, A.M. Luck, C.H. Peck, Conformational changes in the human estrogen receptor observed by 19F NMR, Biochem. Biophys. Res. Commun. 270 (2000) 988–991. [20] J.P. Renaud, N. Rochel, M. Ruff, V. Vivat, P. Chambon, H. Gronemeyer, D. Moras, Crystal structure of the RAR-c ligandbinding domain bound to all- trans retinoic acid, Nature 378 (1995) 681–689. [21] W. Bourguet, M. Ruff, P. Chambon, H. Gronemeyer, D. Moras, Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-a, Nature 375 (1995) 377–382. [22] A. Warnmark, E. Treuter, J.A. Gustafsson, R.E. Hubbard, A.M. Brzozowski, A.C.W. Pike, Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha, J. Biol. Chem. 277 (2002) 21862–21868. [23] E.S. Manas, Z.B. Xu, R.J. Unwalla, W.S. Somers, Understanding the selectivity of Genistein for human estrogen receptor-b using X-ray crystallography and computational methods, Structure 12 (2004) 2197–2207. [24] G.G. Kuiper, B. Carlsson, K. Grandien, E. Enmark, J. Haggblad, S. Nilsson, J.A. Gustafsson, Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptor alpha and beta, Endocrinology 138 (1997) 863–870. [25] M. Murata, C. Gouda, K. Yano, S. Kuroki, T. Suzutani, Y. Katayama, Piezo electric sensor for endocrine-disrupting chemicals using receptor–cofactor interaction, Anal. Sci. 19 (2003) 1355–1357.