Split Gaussia Luciferase-Based Bioluminescence Template for

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Split Gaussia Luciferase-Based Bioluminescence Template for Tracing Protein Dynamics in Living Cells Sung Bae Kim,† Moritoshi Sato,‡ and Hiroaki Tao*,† Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan, and Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan The goal of the present study is to develop a small luminescence template, in which any protein may be incorporated, for illuminating the dynamics of protein signaling. We first determined optimal fragmentation sites of Gaussia princeps-derived luciferase (GLuc). The utility of the template was demonstrated with calmodulin (CaM) and a peptide-linked CaM, which were sandwiched between the fragments of split GLuc dissected at Q105. Living mammalian cells with the probe quickly emitted luminescence in response to endo- and exogenous Ca2+ levels. The applicability of the template was expanded to the visualization of the phosphorylation of the estrogen receptor and interaction of the androgen receptor with a peptide via an intramolecular complementation between GLuc fragments. It also provides the smallest bioluminescence template ever synthesized. Considering that conformational changes of proteins generally occur for signal transductions, the present luminescence template will contribute to the exploration of intracellular molecular events with signaling proteins. Intracellular protein-protein interactions are a central event for the generation of biological regulatory specificity. A majority of bioanalytical methods has been developed for the investigation of protein-protein interactions. Protein-fragment complementation assay (PCA) is one of the major strategies for determining protein-protein interactions in cell lines: Monomeric photoproteins such as GFP and luciferase can be split into two portions with resulting temporally inactive fragments. Any proteins of interest can be fused genetically to the split N- and C-terminal fragments. The interaction between the fused proteins triggers an approximation of the adjacent photoprotein fragments and subsequent recovery of the photoprotein function, which is taken as an analytical signature. Up until now, many functional proteins such as β-lactamase,1 dihydrofolate reductase,2 GFP,3 firefly * Corresponding author. E-mail: [email protected]. † National Institute of Advanced Industrial Science and Technology (AIST). ‡ The University of Tokyo. (1) Galarneau, A.; Primeau, M.; Trudeau, L. E.; Michnick, S. W. Nat. Biotechnol. 2002, 20, 619–622. (2) Pelletier, J. N.; Campbell-Valois, F. X.; Michnick, S. W. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12141–12146. (3) Magliery, T. J.; Wilson, C. G.; Pan, W.; Mishler, D.; Ghosh, I.; Hamilton, A. D.; Regan, L. J. Am. Chem. Soc. 2005, 127, 146–157. 10.1021/ac801658y CCC: $40.75  2009 American Chemical Society Published on Web 12/05/2008

luciferase,4 Renilla luciferase (RLuc),5-7 and Gaussia luciferase (GLuc)8 have been fragmented on the purpose. GLuc is the smallest known luciferase (20 kD) using coelenterazine and generates from 100- to 700-fold higher luminescence intensity than the luciferases from Photinus pyralis (firefly; FLuc) and Renilla reniformis (RLuc).8,9 Marin luciferases like GLuc and RLuc are less stable in light emission, whereas beetle luciferases such as FLuc emit relatively stable luminescence. Especially, the bursting activity of GLuc should be carefully manipulated to relieve the instability by using a microplate reader and an optimal substrate kit such as GAR2 (Nanolight). GLuc catalyzes the oxidation of the substrate, coelenterazine, that emits blue light (λmax ) 480 nm). The substrate, coelenterazine, easily permeates cell membranes and diffuses into all cellular compartments. On the other hand, coelenterazine interacts with multidrug resistance (MDR1)-type P-glycoproteins10 and is disadvantageous for applications in whole animal imaging in vivo owing to the poor tissue permeability of the emission light. The substrate may be somewhat metabolized in the host organisms, e.g., by binding P-glycoproteins. One of the main issues with PCA was the determination of the optimal dissection sites of luciferases for visualizing proteinprotein interactions between two molecules.5,8,11 The previous studies on genetic indicators has reported their optimal dissection sites based on known, positive interactions between two separated proteins. However, our review tests revealed that some of the known optimal cut sites are practically dull for other protein-protein interaction cases. Therefore, it is doubtful whether the known dissection site provides general applicability for illuminating interactions and temporal dynamics between unknown target proteins. (4) Luker, K. E.; Smith, M. C.; Luker, G. D.; Gammon, S. T.; Piwnica-Worms, H.; Piwnica-Worms, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12288– 12293. (5) Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2005, 77, 1295–1302. (6) Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2003, 75, 1584–1589. (7) Paulmurugan, R.; Umezawa, Y.; Gambhir, S. S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15608–15613. (8) Remy, I.; Michnick, S. W. Nat. Methods 2006, 3, 977–979. (9) Tannous, B. A.; Kim, D. E.; Fernandez, J. L.; Weissleder, R.; Breakefield, X. O. Mol. Ther. 2005, 11, 435–443. (10) Pichler, A.; Prior, J. L.; Piwnica-Worms, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1702–1707. (11) Kaihara, A.; Kawai, Y.; Sato, M.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2003, 75, 4176–4181.

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Figure 1. (A) Comparative illustration of the ligand recognition mechanisms of the present bioluminescent probes. Type I probes are characterized as an indicator based on second messenger-recognition. Type II represents a probe based on phosphorylation-mediated protein interactions. Type III models a nonphosphorylation-mediated protein-protein (peptide) binding. (B) Schematic diagram of constructs in the present study. The constructs are categorized in three groups, which, respectively, correspond to the probe types shown in part A. GLuc was dissected into two fragments, and CaM or M13-linked CaM was inserted between the fragments. Abbreviations: kz, Kozak consensus sequence; GLuc-N and -C, N- and C-terminal fragments of GLuc; M13, calmodulin-binding peptide of myosin light-chain kinase; Src SH2, Src homology 2 domain of v-Src, ER LBD, ligand-binding domain of estrogen receptor. (C) A cartoon diagram showing the Ca2+ recognition and conformation changes of SICA-5 and SICAM.

Here, we describe a generally applicable bioluminescence template that quickly visualizes protein dynamics related to cell signaling in living mammalian cells. It provides an optimal template for molecular imaging and is potentially applicable for visualizing (i) any temporal protein-protein interactions or (ii) unknown conformational changes of target proteins: Intracellular signal transduction is briefly catagorized in Figure 1A, where second messenger-mediated signaling (type I), phosphorylation-activated signaling (type II), and exogenous ligand-stimulated signaling (type III) were illustrated. For constructing the measure determining the three types of signaling pathways, we examined dissected fragments of marine copepod Gaussia princeps-derived luciferase (GLuc; 1-185 AA) (Figure 1B,C). We first searched optimal dissection sites for GLuc using a hydrophobicity prediction diagram, previously proposed by Kyte et al.12 This search predicted a hydrophilic region (85-106 AA; Supporting Information, Figure 2) in the middle of GLuc. Highly hydrophobic domains were found before and after the hydrophilic region. Therefore, we examined six cut sites in the hydrophilic region (85-106 AA) of GLuc. CaM (17 kD) was sandwiched between each pair of fragments (Figure 1B). As shown in Supporting Information, Figure 1A, the N- and C-terminal ends of CaM are adjacent in the absence of Ca2+, whereas the ends are spatially sequestered to the opposite sides in a single molecule by Ca2+ addition (single-molecule sandwiched; upper in Figure 1C). On the other hand, an insertion of CaM and a myosin lightchain kinase-derived peptide (M13) between the GLuc fragments gaps the N- and C-terminal fragments of GLuc in the single (12) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105–132.

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molecule. Ca2+-triggered binding between CaM and M13 shrinks the gap and results in the recovery of the luciferase activities (twomolecule sandwiched; bottom in Figure 1C). First of all, we determined the luminescence intensities from COS-7 cells carrying the probes to examine the validity of each dissection site (Figure 2B). These studies reveal that dissection site (Q105) is optimal for making the bioluminescence template in combination with Cam. The general applicability of the present luminescence template was proved by exploring ligand-activated dynamics of CaM, ligand-binding domains (LBDs) of estrogen receptor (ER LBD; 305-550 AA), androgen receptor (AR LBD; 672-910 AA), and glucocorticoid receptor (GR LBD; 527-777 AA). Thus, the results demonstrate that the luminesence template is optimal for potentially illuminating temporal dynamics of even unknown intracellular molecular events. EXPERIMENTAL SECTION Plasmid Construction. The cDNAs of N-terminal (GLuc-N) and C-terminal (GLuc-C) domains were amplified by polymerase chain reaction (PCR) to introduce each unique restriction site (HindIII/KpnI and BamHI/XhoI, respectively) at both ends of the domains using adequate primers and a template plasmid carrying the full-length cDNA of GLuc (pCMV-KDEL-Gluc-1; Nanolight Technology). The secretion signal sequence at the N-terminal region (1-17 AA) was excluded to minimize probe loss after expression. According to the domain length, the cDNA fragments were named GLuc-N1 (18-86 AA), GLuc-N2 (18-89 AA), GLucN3 (18-92 AA), GLuc-N4 (18-99 AA), GLuc-N5 (18-105 AA), GLuc-N6 (18-109 AA), GLuc-C1 (87-185 AA), GLuc-C2 (90-185

Figure 2. (A) Amino acid sequence of GLuc showing the dissection sites. (B) Comparison of luminescence intensities from the cells carrying one of following plasmids: from pSICA-3 to -6 or from pRef-1 to -2. These types of probes lose the luminescence intensities upon increase of the Ca2+ level as shown in the mechanism (see Figure 1C and Supporting Information, Figure 1A). To express the intensities explictly, the values were expressed in RLU (-)/RLU (+). The inset shows absolute luminescence intensities from each probe comprising the GLuc fragments that were cut at the dissection sites from no. 1 to no. 6. Section A shows a Western blot analysis. Lysates carrying SICA-3, -4, -5, and -6 were electrophoresed and blotted with mouse anti-GLuc (Nanolight) or anti-β-actin antibody (Sigma). (C) Time course of luminescence intensities from HeLa cells carrying pSica-5 in response to various concentrations of exogenous Ca2+. (D) Real-time bioluminescence imaging of Ca2+ dynamics in live cells carrying pSica-5. (E) Real-time monitoring of luminescence intensities from COS-7 cells carrying pSicam in response to ATP. (F) Real-time bioluminescence imaging of Ca2+ dynamics in live cells carrying pSicam in response to exogenous Ca2+ or ATP.

AA), GLuc-C3 (93-185 AA), GLuc-C4 (100-185 AA), GLuc-C5 (106-185 AA), and GLuc-C6 (110-185 AA). Two restriction sites (KpnI and BamHI) were introduced by PCR into the 5 and 3 ends of the Xenopus laevis calmodulin (CaM) gene, using a plasmid encoding Yellow Cameleon-3.1 (YC3.1)13,14 as a template. The restricted cDNA product encoding CaM was

ligated and subsequently cloned into the KpnI/BamHI sites of pcDNA 3.1(+) (Invitrogen). The 5 end of CaM was modified with enzymes, HindIII and KpnI, and subsequently fused with the cDNAs of N-terminal fragments of GLuc, whereas the 3 end of CaM was ligated with the cDNAs of C-terminal fragments of GLuc. The plasmids were, respectively, numbered from pSica-1 to pSica-6 Analytical Chemistry, Vol. 81, No. 1, January 1, 2009

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Table 1. Apparent Concentrations of Ca2+ Elevated by ATP in Mammalian Cells (n ) 3)a concentration of host cell average Ca2+ stimulator stimulator (mM) line concentration ATP ATP ATP a

1 1 1

MCF-7 COS-7 HeLa

standard deviation of Ca2+ ion

1.3 × 10-5 M 1.0 × 10-5 M 3.7 × 10-5 M 3.6 × 10-5 M ndb ndb

ATP: adenosine triphosphate. b nd: not determined.

according to the dissection site (Figure 2A). The luminescent probe expressed from each plasmid was, respectively, named SICA-1-6. The cDNA of CaM in pSica-5 was replaced with that of CaM fused to M13, a myosin light-chain kinase-derived peptide, and the subsequent plasmid was named pSicam (Figure 1B). Reference plasmids, pRef-1 and pRef-2, were constructed for comparing the conventional dissection site of firefly luciferase (FLuc) and the present sites of GLuc. It is previously reported that dissection at D415/G416 of FLuc can be a choice for PCA.5,15 cDNAs of CaM or M13-linked CaM were sandwiched between the cDNAs of N- and C-terminal fragments of FLuc dissected at D415/G416. The constructs were, respectively, subcloned in pcDNA 3.1(+) vector and named pRef-1 and pRef-2. To show the general advantage of fragments dissected at site no. 5 (Q105), we designed new constructs containing cDNAs encoding ligand-binding domain (LBD) of representative steroid hormone receptors such as androgen receptor (AR), glucocorticoid receptor (GR), and estrogen receptor (ER). cDNA encoding CaM in pSica-5 was replaced with that encoding leucine-rich peptide-linked AR LBD, LXXLL motif-fused GR LBD, or Src SH2 domain-fused ER LBD. The new plasmids were, respectively, named pSimar, pSimgr, pSimer-1, and pSimer-1m. The specific peptide sequences were summarized in Table 2. The constructed plasmids were sequenced to ensure fidelity with a BigDye Terminator Cycle Sequencing kit and a genetic analyzer ABI Prism310 (Applied Biosystems). Decision on Optimal Dissection Sites of GLuc Applicable to an Integrated-Molecule Format (IMF). We preliminarily examined the recovery of luciferase activities triggered by an approximation of various N- and C-terminal fragments via an intramolecular complementation (Figure 2B). African green monkey kidney fibroblast-derived COS-7 cells were cultured on 24-well plates with Dulbecco’s modified eagle’s medium (DMEM; Sigma) supplemented with 10% steroid-free fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) at 37 °C in a cell incubator maintaining 5% CO2 (Sanyo). COS-7 cells on the 24well plate were transfected with one of the pSica-series plasmids, from pSica-1 to -6 (0.2 µg per well) or pRef series plasmids using a transfection reagent, TransIT-LT1 (Mirus). The cells were extensively incubated in the CO2 incubator for 16 h. The live cells in each well were stimulated for 3 min with vehicle (0.1% PBS) or 1 mM ATP and harvested by trypsinization. The luminescence intensities in the presence of the specific substrate, coelenterazine, (13) Miyawaki, A.; Llopis, J.; Heim, R.; McCaffery, J. M.; Adams, J. A.; Ikura, M.; Tsien, R. Y. Nature 1997, 388, 882–887. (14) Truong, K.; Sawano, A.; Mizuno, H.; Hama, H.; Tong, K. I.; Mal, T. K.; Miyawaki, A.; Ikura, M. Nat. Struct. Biol. 2001, 8, 1069–1073. (15) Kim, S. B.; Kanno, A.; Ozawa, T.; Tao, H.; Umezawa, Y. ACS Chem. Biol. 2007, 2, 484–492.

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were recorded for 15 s with a luminometer (Minilumat LB9506; Berthold). Because luminescence intensities were decreased, the luminescence change was expressed with RLU (-)/ RLU (+), where RLU (+) and RLU (-), respectively, show luminescence intensity in the presence or absence of ATP. Independently, the absolute luminescence intensities from SICA-1 to -6 were examined to evaluate whether the probes are stable in mammalian cells and conserve the enzymatic activity in spite of the fragmentation of GLuc (inset of Figure 2B). The cells carrying each plasmid were lysed with a specific lysis buffer, and the absolute luminescences were determined with the luminometer in the presence of the specific substrate coelenterazine. Calcium Ion Titration Curve of SICA. The response of SICA-5 to varying concentrations of Ca2+ was examined in living mammalian cells (Figure 2C). The results were also shown in dose-response curves (Supporting Information, Figure 1B). COS-7 (African green monkey kidney fibroblast), MCF-7 (human breast carcinoma), or HeLa (human cervical carcinoma) cells were, respectively, cultured in glass bottom plates (black, 24-well) up to 90% confluent. They were, respectively, transfected with pSica-5 and subsequently incubated for 16 h. The cells were washed twice with PBS, and the cells were saturated with 200 µL of PBS supplemented with 2 µM ionomycin (final concentration). Various concentrations of Ca2+ were then simultaneously injected into each well of the 24-well plate. The luminescence intensities on each well were accumulated for 5 s with a biolumunescence plate reader. The negative control was obtained by mockstimulation of the cells with the vehicle. The luminescence changes (∆RLU) according to Ca2+ levels were plotted in the form of a dose-response curve, i.e., relative % of ∆RLU vs Ca2+ levels. Real-Time Bioluminescence Imaging of Ca2+ Fluctuations in Live Cells. Fluctuation of luminescence intensities from cells carrying pSica-5 was observed real-time in the presence of various stimulators (Figure 2C,D). COS-7 cells raised in a glass bottom plate (black, 24-well) were transfected with pSica-5 and subsequently incubated for 16 h. The cells were washed twice with PBS and saturated with 200 µL of HBSS buffer comprising 1.3 mM Ca2+ (final concentration) and coelenterazine. The media were then supplemented with 2 µM ionomycin, and 1 mM histamine (final concentration) or 1 mM ATP (final concentration) was finally added. Changes in luminescence were monitored every 15 s with a bioluminescence plate reader (Mithras LB 940; Berthold). Fluctuation of luminescence intensities by SICAM after sensing Ca2+ was similarly monitored with living COS-7 cells carrying pSicam (Figure 2E,F). Changes in luminescence were recorded every 15 s with the bioluminescence plate reader. Phosphorylation Recognition of SIMER-1. Tyrosine phosphorylation of ER LBD at Y537 was recognized with cofused Src SH2 domain in the present luminescence template (Figure 3A). The COS-7 cells raised in 24-well plates were transiently transfected with pSimer-1 or pSimer-1m and incubated for 16 h. The cells were stimulated for 20 min with varying concentrations of 17β-estradiol (E2) or 4-hydroxytamoxifen (OHT). The cells were then lysed for 10 min with a specific lysis buffer (Promega), and the luminescence intensities were determined with a luminometer (Minilumat LB9506; Berthold) in the presence of the specific substrate coelenterazine.

Figure 3. (A) Ligand sensitivity of SIMER-1 and SIMER-1M recognizing intramolecular phosphorylation. The graphs show dose-response curves of SIMER-1 and SIMER-1 M to E2 and OHT. Inset shows relative ligand sensitivity of SIMER-1. (B) Ligand sensitivity of SIMAR probing steroids. Graphs show dose-response curves of the cells carrying pSimar in response to ligands. (C) Dose-response curves of the cells carrying pSimgr in response to ligands. Table 2. Amino Acid Sequences of Proteins and Peptides Used in the Present Study name

amino acids

myosin light chain kinase-derived M13 leucine-rich peptide for AR LBD GRIP1 NID3-derived LXXLL motif for GR LBD optimal N-terminal fragment of GLuc

KRRWKKNFIAVSAANRFKKISSSGAL 50 PGASLLLLQQ59 742 NALLRYLLDKD752 18 KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICL SHIKCTPKMKKFIPGRCHTYEGDKESAQ105 106 GGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQR CATFASKIQGQVDKIKGAGGD185

optimal C-terminal fragment of GLuc

The relative ligand selectivity of SIMER-1 was estimated with COS-7 cells carrying pSimer-1 (Figure 3A inset). COS-7 cells carrying pSimer-1 were stimulated for 20 min with 10-6 M of E2, OHT, estrone, DHT, and cortisol (final concentration). The cell lysates were supplemented with coelenterazine and the subsequent luminescence intensities determined with the luminometer. Steroid Hormone Sensitivity of SIMAR and SIMGR. Steroid hormone sensitivities of SIMAR and SIMGR were examined with COS-7 cells carrying pSimar or pSimgr (Figure 3B,C). COS-7 cells cultured in 24-well plates were transfected with pSimar or pSimgr and subsequently incubated for 16 h in a cell incubator. The cells were stimulated for 20 min with varying concentrations of steroids, and the recovered luciferase activities were recorded with the luminometer after supplementation of the substrate coelenterazine. RESULTS AND DISCUSSION Consideration of the Reversibility of the Present IMF Probes. Our key criterion of design of the present probe rendering an intramolecular protein complementation was that it should be reversible so that Ca2+ levels can be quantitatively measured. The Ca2+-induced CaM-M13 binding itself is a naturally occurring reversible interaction. However, we should consider that modification of them with GLuc fragments in a single molecule may affect the sensing property to Ca2+. The molecular mechanisms of protein complementation between GLuc fragments were previously demonstrated by Michnick et al.8,16 They discussed two models to explain how protein complementation assays (PCA) would be reversible. In their most likely model, the possible equilibrium distribution states of the PCA are in a complete cycle, where the free-energy landscape among the states changes. An irreversible association state of the (16) Michnick, S. W.; Ear, P. H.; Manderson, E. N.; Remy, I.; Stefan, E. Nat. Rev. Drug Discovery 2007, 6, 569–582.

PCA is highly unlikely as it would require the spontaneous folding between the GLuc fragments. According to our observation, the GLuc fragments in the present IMF probes did not exhibit a spontaneous complementation and did make a robust doseresponse curve to stimulations. This observation suggests that a basal intramolecular complementation itself would not invade the reversibility and quantitativity of the host probe. In addition, it is previously demonstrated that the orientation and distance between reporter fragments should be matched within 8 Å for protein complementation (for lighting “on”).17 This gap is too short to concern a nonspecific protein complementation even in an IMF probe. The gap between N- and C-termini of CaM is enlarged up to 32.51 Å upon Ca2+ binding (for lighting “off”). Considering the large gap and the rigidity of R-helical CaM, the present IMF probe should admit a large spatial margin enough to exert an efficient on-off system. Optimal Dissection Site for the Present Luminescence Template in the Interface between the Hydrophobic and Hydrophilic Sequences of GLuc. The general applicability of the present probes was first examined with GLuc and CaM. The distance between the N- and C-terminal ends of CaM varied, depending on the presence or absence of Ca2+ (Figure 1C and Supporting Information, Figure 1A). To make use of the conformational change of CaM in real-time imaging, we first examined six-dissected fragment pairs of GLuc that provide stable and reproducible luminescence via intramolecular complementation (Figure 2A,B). The dissection sites were decided with a hydrophobicity prediction search reporting a hydrophilic region (85-110 AA; Supporting Information, Figure 2) in the middle of GLuc. Highly hydrophobic domains were found before and after the hydrophilic region. The probes for illuminating cytosolic Ca2+ were named single-molecule-format bioluminescent indicator for imaging calcium ions (SICA). (17) Michnick, S. W. Curr. Opin. Struct. Biol. 2001, 11, 472–477.

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The experiment in the inset of Figure 2B showed no restoration of luminescence intensity from SICA-1 to -3, whereas recovery of high luminescence was exhibited from SICA-4 to -6. The probe levels were examined after the overexpression in COS-7 cells. Apparent variance in the expression levels of probes was not observed with a Western blot analysis as shown in Figure 2B. These results suggest (i) how dissection sites are significant in recovery of luciferase activity in the IMF probes, and (ii) additional studies may reveal the reason of the present all-or-none nature of the luciferase activity. An experiment for ligand sensitivity of SICA-5 revealed that fragments dissected at the Q105/G106 of GLuc (site no. 5) exhibited maximum variance of luminescence intensities in the presence or absence of ATP. On the other hand, the previously known fragments of FLuc (D415/G416)15 did not exert notable luminescence variance upon fusion with CaM or a CaM-binding peptide of myosin light chain kinase (M13)-linked CaM (see REF-1 and -2 in Figure 2B). The background luminescence intensities by REF-1 and -2 were approximately 15 times higher than those with SICA-5. These results may be interpreted thus: Although the fragments of FLuc were intercalated by CaM, a basal complementation may be easy to occur, and thus appeared in high background luminescence. On the other hand, the fragments of GLuc were small enough not to invade the proper binding property between CaM and M13. In addition to the present nonoverlapping fragments (dissecting at Q105), several overlapping fragments of luciferases were introduced by PiwnicaWorms and Gambhir et al.18,19 Some of the fragments were examined similarly but did not exhibit a considerable merit for the present IMF probes (data not shown). The results in Figure 2B provide new insights on decisions regarding dissection sites of GLuc. Dissection site no. 6 (G109/ E110) was previously proven to be the most potential cutting point in GLuc upon determination of interactions between two molecules.8 The authors showed that even a trivial shift of the dissection site dramatically decreases the luminescence intensities. Their optimal dissection site was surprisingly not suitable for our IMF probing system, apparently owing to the high background luminescence intensities. On the basis of these results, we compared the hydrophobicity of the peptides near the dissection sites (Supporting Information, Figure 2). Dissection site no. 6 is in the middle of a hydrophobic region, whereas the present site no. 5 is just in the interface between the hydrophilic and hydrophobic regions. A previous optimal dissection site of FLuc was also in the interface of the regions.15 There is no doubt that the interface region is critical in the recovery of luminescence intensity. The high background luminescence of SICA-6 may be explained by a nonspecific interaction between the hydrophobic regions at the dissection sites of the fragments. Dynamics of Cytosolic Calcium Ions Immediately Traced with SICA-5. The sensitivity of SICA-5 to exogenous Ca2+ was examined in various mammalian cells (Figure 2C and Supporting Information, Figure 1B). The culture cell lines, HeLa, COS-7, and MCF-7 cells were first saturated with PBS comprising coelenterazine. An addition of 2 µM ionomycin alone did not influence luminescence intensities from the cells carrying pSica-5. However, subsequent (18) Luker, K. E.; Smith, M. C.; Luker, G. D.; Gammon, S. T.; Piwnica-Worms, H.; Piwnica-Worms, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12288– 12293. (19) Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2005, 77, 1295–1302.

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supplementation of Ca2+ ion quickly changed the luminescence intensities in a dose-dependent manner. The detectable concentration range was from 10-6 to 10-3 M Ca2+(Supporting Information, Figure 1B). The dose response curves, [Ca2+] vs ∆RLU, exhibited a high similarity with the characteristic biphasic Ca2+-binding curves of previous FRET-based studies.13,14 The apparent effective concentrations 50% (EC50) of SICA-5 were 0.25 × 10-4 M (COS-7) and 0.50 × 10-4 M (HeLa). The apparent dissociation constant (Kd) between CaM and Ca2+ in HeLa cells was 0.6 × 10-17 M4 (i.e., average of 5.0 × 10-5 M). This Kd value is included in the reported range by previous probes, i.e., from ∼0.2 µM to 0.5 mM.20 Variation among fusion protein probes may be caused by the modification of CaM with split-reporter proteins. It has been previously proven that Mg2+, pH, ionic strength, and hydrophobic proteins such as serum albumin do not alter the high sensitivity of CaM to Ca2+. 13,21 On the basis of the calibration curves, we determined endogenous concentrations of Ca2+ elevated by ATP in living mammalian cells (Table 1). ATP is a purinergic agent elevating endogenous Ca2+ levels via ATP receptors on the plasma membrane.13,22 This evaluation was possible because an addition of ATP quickly activates the ATP receptor on the plasma membrane of COS-7 cells. The subsequent elevation of the inositol trisphosphate (IP3) level triggers the outflux of Ca2+ from the endoplasmic reticulum (ER) to the cytosol. The elevation of Ca2+ was finally recognized by SICA-5 emitting bioluminescence. Up until now, Ca2+ levels have been determined with fluorescence microscopes at a specific site of a single cell. The present bioluminescent probe determined 1.3 × 10-5 M Ca2+ in MCF-7 and 3.7 × 10-5 M Ca2+ in COS-7 after stimulation with 1 mM ATP. The present values are consistent with conventionally determined Ca2+ levels ranging from 0.3-1.5 × 10-5 M after stimulation.23,24 Exogenous Ca2+ alone did not influence luminescence variance (Figure 2D). The addition of ionomycin induced the influx of exogenous Ca2+ into the cytosol and thus changed conformation of CaM inside SICA-5 triggering a dissociation of the basal fragment complementation. The luminescence intensities decreased again with a subsequent addition of histamine or ATP. Corresponding experiments were also conducted with a plasmid comprising two components, i.e., M13-linked CaM, between the fragments of the optimal GLuc template, i.e., pSicam (Figure 2E,F). Stimulation with 1 mM ATP enhanced the luminescence intensities from COS-7 cells carrying pSicam. The intensities reached to the plateau in ∼2.5 min. The results are summarized thus: (i) both SICA-5 and SICAM recognized exo- and endogenous Ca2+ levels and emitted bioluminescence in a dose-dependent manner, and (ii) the fragments of GLuc dissected at Q105 are useful for visualizing ATP-triggered Ca2+ dynamics upon combination with either CaM (single component) or M13-fused CaM (two components). (20) Zou, J.; Hofer, A. M.; Lurtz, M. M.; Gadda, G.; Ellis, A. L.; Chen, N.; Huang, Y.; Holder, A.; Ye, Y.; Louis, C. F.; Welshhans, K.; Rehder, V.; Yang, J. J. Biochemistry 2007, 46, 12275–12288. (21) Falke, J. J.; Drake, S. K.; Hazard, A. L.; Peersen, O. B. Q. Rev. Biophys. 1994, 27, 219–290. (22) Smit, M. J.; Leurs, R.; Bloemers, S. M.; Tertoolen, L. G.; Bast, A.; De Laat, S. W.; Timmerman, H. Eur. J. Pharmacol. 1993, 247, 223–226. (23) Ganitkevich, V.; Hirche, H. Cell Calcium 1996, 19, 391–398. (24) Ganitkevich, V. Cell Calcium 1998, 23, 313–322.

Tyrosine Phosphorylation of ER LBD at Y537 Recognized with Its Cofused Src SH2 Domain Inside the Luminescence Template. The general applicability of the luminescence template was examined by replacing CaM in SICA-5 with Src SH2-fused ER LBD (SIMER-1; Figure 3A). 17β-estradiol (E2) and 4-hydroxytamoxifen (OHT) can phosphorylate ER LBD at Y537. As a negative control, a probe comprising a mutated form of ER LBD (Y537F) was examined in parallel (SIMER-1M). SIMER-1 sensitively elevated the luminescence intensities in response to E2 and OHT. The luminescence intensities by OHT were notably increased from 10-8 M, plateauing at 10-6 M. E2 also enhanced the luminescence intensities, which were especially sharp between 10-6 and 10-5 M. On the other hand, point mutation of ER LBD at Y537 significantly reduced the ligand sensitivity of SIMER-1 to both E2 and OHT. The relative selectivity of SIMER-1 to ligands was compared at 10-6 M (Figure 3A, inset). Selectivity in decreasing order was as follows: OHT > E2 > estrone > cortisol ) 5R-dihydrotestosterone (DHT). These results show that (i) phosphorylation of a protein can be imaged with the present luminescence template through intramolecular complementation and that (ii) ligand-activated ER phosphorylation at Y537 can be discriminated by SIMER-1. Whether ligands phosphorylate ER has been the subject of debates. Arnold et al. reported that (i) phosphorylation of Tyr537 of hER is regulated by c-Src and occurs independent of estrogen and that (ii) phosphorylation of Tyr537 increases estradiol-binding capacity.25,26 On the other hand, Migliaccio et al. demonstrated that E2 phosphorylates ER and stimulates the Src/Ras/Erk pathway of cancer cell lines.27 Here, we give evidence that both OHT and E2 can phosphorylate ER LBD at Y537 with a similar efficiency. Steroids Recognized with Specific Receptors Inside the Luminescence Template. To demonstrate the general usefulness of the present template, we replaced CaM with AR LBD or GR LBD in the backbone of SICA-5 (Figure 3B,C). COS-7 cells carrying pSimar or pSimgr, respectively, exhibited 10 or 14 times enhanced bioluminescence upon stimulation of the agonist. SIMAR and SIMGR exhibited characteristic electivity to ligands and sensitivity to the 10-7 M agonist. These results show that the dissection point, Q105, in GLuc is generally useful for constructing IMF probes. Insertion of One or Two Components between GLuc Fragments. The present Sica series probes comprise one component between the fragments of GLuc. In addition, the general applicability of the luminescence template used in SICA-5 was examined with a new probe, in which two components, M13linked CaM, were included between the fragments of GLuc (Figure 2E,F). In contrast to the Sica series probes, SICAM comprising two internal interacting components enhanced luminescence intensities in response to exo- and endogenous Ca2+. This type of probe can minimize interferences by cytosolic proteins because a protein in the probe interacts preferentially with its adjacent protein component. The intensities reached a plateau in (25) Arnold, S. F.; Vorojeikina, D. P.; Notides, A. C. J. Biol. Chem. 1995, 270, 30205–30212. (26) Arnold, S. F.; Melamed, M.; Vorojeikina, D. P.; Notides, A. C.; Sasson, S. Mol. Endocrinol. 1997, 11, 48–53. (27) Migliaccio, A.; Castoria, G.; Di Domenico, M.; de Falco, A.; Bilancio, A.; Lombardi, M.; Bottero, D.; Varricchio, L.; Nanayakkara, M.; Rotondi, A.; Auricchio, F. J. Steroid Biochem. Mol. Biol. 2002, 83, 31–35.

2.5 min. This response time includes all the time for (i) ligand sensing, (ii) elevation of IP3 and Ca2+ levels, and (iii) rearrangement of the internal components, including intramolecular complementation inside the probe as illustrated in Figure 1C. This response time is exactly the same as the 2.5 min of Yellow Cameleon-3er (YC3er) comprising CaM and M13 (FRET-based; located in the cytosol)13 and is more rapid than the 4.3 min of Fllip containing PIP3-recognition domain (FRET-based; localized on the plasma membrane).11 The rapid response time in case of SICA-5 to ATP, less than 1 min, should be also noted as a merit of this probe. It may be benefited by the simpler structure of SICA-5. Inter- or Intramolecular Protein Complementation. Most of the recent studies on protein complementation have proved its merits with known positive interactions between two separated proteins.5,8,11,15 For instance, it was demonstrated previously that GLuc has an optimal dissection site at G109.8 However, because they used positively interacting proteins, it is still unclear whether their dissection sites provide general applicability for illuminating protein-protein interactions of unknown target proteins. At least in our review studies on its application to intramolecular complementation, their optimal fragmentation site caused high background luminescence, even in a basal condition, and subsequent poor signal-to-background ratios (compare SICA-5 and -6 in Figure 2B). This comparison may be explained as follows: Interactions between two separated proteins need a relatively strong affinity to recognize each other in the complex context of mammalian cells. However, two components inside a single fusion protein are adjacent. Therefore, their binding affinity should be weak enough to suppress the basal interactions. It means that optimal dissection sites between single- and two-molecule-format probes cannot be equal. There is another example for this issue. Paulmurugan et al. previously suggested several potential dissection sites for FLuc on the basis of a positive interaction between two separated proteins.5 However, one of their worst dissection sites (D415) was practically found to be an optimal cut point for intramolecular complementation.15 The two-molecule-format probes provide maximal stoichiometric efficiency in sensing ligands when the two components are equally expressed beforehand. Biased expressions of the fusion proteins inevitably cause inefficiency. On the other hand, singlemolecule-format bioluminescent probes are a characteristic recombinant protein, in which all the components for ligand sensing and signal development were integrated. This method provides a straightforward measure of protein-protein interactions with an exact stoichiometry and relieved probability of nonspecific binding. It is unclear whether two-molecule-format probes illuminate protein-protein interactions even in cell-free conditions. At least nuclear trafficking of a target protein, previously explored with a two-molecule-format probe,28 is impossible to be imaged in a cellfree condition. On the other hand, the present IMF probe comprises all the components for signal sensing and light emission inside the fusion. Thus, a conformational change of the fusion protein is directly connected to light emission. The intrinsic merit of the present IMF probe may enable researchers to image an (28) Kim, S. B.; Ozawa, T.; Watanabe, S.; Umezawa, Y. Proc. Natl. Acad. Sci.U.S.A. 2004, 101, 11542–11547.

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intracellular protein-protein recognition process, even in cell-free conditions. This property of an IMF probe was explicitly demonstrated in a previous paper with a bioluminescence strip sensing estrogenicity of chemicals.29 Protein Size Issue. The present approach provides the smallest bioluminescent probe ever developed. The total molecular weight (MW) of SICA-5 is merely 41 kDa, which is even smaller than the single molecule of click beetle luciferase (64 kDa) or firefly luciferase (61 kDa). Molecular probes based on FRET should comprise two GFP variants weighing each 27 kDa. The small size of GLuc minimizes steric hindrance to the adjacent target proteins. In addition, the compact size of the present probes is beneficial for targeting precisely specific intracellular locations and for distributing uniformly in both cytosolic and nuclear compartments, as expected of a protein less than 60 kDa to distribute freely into the nucleus. Taken together, we presented the synthesis of a luminescence template that quickly visualizes protein dynamics in living mammalian cells. The present imaging system provides an optimal luminescent measure with general applicability for visualizing the temporal dynamics, protein-protein interactions, and conformational change of proteins of interest, which was exemplified with an intracellular Ca2+ fluctuation and steroid-receptor binding. We took advantage of intracellular complementation as its on-off system, in addition to the general merits of GLuc: the probes were stable in mammalian cells and provided high-efficiency recognition (29) Kim, S. B.; Umezawa, Y.; Kanno, K. A.; Tao, H. ACS Chem. Biol. 2008, 3, 359–372.

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to ligands. These probes are characterized as a bioluminescent indicator where all component proteins for ligand sensing and signal development were integrated in the single molecular backbone. With this study, we achieved several important advances in molecular imaging technology: (i) a generally applicable IMF probe template was constructed for illuminating conformation changes of proteins; (ii) dynamics of cytosolic Ca2+ was traced with a bioluminescent probe in living mammalian cells; (iii) the smallest ever bioluminescent probe was developed with enhanced luminescence intensity and signal-to-noise ratio; (iv) potential dissection sites for IMF probes were specified; and (v) endogenous Ca2+ levels in living mammalian cells were specified. All human sense organs comprise highly specialized cell lines for recognizing outer signals such as rod photoreceptor cells and olfactory receptor neurons. Considering that conformational changes of proteins are general for signal transductions, for instance, in the plasma membrane of sense organ cells, the present luminescence template will contribute to the exploration of intracellular molecular events with signaling proteins. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 9, 2008. Accepted October 23, 2008. AC801658Y