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A Bioluminescent Indicator for Highly Sensitive Analysis of Estrogenic Activity in a Cell-based Format Osamu Takenouchi, Akira Kanno, Hideo Takakura, Mitsuru Hattori, and Takeaki Ozawa Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00466 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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Bioconjugate Chemistry

A Bioluminescent Indicator for Highly Sensitive Analysis of Estrogenic Activity in a Cell-based Format

Osamu Takenouchi†, Akira Kanno†, Hideo Takakura†, Mitsuru Hattori† and Takeaki Ozawa†

† Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

*Correspondence should be addressed to T. O. Email: [email protected] Tel.: 81-3-5841-4351 Fax: 81-3-5802-2989

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Abstract Estrogens regulate different physiological systems with wide ranges of concentrations. The rapid analysis of estrogens is crucially important for drug discovery and medical diagnosis, but quantitation of nano-molar estrogens in live cells persists as an important challenge. We herein describe a bioluminescent indicator used to detect low concentrations of estrogens quantitatively with a high signal-to-background ratio. The indicator comprises a ligand-binding domain of an estrogen receptor connected with its binding peptide, which is sandwiched between split fragments of a luciferase mutant. Results show that the indicator recovered its bioluminescence upon binding to 17β-estradiol at concentrations higher than 1.0 × 10-10 M. The indicator was reactive to agonists but did not respond to antagonists. The indicator is expected to be applicable for rapid screening estrogenic compounds and inhibitors, facilitating the discovery of drug candidates in a high-throughput manner.

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Introduction Estrogens are small chemical compounds responsible for the development and maintenance of various tissues.1–3 The distribution of estrogens varies depending on the tissues and their local concentration changes periodically.4 Therefore, temporal and quantitative analyses of estrogens are crucially important for the diagnosis and treatment of estrogen-related diseases.5–7 Nevertheless, because of the change of estrogen concentrations over a wide dynamic range from 1.0 × 10-11 to 0.2 × 10-9 M8, it is difficult to analyze estrogens in low physiological concentrations in living systems. Moreover, many compounds originated from plants and industrial materials bind to the estrogen receptor (ER), thereby activating or inhibiting estrogenic effects.9–11 Evaluation of such agonistic and antagonistic effects, particularly discrimination between agonists and antagonists, is often necessary for biochemical studies and medical diagnosis. To detect estrogenic activity in living cells, researchers often use luciferase reporter gene assays.12 The luciferase catalyzes the oxidization reaction of luciferin, which leads to bioluminescence emission. The use of luciferases provides important benefits for quantifying and monitoring target biological events in living cells and animals.13 The assays using luciferases do not need excitation light, contrary to those using fluorescent proteins. For that reason, highly sensitive detection is possible with quite low background noise and low light toxicity to the tissues of interest. These assays have an advantage to detect low estrogenic activity in living cells. However, they need 16-24 hours to express from the luciferase gene to its corresponding protein.12 When we consider a large

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scale of chemical libraries, shorter time of detection after ligand stimulation is desirable for more high-throughput and efficient screening. To improve such issues of reporter gene assays, we and other groups have developed many intramolecular indicator using luciferase fragment complementation assay technique.14–19 Reportedly, an indicator for estrogens has been developed using a ligand binding domain (LBD) of ER fused to split firefly luciferase fragments or Gaussia princeps-derived luciferase (GLuc).18,19 These indicators clearly showed bioluminescence signals upon short time stimulation. However, the sensitivity was insufficient; the indicators were reactive over 1.0 × 10-9 M of estrogen concentrations. Better sensitivity of less than 1.0 × 10-9 M is requisite for quantitative evaluation for endogenous level of estrogen and estrogenic compounds with quite low activities.8 In addition, these indicators were reactive to antagonist, which hampers the discrimination between agonists and antagonists. Here, we present significant improvement of the bioluminescence indicator for estrogens with high sensitivity and rapidity for large scale screening of testing compounds. The indicator comprises LBD of ER, a short amino acid sequence (LXXLL motif), and split fragments of emerald luciferase (ELuc) derived from Brazilian Pyrearinus termitilluminans.20 An important benefit of the use of ELuc is that it emits much brighter luminescence than firefly luciferase does. In addition, the ELuc spectrum is independent of pH in a range from 6 to 8.21 The indicator senses estrogenic activity for ER, the properties of which are also beneficial for discriminating between agonist and antagonist. We herein demonstrate the applicability of the new indicator for rapid chemical screening with a

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multi-well plate format.

Materials and methods Materials and constructs Estrone (E1), estriol (E3), testosterone (T), bisphenol-A (BPA), genistein (Gen), and daidzein (Dai) were purchased from Wako Pure Chemical Industries Ltd. (Japan). 17β-Estradiol (E2), Diethylstilbestrol (DES) and 4-hydroxytamoxifen (OHT), and raloxifene (RAL) were obtained from Sigma-Aldrich Japan. Androstenedione (AND) was from Tokyo Chemical Industry Co., Ltd. (Japan). ICI 182,780 (ICI) were obtained from Nacalai tesque Inc. (Japan). Click beetle cDNA was obtained from Toyobo Co. Ltd. (Japan). The cDNA of a ligand binding domain of estrogen receptor connected with LHRLL sequence and split luciferase fragments was inserted in the restrict enzyme sites of NheI and XhoI in pcDNA3.1 V5/His (B) (Invitrogen Corp., Carlsbad, CA). Randomly mutated LXXLL sequences were generated using a PCR technique. Cell culture and transfection African green monkey kidney (COS-7) cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) with high glucose supplemented with 10% fetal bovine serum (FBS), 100 unit/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2. Cells were transfected with the vectors coding the estrogen indicator with the vector coding full length of Renilla luciferase (RLuc) using TransIT-LT1 (Mirus, Madison USA). The RLuc was used as an internal control to normalize the transfection

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efficiency and the number of cells. In addition, the pET vector (Novagen Inc., Darmstadt, Germany) was used for expression in Escherichia coli (E. coli). Luciferase assay using COS-7 cells After 24 h from transfection, cells were treated with each ligand of ER for 1 h. Cells were lysed using a passive lysis buffer (Promega Corp., Madison, WI). After 15-mins treatment, the suspension was mixed in single tubes with the Dual-Glo luciferase assay reagent (Promega Corp.). The luminescence intensity was measured using a luminometer (Lumat LB9507; Berthold Technologies GmbH and Co. KG, Germany). In high-throughput screening using 96-well microtiter plates, the Dual-Glo luciferase assay reagent was added directly into each well according to the manufacturer’s protocol. The luminescence intensity was measured using a 96-well microplate reader (TriStar LB941; Berthold Technologies GmbH and Co. KG). All measurements were performed three times with different wells. Each luminescence intensity was shown as a ratio of ELuc to RLuc (ELuc/RLuc) and normalized against luminescence intensity upon stimulation of DMSO. The luminescence intensity data are shown as averages with standard deviations. Luciferase assays using E. coli expression system The cDNA of the indicator was transformed in E. coli (BL21) and incubated overnight on LB plate containing ampicillin. Each E. coli colony expressing the indicator was cultured in a 2 ml tube at 37 °C until OD600 reached 0.4–0.5. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to each tube and incubated further for 12 h at 15°C. After sonication of E. coli, the suspension was mixed

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with 1.0 × 10-6 M E2 or DMSO for 15 min. Finally, 100 µl emerald luciferase assay reagent (Toyobo Co. Ltd., Japan) was added to each well. The luminescence intensity was measured using a 96-well microplate reader.

Results Development of a high-sensitive indicator for 17β-estradiol. Reportedly, ER undergoes different conformational changes depending on the binding mode of agonists or antagonists.22 For agonists, the LBD of ER interacts with a coactivator with a LXXLL motif to form the ER-coactivator complex,23 which activates transcription of particular DNAs thereafter (Figure S1). In contrast, upon binding of ERs to antagonists, an α-helix (H12) in the LBD inhibits interaction of the coactivator with ER. Based on such properties of structural changes in ER, we constructed an indicator composed of the LBD of ER (305–550 amino acids), split ELuc fragments (the N-terminus; 2–408, the C-terminus; 407–542 amino acids) and LHRLL sequence derived from steroid receptor coactivator 1 (named HR-ELuc, Figure 1a). Upon stimulation of the indicator with estrogens, the LHRLL sequence stabilizes the LBD by strong interactions between LBD and the LHRLL sequence. The structural change in the LBD connected with the LHRLL sequence induces reconstitution of the ELuc fragments into an active form, resulting in the recovery of bioluminescence (Figure 1b). Although the LBD forms antagonist-specific conformation upon binding to antagonist, the proximity of luciferase fragments is inhibited due to the presence of the

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LXXLL sequence.

a) H2N

ELuc_N

LXXLL

LBD of ER

b) ELuc_N

ELuc_C

COOH

Luminescence

ELuc_C Estrogen

LXXLL LBD of ER Figure 1. Principle of the indicator for detecting estrogenic compounds. a) Schematic structure of the estrogen indicator. The indicator comprises the N-terminal fragment of ELuc (Eluc_N), Leu-His-Arg-Leu-Leu sequence (LHRLL), the ligand binding domain (LBD) of ER, and the C-terminal fragment of Eluc (ELuc_C). b) Basic principle of estrogen detection. The indicator changes its conformation upon binding to an estrogenic compound, which brings the Eluc fragment in proximity allowing for luminescence recovery.

To characterize the indicator in living cells, COS-7 cells expressing the HR-ELuc were treated with different concentrations of 17β-estradilol (E2) for 1 h. Then, the luminescence intensities were analyzed using a luminometer. Results show that the luminescence intensities increased concomitantly with increasing concentrations of E2. The maximum response upon stimulation with 1.0 × 10-6 M E2 was 11-fold higher than the background luminescence (Figure S2). No change in the luminescence intensity was found for E2 concentrations lower than 1.0 × 10-10 M, of which the sensitivity was the same as that reported previously. To improve the sensitivity, we introduced amino acid mutations randomly at the sites of His and Arg in the LHRLL sequence using a random mutagenesis technique. After transformation of the cDNAs which encode the indicator with the randomly mutated LXXLL motif, we mixed the 8


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suspensions of E. coli with 1.0 × 10-6 M E2 including D-luciferin. Of more than 500 colonies screened, we obtained three sequences that represented a high signal-to-background ratio (Leu-Thr-Asn-Leu-Leu; LTNLL, Leu-Glu-Ser-Leu-Leu; LESLL and Leu-Arg-Leu-Leu-Leu; LRLLL) (Figure S3). To further investigate the bioluminescence property in mammalian cells, we analyzed the luminescence intensities of these indicators in COS-7 cells upon E2 stimulations (Figure 2a). When the cells were stimulated with 1.0 × 10-6 M E2, indicators with LTNLL, LESLL, and LRLLL motifs showed 40, 59, and 61-fold increases in the luminescence, respectively. The most sensitive one was the indicator with the LRLLL motif (named RL-ELuc), which was used for additional experiments. Next, we optimized the dissection sites of ELuc fragments. The lengths of N-terminal and C-terminal fragments were changed one by one (N-terminus; 12 fragments from 2–406 to 2–417 amino acids and C-terminus; 25 fragments from 389–542 to 413–542 amino acids) and 300 pairs of luciferase fragments were generated. We tested the luminescence recovery in the presence of 1.0 × 10-6 M E2 using E. coli and COS-7 cells. The best sensitive detection was obtained when we used the indicator with the pair of luciferase fragments (2–408 and 407–542 amino acids, Figure S4). Reportedly, a combination of the N-terminal fragment of ELuc and the C-terminal fragment of a mutated click beetle red luciferase (McLuc1; 395–541 amino acids) showed much higher luminescence intensity than a pair of ELuc.16 Using the pair of ELuc and McLuc1, we compared the bioluminescence recovery with the indicator using a pair of ELuc fragments. The absolute photon

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counts of the indicator using McLuc1 was three-fold higher than the indicator composed of ELuc fragments (Figure S5). We then prepared 12 N-terminal fragments of ELuc (from 2–406 to 2–417 amino acids) and 25 C-terminal fragments of McLuc1 (from 386–541 to 410–541 amino acids). After we developed 300 indicators in all, we evaluated the sensitivity of each upon stimulation with 1.0×10-6 M E2 (Figure S6). A pair of 2–406 amino acids of ELuc and 399–541 amino acids of McLuc1 showed the highest luminescence intensity. Next, we verified the response of the indicators with the sensitive pair of luciferase fragments (named RL-EMcLuc) in COS-7 cells (Figure 2b). The indicator showed 80-fold increases in the luminescence in the presence of 1.0 × 10-6 M E2.

Figure 2. The luminescence intensities from the modified estrogen indicators a) Comparison of the luminescence intensities between different LXXLL sequences. Luminescence increases for the mutated indicators in COS-7 cells. Cells were transfected with the indicators including the LXXLL sequences and incubated for 24 h. The cells were stimulated to 1.0×10-6 M E2 or DMSO for 1 h. The luminescence intensities were measured 5 s using a luminometer. The luminescence intensities were normalized against the luminescence intensity upon stimulation of DMSO. b) Optimization of the dissection sites in the luciferases. Verification of optimal dissection sites was measured in COS-7 cells. The COS-7 cells transfected with the indicator were exposed to 1.0×10-6 M E2 or DMSO for 1 h. The luminescence intensities were measured for 15 s using a luminometer.

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Furthermore, we confirmed inhibitory effect of antagonist ICI on the bioluminescence upon stimulation of 1.0 × 10-6 M E2 (Figure S7). The bioluminescence decreased with increasing the concentration of ICI. This result suggests that the bioluminescence from RL-EMcLuc was induced by interaction between the LBD and the LXXLL motif. In addition, we compared the response to the various concentrations of E2 between the RL-ELuc and RL-EMcLuc in the COS-7 cells (Figure 3). The RL-EMcLuc showed luminescence stronger than the RL-ELuc. The luminescence upon addition of 1.0 × 10-10 M E2 increased 2.4-fold higher than that treated with DMSO. The lowest detection limit was calculated as the concentration that equals the signal of 3 standard deviation of signal from DMSO-treated cells and was 1.1 × 10-10 M.

Figure 3. Concentration dependence of different chemicals on luminescence intensities. Luminescence intensity changes upon stimulation of different concentration of E2. The COS-7 cells expressing the RL-ELuc (Light blue) or RL-EMcLuc (Deep blue) were stimulated with various concentrations of E2 for 1 h. The luminescence activities were measured for 15 s. The luminescence intensities were normalized against the luminescence intensity upon stimulation of DMSO.

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In addition, we confirmed similar expression levels of the indicators (Figure S8). Taking these results together, we concluded that the RL-EMcLuc enables highly sensitive and quantitative detection of E2 in such low concentration ranges. Furthermore, the luminescence recovery was sufficiently detected in 5 minutes (Figure S9), indicating that fast responses of the indicator are a significant advantage compared to reporter gene assays.

Agonist-dependent luminescence recovery using a 96-well microtiter plate reader To demonstrate the usefulness of RL-EMcLuc for the screening of estrogenic compounds, we evaluated the response to seven known estrogenic compounds: three endogenous estrogens of E1, E2, and E3, an artificial estrogen of DES, and the three botanical estrogenic compounds of Gen, Dai, and BPA (Figure 4a). The COS-7 cells were transfected with the cDNA encoding the RL-EMcLuc in a 96-well microtiter plate. Cells were exposed to the estrogenic compounds for 1 h. Then bioluminescence intensities were measured using a plate reader. The RL-EMcLuc showed strong luminescence recovery with increasing concentrations of all agonists, although the responses to Dai and BPA were lower than that to other agonists. In contrast, no change in the luminescence was obtained for the response to 1.0 × 10-6 M antagonists (RAL, OHT, ICI) and estrogen precursors (AND and T) (Figure 4b), demonstrating that the RL-EMcLuc senses agonistic compounds but it does not respond to antagonists.

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Figure 4. Luminescence intensity upon stimulation of different ligands. The COS-7 cells were transfected with the vectors encoding the RL-EMcLuc and cultured for 24 hr. Cells were stimulated with different concentrations of ligands, such as estron (E1), 17β-estradiol (E2), estriol (E3), diethylstilbestrol (DES), bisphenol-A (BPA), genistein (Gen), and daizein (Dai) for 1 h and then treated with the substrate. Luminescence intensities from each well were measured for 5 s. Luminescence intensities were normalized against that upon the stimulation of DMSO. b) Reactivity of the indicator to antagonists and a precursor of estrogen. The COS-7 cells expressing RL-EMcLuc were stimulated with 1.0×10-6 M chemicals such as raloxifene (Ral), 4-hydroxytamoxifen (OHT), ICI 182,780 (ICI), androstenedione (AND), and testosterone (T) for 1 h. The luminescence intensities were normalized against the luminescence intensity upon stimulation of DMSO.

Next, we calculated the concentrations whereby each ligand induced five-fold increases in the luminescence intensity (EC5) and compared the results with the binding affinity using the radioactive ligands reported previously from two different groups (Table S1 and Figure 5).24, 25, 26 The EC5 values were significantly correlated with binding affinities (R2=0.98 and 0.95), suggesting that the luminescence intensity of the indicator is originated from the binding property of a targeting compound in the case of agonistic compounds.

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Figure 5. Relationship between binding affinity and estimated concentration inducing 5-fold increase of luminescence intensity. Relative binding affinity to ERα calculated using radioactive estrogens and the estimated concentration inducing 5-fold increase of luminescence intensity (EC5) were plotted. The relative binding affinity values were based on a) Fang et al., 2001 and b) Kuiper et al., 1997 and 1998. The tested compounds were shown as estron (E1), 17β-estradiol (E2), estriol (E3), diethylstilbestrol (DES), bisphenol-A (BPA), genistein (Gen), and daizein (Dai).

Discussion We developed a novel bioluminescent indicator for highly sensitive detection of estrogenic compounds based on the luciferase complementation assays. To date, several indicators for estrogen have been developed using the LBD of ER and fragments of split luciferases.18,19 Of the various indicators, we herein improved the sensitivity and selectivity for estrogens by the random mutation in LXXLL sequence and optimized the dissection sites of luciferases sufficient to detect lower concentration ranges of estrogens. Three pairs of successive amino acids (threonine - asparagine, glutamic acid - serine, and arginine - leucine) were newly identified in the XX motif showing high sensitivity for E2. The three LXXLL sequences that we identified are new motifs and are not contained in known coactivators. Although some LXXLL sequences have been reported to disrupt

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transcription activity27, we believe that the inhibitory effect of the LXXLL sequence on endogenous ERs is quite small because the LXXLL sequences interact preferentially with the ER of the indicator by intramolecular structural changes. The sensitive response to E2 was achieved by lowering intensities of background luminescence in the absence of E2. Taken together, these data indicate that amino acids in the LXXLL are extremely important for the sensitive and selective detection of agonists over antagonists, possibly because the basal affinities of ER might differ among LXXLL motifs. In other cases, indicators using the fluorescence resonance energy transfer (FRET) principle have been reported28-30. The detection limit of the FRET indicators was from 0.64 × 10-10 to 2.2 × 10-10 M28-30, which suggest that the sensitivity of our indicator is almost the same as the FRET indicators. However, the dynamic range of FRET signals was much smaller than that of the bioluminescent indicator. Therefore, the present indicator is suitable for detection of different estrogenic compounds. A salient benefit of the present indicator is selectivity for agonists. The indicator showed no luminescence recovery for antagonists or estrogen precursors. Similar indicators reported previously have a property of response to E2 and DES (approx. 1.0 × 10-6 M).18 However, the indicators also showed a similar level of luminescence recovery upon the stimulation of antagonists (1.0 × 10-6 M) such as the OHT and RAL attributable to a property of detecting the conformational change of ER. Another indicator by Kim et al. was composed of a Src homology 2 domain of v-Src which interacts with LBD of ER.19 A treatment of antagonist, OHT, also induced luminescence recovery because of

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the phosphorylation of LBD. Contrary to such previous indicators, modification of LXXLL sequence for the present indicator enabled to discriminate between agonists and antagonists. Therefore, it will be possible to discriminate between agonistic and antagonistic effects on ER when both present and previous indicators are applied for the screening of chemicals. Aside from such an advantage, a short response time would allow for high-throughput screening of artificial estrogenic compounds from a large chemical library for pharmacological purposes or rapid verification of estrogenic activity in botanical food and industrial products. In this study, we demonstrated a significant improvement for sensing low concentration of estrogen. However, the detectable concentration of the indicator did not cover the whole range of physiological concentration of estrogens. Therefore, for further application our indicator to diagnosis of estrogen-related diseases using blood and urine samples, some pretreatments such as concentration or dialysis will be needed because the physiological concentrations of estrogen in practical samples is less than those detectable by the present indicator. In addition, many inhibitory factors for the indicator are included in practical samples, which may hamper accurate evaluation of estrogen concentrations. In conclusion, we developed an estrogen indicator based on luciferase complementation assays using the fragments of ELuc and McLuc1. We identified optimal lengths of Eluc and McLuc1 fragments and demonstrated that the modification of the LXXLL sequence is effective to improve the indicator sensitivity and selectivity. This indicator will be applicable for high-throughput screening

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of chemical compounds possessing estrogenic activity. The fundamental principle presented in this study is expected to present the possibility of developing different steroid hormone indicators using the corresponding ligand binding domains and a peptide sequence of their coactivators.

Acknowledgements This work was supported by the Japan Society for the Promotion of Science (JSPS, No. 26220805). Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org.

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Luciferase-Fragment 82, 9306–9313. (17) Misawa, N., Kafi, A. K. M., Hattori, M., Miura, K., Masuda, K., and Ozawa, T. (2010) Rapid and high-sensitivity cell-based assays of protein-protein interactions using split click beetle luciferase complementation: An approach to the study of G-protein-coupled receptors. Anal. Chem. 82, 2552–2560. (18) Paulmurugan, R., and Gambhir, S. S. (2006) An intramolecular folding sensor for imaging estrogen receptor-ligand interactions. Proc. Natl. Acad. Sci. U. S. A. 103, 15883–15888. (19) Kim, S. B., Sato, M., and Tao, H. (2009) Split Gaussia luciferase-based bioluminescence template for tracing protein dynamics in living cells. Anal. Chem. 81, 67–74. (20) Nakajima, Y., Yamazaki, T., Nishii, S., Noguchi, T., Hoshino, H., Niwa, K., Viviani, V. R., and Ohmiya, Y. (2010) Enhanced beetle luciferase for high-resolution bioluminescence imaging. PLoS One 5. (21) Viviani, V. R., Arnoldi, F. G. C., Neto, A. J. S., Oehlmeyer, T. L., Bechara, E. J. H., and Ohmiya, Y. (2008) The structural origin and biological function of pH-sensitivity in firefly luciferases. Photochem. Photobiol. Sci. 7, 159−169. (22) Celik, L., Lund, J. D. D., and Schiøtt, B. (2007) Conformational dynamics of the estrogen receptor alpha: molecular dynamics simulations of the influence of binding site structure on protein dynamics. Biochemistry 46, 1743–1758. (23) Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997) A signature motif in

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a) H2N

ELuc_N

LXXLL

LBD of ER

b) ELuc_N

ELuc_C

COOH

Luminescence

ELuc_C Estrogen

LXXLL LBD of ER Figure 1. Takenouchi et al.



a)

b)

Relative Luminescence Intensity


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80 60 40 20 0

LXXLL LHRLL LTNLL LESLL LRLLL

80 60 40 20 0 396 397 398 399

408 407 406

Figure 2. Takenouchi et al.


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80 6

60

4 2

40

0 DMSO

-10

-9

20 0

Log E2 [M]

Figure 3 Takenouchi et al.



a)

b)

BPA Dai Gen 10

E1

E2 E3 DES 1 -11

-10

-9 -8 Log ligand [M]

-7

-6

10 8 6 4 2 0

Ral OHT ICI AND T

100


Relative luminescence intensity

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Figure 4 Takenouchi et al.


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b) 1000

DES

100

R² = 0.9809 E2 E3

10

E1

1

Gen

0.1 Dai

0.01

BPA

0.001 1

100 10000 -10 EC5 (×10 M)

Relative binding affinity

a)

Relative binding affinity

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1000

DES

100

R² = 0.9545 E1

E2

10

E3

Gen

1 0.1

Dai BPA

0.01 1

100 10000 -10 EC5 (×10 M) Figure 5 Takenouchi et al



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TOC graphic Luminescence

Estrogen Indicator ELuc_N

Agonist

ELuc_C

LXXLL sequence

Antagonist Ligand binding domain of estrogen receptor

Takenouchi et al.


Bioluminescent Indicator for Highly Sensitive ... - ACS Publications

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