Chemically Modified Firefly Luciferase Is an Efficient Source of Near

Bioconjugate Chem. 2010, 21, 2023–2030

2023

Chemically Modified Firefly Luciferase Is an Efficient Source of Near-Infrared Light Bruce R. Branchini,* Danielle M. Ablamsky, and Justin C. Rosenberg Department of Chemistry, Connecticut College, New London, Connecticut 06320, United States. Received June 5, 2010; Revised Manuscript Received September 14, 2010

Bioluminescence and bioluminescence resonance energy transfer (BRET) are two naturally occurring light emission phenomena that have been adapted to a wide variety of important research applications including in vivo imaging and enzyme assays. The luciferase enzyme from the North American firefly, which produces yellow-green light, is a key component of many of these applications. Recognizing the heightened interest in the potential of nearinfrared (nIR) light to improve these technologies, we have demonstrated that spectral emissions with maxima of 705 and 783 nm can be efficiently produced by a firefly luciferase variant covalently labeled with nIR fluorescent dyes. In one case, an outstanding BRET ratio of 34.0 was achieved emphasizing the importance of selective labeling with fluorescent dyes and the efficiency provided by the intramolecular BRET process. Additionally, we constructed a biotinylated fusion protein that similarly produced nIR light. This novel material was immobilized on solid supports containing streptavidin, demonstrating, in principle, that it may be used for receptor-based imaging. Also, the matrix-bound labeled fusion protein was used to measure factor Xa activity at physiological concentrations in blood. We believe this to be the first report of bright nIR light sources produced by chemical modification of a beetle luciferase.

INTRODUCTION A wide variety of biological organisms emit light spanning the visible spectrum. Bioluminescence emission maxima ranging from 450 to 625 nm have been recorded (1) for species of crustaceans and beetles. Moreover, the 705 nm peak luminescence emanating from the light organs of several dragonfish (2) extends bioluminescence beyond human vision into the near-infrared (nIR) 700-1000 nm. In the dragonfish, the emitter is likely a secondary pigment (2) excited via bioluminescence resonance energy transfer (BRET), the natural process responsible for the green glow of the aequorin-GFP complex (3, 4). BRET-based assays, in which a bioluminescence donor excites a fluorescent acceptor, have been developed for a variety of applications (5) including measuring protease activity (6) and detecting protein-protein interactions (7-9). In the field of in vivo imaging, there is heightened interest in the nIR region because it can enable deeper imaging owing to lower background and minimized signal loss from light absorption and scattering (10). Promising BRET-based imaging results have been reported with quantum dot (QD)-Renilla luciferase (rLuc) conjugates (11, 12), including a recently described bioinorganic hybrid structure that emits at ∼800-1050 nm (13), and with a Cypridina luciferase (cLuc) containing fusion proteins modified with an indocyanine dye emitting at 675 nm (14). Our interest is in developing new biomaterials with photonic properties that may be suitable for improved biomarker and biosensor applications focusing on the firefly luciferase system from Photinus pyralis (Luc). The firefly enzyme produces yellow-green light (Figure 1a) through a series of reactions that require substrates firefly (beetle) luciferin (LH2), Mg-ATP, and oxygen. This process is generally considered to have the * Corresponding author. Bruce Branchini, Department of Chemistry, Connecticut College, 270 Mohegan Avenue, New London, CT 06320. Ph: +1 860 439 2479, Fax: +1 860 439 2477, E-mail: brbra@ conncoll.edu.

highest known quantum yield (41 ( 7.4%) (15) of any bioluminescence system based on the conversion of substrate (luciferin) into photons. Additional advantages of working with Luc are the superior stability and relatively low cost of LH2. While notable applications of BRET-based red sources have been reported (11, 12, 14), we have developed Luc-based BRET systems with greater BRET efficiencies that provide improved signal-to background ratios. The efficiency of BRET is dependent on the spectral overlap, relative orientation, and the distance between the bioluminescence donor and the fluorescence acceptor. We set out to determine whether the Luc system could be used to produce efficient BRET-based sources of nIR light. Our strategy was to optimize spectral overlap with the longwavelength acceptor absorption spectra typical of nIR fluorescent dyes by starting with a recombinant P. pyralis luciferase containing the mutations Thr214Ala, Ala215Leu, Ile232Ala, Ser284Thr, Phe295Leu, Arg330Gly, Ile351Val, and Phe465Arg (Ppy RE8), our recently developed (16) thermostable Luc variant, which emits red light (617 nm) (Figure 1b). We investigated two acceptors, the Alexa Fluor nIR dyes AF680 and AF750 with excitation maxima of 680 and 750 nm and emission maxima of 705 and 780 nm, respectively. Using versions of these dyes containing maleimide groups, we covalently attached the nIR dyes to Ppy RE10, a variant of Ppy RE8 containing two surface Cys residues at positions 169 and 399, and achieved highly efficient nIR emission by an intramolecular BRET process that minimized donor-acceptor distance. We report here the design, construction, and characterization of soluble and immobilized Luc-based sources of nIR light. Additionally, we demonstrate that the novel materials can be applied to the assay of factor Xa activity and we provide information from model studies with blood designed to assess the potential advantage of nIR transmittance. The measurement of factor Xa activity has become increasingly common in clinical laboratories, because it is an effective means to monitor heparin

10.1021/bc100256d  2010 American Chemical Society Published on Web 10/11/2010

2024 Bioconjugate Chem., Vol. 21, No. 11, 2010

Figure 1. Normalized bioluminescence and BRET emission spectra of (a) Luc, (b) Ppy RE8, and Ppy RE10 labeled with (c) AF680 or (d) AF750. Reactions were initiated by the addition of LH2 and Mg-ATP at pH 7.8. Ribbon diagrams are based on a Luc-DLSA crystal structure (Branchini, B. R., Southworth, T. L., and Gulick, A. M. Unpublished results) (25) showing mutated residues (green) and DLSA as CPK models.

anticoagulant therapy used for treatment and prevention of thromboembolic disease (17).

EXPERIMENTAL PROCEDURES Materials. The following materials were obtained from the sources indicated: Mg-ATP (bacterial source) from SigmaAldrich (St. Louis, MO); Alexa Fluor 680 C2-maleimide (AF680) and Alexa Fluor 750 C5-maleimide (AF750) dyes from Invitrogen (Carlsbad, CA); NanoLinK streptavidin magnetic microspheres from SoluLinK Biosciences (San Diego, CA); and Streptavidin agarose resin from Thermo Scientific (Rockford, IL). The recombinant GST-fusion proteins Ppy RE8 (16) and Ppy RE10 were expressed and purified as previously reported (16, 18-21). LH2 was a generous gift from Promega (Madison, WI), and the pET-KBPT-Luc plasmid (22) was a generous gift from E. S. Yeung, Iowa State University. General Methods. Concentrations of unlabeled proteins were determined using the Bio-Rad Protein Assay system using BSA as the standard. Unless otherwise specified, specific activity measurements, heat inactivation studies, and steady-state kinetics constants were determined as previously reported (18, 19, 21, 23). Mass spectral analyses were performed by tandem HPLCelectrospray ionization mass spectrometry (LC/ESMS) using a ThermoFinnigan Surveyor HPLC system and a ThermoFinnigan LCQ Advantage mass spectrometer. The found molecular masses (Da) of the newly reported luciferase variants were within the allowable experimental error (0.01%) of the calculated

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values (in parentheses): Ppy RE8-Thr169Cys, 61000 ( 6 (60998); Ppy RE8-Phe368Cys, 60961 ( 6 (60956); Ppy RE8Ser399Cys, 61021 ( 6 (61016); and Ppy RE10, 61020 ( 6 (61014). Covalent Labeling of Luciferase Enzymes with Alexa Fluor Maleimide Dyes. Stock solutions (10 mM, determined by UV-vis spectroscopy) of the AF680 (ε684 nm ) 175 000 M-1 cm-1) and AF750 (ε753 nm ) 290 000 M-1 cm-1) dyes (24) were prepared in sterile deionized water, divided into 30 µL aliquots, lyophilized, and stored at -20 °C. All labeling reactions were performed at 10 °C in 20 mM sodium phosphate buffer, pH 7.2 containing 150 mM NaCl, 5 mM EDTA and 0.8 M ammonium sulfate (PBSA buffer). Using Slide-A-Lyzer 10K MWCO dialysis cassettes (Thermo Scientific, Rockford IL), stock luciferase solutions (∼2 mg/mL) in 20 mM Tris-HCl (pH 7.0) containing 150 mM NaCl, 1 mM EDTA, and 1 mM DTT were dialyzed against PBSA buffer (5 changes, 1 L each) to remove DTT and introduce 0.8 M ammonium sulfate. The inclusion of ammonium sulfate in PBSA effectively reduced noncovalent dye incorporation that can result from hydrophobic interactions between the dyes and the proteins. Labeling reactions were initiated by the addition of 1 mL of 30 µM enzyme in PBSA buffer to a lyophilized aliquot of AF680 or AF750 (final concentration 300 µM) and gently mixed for 15 min. Reactions were quenched by addition of 20 µL of 100 mM glutathione in PBSA buffer, incubated for 15 min, and dialyzed against PBSA buffer (8 changes, 1 L each). The nIR dye-labeled proteins could be stored in PBSA buffer at 5 °C for at least 6 weeks without the loss of more than 10% activity. For long-term storage, labeled and unlabeled proteins were dialyzed into 20 mM sodium phosphate buffer, pH 7.2, containing 150 mM NaCl and 5 mM EDTA, flash-frozen in liquid N2, and stored at -80 °C with complete retention of activity. Determination of Fluorescent Dye Labeling Stoichiometry. The degree of nIR dye labeling was estimated by UV-vis spectroscopy by measuring the micromoles of dye (using the molar absorptivity coefficients above) bound to micromole samples of labeled luciferases. A Micro BCA Protein Assay Kit (Thermo Scientific, Rockford IL) was used to determine the quantity of enzyme with readings taken at 540 nm to avoid interference with dye absorption bands. Additionally, LC/ESMS analysis of the labeled luciferases was performed using a BioBasic-C4 (100 × 1 mm) column eluted at a flow rate of 50 µL/min using an acetonitrile gradient of 10%/min. Elution was monitored by absorbance at 260 and 680 nm or 750 nm. With all labeled enzymes, long-wavelength absorption associated with the Alexa Fluor dyes was only observed coeluting with protein indicating the absence of any noncovalently attached label. If present, fluorescent dyes not covalently attached to protein would be observed eluting prior to the covalently labeled enzymes (Supporting Information Figure S1). Dye/protein incorporation ratios were determined from the observed masses of each covalently labeled protein. Protein mass increases of 979 Da (AF680) and 1048 Da (AF750) were taken as 1:1 dye/ protein incorporation ratios, while mass increases of 1958 Da (AF680) and 2096 Da (AF750) corresponded to 2:1 dye/protein incorporation ratios. Postrun data analysis was performed using ThermoFinnigan BioWorks Browser 3.0 deconvolution software. Identification of Ppy RE10 Residues Labeled with Fluorescent Dyes. Samples (200 µg) of Ppy RE10 labeled as described above with AF680 or AF750 were digested with thermolysin (protease/protein, 1:10, w/w) in 50 mM ammonium bicarbonate, pH 8.0, for 6 h at 37 °C. Peptides were separated on a Gemini-NX 5 µ C18 110 Å column (50 × 2.00 mm) at a flow rate of 50 µL/min using 0.1% aqueous TFA containing linear gradients of acetonitrile: 5% (v/v) initially for 5 min, 50%

Near-Infrared BRET with Firefly Luciferase

after 90 min, and 95% after 110 min. Elution was monitored by absorbance at 214 nm, 260 nm, and either 680 or 750 nm for AF680 or AF750 labeled peptides, respectively. Each digest of nIR dye-labeled enzyme produced two major peaks with longwavelength absorbance attributable to the dyes. For the AF680labeled luciferase, masses of 1199 ( 0.1 and 1344 ( 0.1 Da were found corresponding to the mass of the dye plus Val168Cys169 and Ile397Met398Cys399, respectively. For the AF750-labeled enzyme, masses of 1268 ( 0.1 and 1413 ( 0.1 Da were observed that were consistent with the mass of the dye plus Val168Cys169 and Ile397Met398Cys399, respectively. While the presence of Val80Cys81 is a potential source of ambiguity, Ppy RE8, which lacks Cys at position 169, did not incorporate the Alexa Fluor dyes. Additionally, the labeled peptide sequences were confirmed by MS/MS analysis in which masses of 1100 ( 0.1 and 1169 ( 0.1 Da, corresponding to Cys covalently bound to AF680 and AF750, respectively, were observed. Upon prolonged standing at room temperature, proteolysis mixtures produced additional peaks corresponding to mass additions of 18, suggesting hydrolytic ring-opening of the maleimide group. Since the dyes are presumably attached through thioether linkages, maleimide ring-opening would not release the dyes. Bioluminescence and BRET Emission Spectra. Bioluminescence emission spectra were obtained using a Horiba JobinYvon iHR imaging spectrometer equipped with a liquid N2 cooled CCD detector and the excitation source turned off. Data were collected at 25 °C (in a 0.8 mL quartz cuvette) over the wavelength range 400-935 nm with the emission slit width set to 25 nm and were corrected for the spectral response of the CCD using a correction curve provided by the manufacturer. Reactions (0.525 mL final volume) were initiated by addition of 5 µL aliquots of luciferase in PBSA buffer (0.2-0.3 µM final concentration) to solutions containing LH2 (150 µM) and Mg-ATP (2 mM) in 25 mM glycylglycine buffer, pH 7.8. The pH values of the reaction mixtures were confirmed before and after spectra were obtained. BRET ratios were calculated from emission spectra by dividing the area under the BRET emission peak by the area under the residual bioluminescence peak. Immobilization of BXRE-680 onto Streptavidin Supports. Biotinylated BXRE10, the fusion protein consisting of an N-terminus hexa-His tagged biotin binding domain (BBD) joined to Ppy RE10 through the peptide linker GSGSIEGRGSGS, was chemically modified with AF680 as described above yielding BXRE-680, the corresponding biotinylated fusion protein with AF680 covalently bound in a 2:1 dye/protein ratio. Streptavidin-coated magnetic microspheres and agarose resin were each prepared by aliquoting 0.1 mL portions of bead slurry, separating and removing the supernatant (via magnet for magnetic beads, quick desktop centrifugation for agarose resin), and washing three times with 0.1 mL portions of PBSA buffer. The supernatant was removed, and the beads were stored on ice. BXRE-680 was bound to the supports by adding 0.1 mL of an ∼1.5 mg/mL protein solution in PBSA to the prewashed beads, which were then incubated with gentle mixing for 10 min on ice. The supernatant was removed, and the immobilized BXRE-680 beads were washed five times with 0.1 mL portions of PBSA buffer. The materials were resuspended in PBSA buffer to a total volume of 0.1 mL and stored in PBSA buffer at 5 °C. An equally effective procedure to prepare immobilized BXRE680 was to add the streptavidin-coated supports to labeling mixtures of nIR dye and BXRE10 (see above), followed by washing with ten 0.1 mL aliquots of PBSA. Excess label and unreacted glutathione were effectively removed while avoiding the exhaustive dialysis step. Alternatively, biotinylated BXRE10 can be bound to the insoluble supports and then labeled with AF680 followed by repeated washing with PBSA buffer.

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Estimation of Specific Activities of Immobilized Fusion Proteins. Integrated specific activities of bead-bound fusion protein samples were estimated using a Horiba Jobin-Yvon Fluorolog-3 with the excitation source shunted. A “flea”-sized magnetic stir bar was used to suspend the immobilized material throughout the 15 min measurement. A volume of bead solution equivalent to ∼2 µg of protein was mixed with 0.4 mL of 0.6 mM LH2 in a 0.8 mL quartz cuvette. The reactions were initiated at 25 °C by injection of 120 µL of 18 mM Mg-ATP solution through a modified lid. Signal intensity was measured over 15 min by centering the emission monochromator to the appropriate peak wavelength (617 nm for BXRE10, 706 nm for BXRE680). The emission slit width was set to the maximum of 29.4 nm, and emission intensities were corrected for the spectral response of the R928 photomultiplier tube, and for the relative percentage of the overall emission profile observable through the 29.4 nm window. Transmittance of Bioluminescence through Blood. Solutions (5 mL) of 2 mM Mg-ATP and 0.60 mM LH2 in PBS, pH 7.3, were diluted with equal volumes of PBS or citrated human blood, and the pH of each was adjusted to 7.3. Aliquots (520 µL) of PBS- or blood-containing solutions were added to cuvettes, and bioluminescence was initiated by addition of 3 µg of luciferase at 25 °C. After 10 s, bioluminescence emission was recorded with a CCD detector as described above. Percent light transmittance through 50% blood solution was calculated from the ratio of the integrated intensities of the signals transmitted through blood and PBS. Factor Xa Assay. Assays of factor Xa activity from a commercial sample of authentic material were performed in either PBSA buffer or citrated human blood. The specific activity of the sample was confirmed by assay with the chromogenic substrate Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA · HCl (Kabi Diagnostica, Stockholm, Sweden). A sample of BXRE-680 immobilized onto agarose beads (Agarose-SA:BXRE-680), prepared as described above, was divided into 0.1 mL aliquots that were briefly centrifuged. The supernatants were removed and replaced with 0 to 20 µg/mL of factor Xa in 0.1 mL of either PBSA or blood. The solutions were resuspended and gently mixed while incubating at 25 °C. At various time intervals, the suspensions were briefly centrifuged, 5 µL aliquots were withdrawn, and incubation was continued after the mixtures were resuspended. To simulate performing the bioluminescence measurements in blood, assay solutions containing 0.25 mL of blood and 0.25 mL of 1.0 mM Mg-ATP and 0.3 mM LH2 in PBS were used. Light reactions were initiated by the addition the 5 µL aliquots of supernatant and were monitored with a liquid N2 cooled CCD as described above. Emission intensity was determined by integrating the resulting bioluminescence emission spectra. Light reactions performed in PBSA (no added blood) were monitored using a photomultiplier tube as described above.

RESULTS AND DISCUSSION Ppy RE8 Cysteine Variants. With the goal of developing efficient intramolecular BRET-based sources of nIR light using the firefly luciferase system, we initiated our studies with the recently developed thermostable Luc variant Ppy RE8 (16), which emits red light (617 nm) (Figure 1b) with 63% bioluminescence specific activity compared to wild-type Luc (Ppy WT) (Table 1). The red-shifted emission of Ppy RE8 provided better donor-acceptor overlap with the nIR fluorescent dyes AF680 and AF750 than was possible with Luc, 59% with AF680 and 29% with AF750 compared to 40% and 15%, respectively (Figure 2). The commercially available AlexaFluor dyes afford nIR fluorescence with good quantum yields of 0.36 (AF680) and 0.12 (AF750) (24), and excellent labeling specificity for Cys residues provided by the appended maleimide groups.


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Table 1. Properties of Firefly Luciferase Variants Km (µM) enzyme

relative specific activitya

decay timeb (min)

LH2

Mg-ATP

bioluminescence λmax (nm, fwhmc)

thermal inactivationd (h)

Ppy WT Ppy RE8 Ppy RE8/C169 Ppy RE8/C368 Ppy RE8/C399 Ppy RE10

100 63 78 84 94 97

0.10 0.26 3.10 3.90 2.30 1.70

15 ( 2 77 ( 8 138 ( 9.6 105 ( 13 113 ( 13 175 ( 16

160 ( 20 391 ( 39 450 ( 45 523 ( 50 517 ( 48 422 ( 41

560 (73) 617 (73) 617 (72) 617 (72) 617 (71) 617 (71)

0.26 3.50 4.00 1.10 3.50 3.60

a Specific activity based on 15 min integration assays performed as previously described (23). The error of the triplicate measurements was less than 10% of the measured value. b Time for maximum signal intensity to decay to 15% of the initial value. c Full width at half-maximum intensity. d Time for the maximum initial activity to decay to 50% at 37 °C.

Figure 2. Overlap of normalized bioluminescence emission spectra of Luc (solid line), Ppy RE8 (dashed line), and Ppy RE10 (dashed line) with the normalized excitation (absorption) spectra of AF680 (closed circles) and AF750 (open circles).

Although Ppy RE8 contains four native Cys residues, molecular modeling based on a crystal structure (1) of Luc in complex with the inhibitor DLSA (25), an N-acylsulfamate analogue of LH2-AMP, indicated that the thiols were not in solvent-exposed surface positions. As anticipated, control experiments with Ppy RE8 and the AlexaFluor nIR dyes produced neither measurable BRET signals nor any covalent incorporation of dye as determined by LC/ESMS (data not shown). Next, molecular modeling was used to select residues Thr169, Ser185, Ser307,

Phe368, and Ser399 for mutation to Cys based on their surface locations, proximity to (but not part of) the active site, and, with the exception of Phe368, structural similarity to Cys. The single Cys Ppy RE8 variants were obtained as pure proteins in yields of 5-10 mg/0.25 L culture, and the characterization of the bioluminescence properties of the three most promising ones is presented in Table 1. Significantly, the introduction of a single Cys residue at positions 169, 368, or 399 produced luciferases with bioluminescence emission maxima of 617 nm, a value identical to that of the template enzyme Ppy RE8. The Km values of the single Cys mutants were slightly elevated up to 2.3-fold for LH2 and 1.3-fold for Mg-ATP, while the specific activities determined from 15 min integrated light emission were somewhat enhanced. Since the overall stability of the Cys368 enzyme, estimated from 37 °C thermal inactivation data (Table 1), was significantly lower than that of Ppy RE8 and the other Cys variants, we did not study it further. Instead, we added both the T169C and S399C mutations to Ppy RE8 and expressed and purified the resulting Ppy RE10 protein obtaining 7 mg from 0.25 L culture. The protein has 1.4-fold enhanced specific activity and is otherwise quite similar to Ppy RE8. Covalent Labeling of Luciferases with Alexa Fluor Maleimide Dyes Produces nIR BRET Emission. The Ppy RE8 Cys169 and Cys399 enzymes along with Ppy RE10 were covalently labeled with AF680 and AF750 by incubating 30 µM protein solutions in PBSA buffer with a 10-fold molar excess of dye for 15 min at 10 °C. Ppy RE8/C169 and Ppy RE8/C399 have similar activity and thermostability properties (Table 1) and nIR dye labeling produced BRET emission at ∼705 nm (with AF680) and ∼780 nm (with AF750). However, the BRET efficiencies obtained with these proteins were quite different (Table 2). The BRET ratios of the position 399 labeled enzymes were ∼5- to 9-fold greater than those of the corresponding position

Table 2. Properties of Luciferase Variants and Fusion Proteins Labeled with Alexa Fluor nIR Dyes enzymea

relative specific activityb

decay timec (min)

thermal inactivationd (h)

BRET λmax (nm, fwhme)

BRET ratiof

labeling stoichiometryg

Ppy WT Ppy RE8 Ppy RE10 BXRE10h Ppy RE8/C169-AF750 Ppy RE8/C399-AF750 Ppy RE10-AF750 Ppy RE8/C169-AF680 Ppy RE8/C399-AF680 Ppy RE10-AF680 BXRE-680 MagSphere-SA:BXRE-680 Agarose-SA:BXRE-680

100 63 97 60 19 86 41 132 180 164 115 34 76

0.10 0.26 1.7 2.0 5.3 2.4 4.4 2.4 1.5 7.4 5.7 13.6 13.0

0.26 3.50 3.60 3.5 2.3 2.8 3.0 3.3 1.3 3.5 1.5 -

778 (71) 780 (70) 783 (60) 704 (59) 705 (60) 705 (57) 706 (49) 706 (49) 708 (48)

0.5 2.1 4.0 4.5 10.0 34.0 14.0 11.0 13.7

1.0 (1:1) 1.3 (1:1) 1.8 (2:1) 1.1 (1:1) 1.4 (1:1) 2.1 (2:1) 2.0 (2:1)i -

a AF750 and AF680 refer to the fluorescent dyes covalently attached to the respective enzymes. b Specific activity based on 15 min integration assays performed as previously described (23). The error of the triplicate measurements was less than 10% of the measured value. c Time for maximum signal intensity to decay to 15% of the initial value. d Time for the maximum initial activity to decay to 50% at 37 °C. e Full width at half-maximum intensity. f BRET ratios were calculated from emission spectra by dividing the area under the BRET emission peak by the area under the residual bioluminescence peak. g The number of dye molecules incorporated per molecule of enzyme was calculated by UV-vis absorption as described in the Experimental Procedures. The error in the UV-vis based determination is ∼20%. The ratios in parentheses are based on the masses associated with the only peaks detected using LC/ESMS. h Contains bound biotin. i The BXRE-680 LC peak contained a second minor mass indicative of 1:1 dye/protein labeling.



Figure 3. BRET emission spectrum of BXRE-680 immobilized on Agarose-SA. The fusion protein contains biotin bound to a biotin binding domain that is connected through a 12 amino acid linker (containing a factor Xa protease site) to Ppy RE10 labeled with AF680. The emission spectrum was recorded by suspending an aliquot of the immobilized material in a pH 7.8 buffer containing LH2 and Mg-ATP.

Figure 4. Visible and nIR light emission produced by unlabeled and labeled immobilized fusion proteins. In each panel, the tubes contain ∼20 µL of Agarose-SA bound substrate (right tubes, BXRE-680; and left tubes, the immobilized fusion protein with no AF680 attached) that settled from 70 µL pH 7.8 buffered suspensions after brief centrifugation. The protein concentrations in the 20 µL of settled resin are ∼40 µM. In panels (a) and (b), photographs were taken with a Nikon D80 equipped with a stock IR filter (a) in ambient light prior to addition of LH2 and Mg-ATP and (b) in the dark at 15 s exposure following addition of the substrates and showing only visible light emission. In panels (c) and (d) photographs were taken with an iGen NV 20/20 camera with night vision (infrared) capability (c) capturing total light emission; and in (d) using a Schott 695 nm long-pass filter showing only nIR emission.

169 enzymes (Table 2). Since the dyes attached to Cys399 are estimated to be 15 Å closer (20 Å versus 35 Å) to the emitter oxyluciferin, the higher BRET ratios associated with the labeling of Cys399 are expected, because resonance energy transfer efficiency is inversely proportional to and heavily weighted by the distance between the donor and the acceptor. We reproducibly and quantitatively labeled milligram quantities of Ppy RE10 by attaching AF680 or AF750 to both the Cys169 and Cys399 residues (Table 2). Thermolysin proteolysis and LC/ESMS analyses verified the covalent attachment of the nIR dyes to the expected Cys169 and Cys399 residues (Ex-

perimental Procedures), presumably through thioether linkages. The dye-modified luciferases displayed emission maxima shifted to 705 and 783 nm with BRET ratios of 35.0 and 4.1, respectively (Figure 1c and d). Integrated light emission was 4.0-fold greater for Ppy RE10 labeled with AF680 compared to the AF750 labeled enzyme, representing a 1.7-fold increase and 2.4-fold decrease with respect to the Ppy RE10 template. The nIR dye-labeled Ppy RE10 enzymes provided an excellent combination of high BRET ratios, retention of enzyme activity, minimal nonspecific dye incorporation, long-wavelength emission maxima, and extended glow kinetics. Importantly, the two additional engineered Cys residues did not alter the 617 nm bioluminescence maximum of precursor Ppy RE8, nor did they adversely affect specific activity. While the BRET ratio of 4.0 with the attached AF750 label is very good and is similar to results obtained with quantum dot (QD)-rLuc mutant conjugates (12), the 34.0 value obtained with AF680 is extraordinary. Moreover, the bioluminescence maximum of Luc has been red-shifted 223 nm by the combined effects of mutagenesis (57 nm) and BRET (166 nm) in the AF750 labeled enzyme. The nIR dye-labeled Ppy RE10 enzymes were stable for at least 6 weeks when stored in solution at 5 °C, and they could be flash frozen, stored at -80 °C, and thawed without loss of activity. Additionally, the BRET activity was retained at levels greater than 50% for ∼3 h at 37 °C. To the best of our knowledge, this is the first report of bright nIR light sources produced by chemical modification of a beetle luciferase. The outstanding BRET ratio (34.0) achieved with AF680 labeling of Ppy RE10 emphasizes the importance of selective labeling with fluorescent dyes. We considered the basis of the appreciably different labeling results, particularly the 4.0-fold higher specific activity and 8.5fold greater BRET ratio obtained with Ppy RE10 labeled with AF680 and AF750, respectively (Table 2). Since the dyes are approximately the same size, have similar chemical structures (preliminary structure assignments based on HRMS and NMR) and are attached to the same Ppy RE10 thiols, distances from the emitter can be ruled out, although differences in spatial


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Figure 5. Bioluminescence assays of factor Xa activity in whole blood. Stirred suspensions of aliquots of Agarose-SA:BXRE-680 in whole blood (0.1 mL) were incubated at 20 °C with factor Xa: (filled squares) 0 µg/mL; (open circles) 2.5 µg/mL; and (filled circles) 10 µg/mL. Aliquots (5 µL) were withdrawn and assayed at the times shown as described in detail in the Experimental Procedures. The inset shows bioluminescence end point assay data collected 30 min after incubation of factor Xa in stirred substrate suspensions of Agarose-SA:BXRE-680.

orientation of the dyes may be a contributing factor. Instead, it is likely that the variation in the specific activities can largely be accounted for by the 3-fold higher fluorescence quantum yield of AF680 (0.36) compared to AF750 (0.12) (24). That is, even if the energy transfer efficiencies were identical, the AF680 labeled enzyme would be expected to produce ∼3-fold more BRET emission than the AF750 labeled protein. While the difference in the quantum yields of the nIR dyes also contributes to the superior BRET ratio of the AF680 labeled Ppy RE10, the 2-fold greater spectral overlap of Ppy RE10 bioluminescence with the AF680 excitation spectrum is also an important contributing factor. Additionally, the AF680 labeled enzymes produced integrated specific activities (based on total photons emitted) that were greater than that of Ppy WT (Table 2). This is most likely due to improved turnover. Under the assay conditions employed in the measurement of the integration-based activities, Luc is inhibited by the formation of dehydroluciferylAMP, a side product naturally formed during the oxidation of LH2 (26-28), that binds tightly and dissociates very slowly (KD ) 0.5 nM) from the active site. Possibly, the binding of AF680 on the surface of the Luc variants results in conformational changes that caused an increase in the KD of the inhibitor. Immobilized AF680-Labeled Ppy RE10 Fusion Proteins. To demonstrate that a Luc-based BRET source could be applied where nIR emission would be advantageous, e.g., in receptorbased imaging and for enzyme assays in whole blood, we constructed the fusion protein BXRE-680 and immobilized it to streptavidin (SA) bound to agarose or magnetic microspheres (Figure 3). Modeled after a similar fusion protein containing Luc immobilized on magnetic beads (22), BXRE-680 contains biotin bound to a biotin binding domain connected to AF680labeled Ppy RE10 through a12 amino acid residue linker sequence that includes the IleGluGlyArg factor Xa protease site. Reaction of AF680 with the biotinylated fusion protein BXRE10 produced BXRE-680 with an ∼2-fold greater specific activity, likely resulting from the slower decay of luminescence (Table 2). The linker peptide and BBD do not contain Cys residues and LC/ESMS analysis of BXRE-680 confirmed the incorporation of 2 nIR dyes/fusion protein. After attachment to both agarose and magnetic microspheres through the strong streptavidin (SA)-biotin interaction (29), matrix-bound BXRE-680 produced BRET ratios of 13.7 (708 nm maximum) and 11.0 (706 nm maximum), respectively (Table 2). While immobilization reduced specific activity 1.5- and 3.4-fold for the agarose and magnetic bead bound materials, respectively, the materials

are bright sources of red and nIR light as illustrated with Agarose-SA:BXRE-680 (Figure 4). Yeung and co-workers reported (22) making a biotinylated fusion protein of Luc with an attached BBD that was then immobilized on SA-containing beads. The modified beads were attached to cell surfaces and employed to image ATP release from live astrocyte cells upon addition of LH2 using a CCD camera (22). The construction of the biotinylated BXRE-680 fusion protein and demonstration that it efficiently emits nIR light while immobilized through the streptavidin-biotin interaction (Figures 3 and 4) established, in principle, that our material can be applied similarly for receptor imaging of genetically encoded streptavidin sites. However, one drawback of the chemical labeling approach used to produce our materials is that they cannot be genetically encoded, thereby limiting their usefulness for in vivo applications. Nevertheless, Ohmiya and co-workers recently demonstrated (14) the targeting and tumor imaging capabilities of a biotinylated nIR dye labeled-cLuc conjugate in live mice. This was accomplished by first linking the labeled cLuc conjugate to an avidin-monoclonal antibody against a cell surface tumor marker. Injection of this biotin-avidin complex, followed by injection of the cLuc substrate coelenterazine produced BRET signals (λmax ) 675 nm) that enabled tumor detection and imaging (14). These interesting results strongly support the feasibility of applying BXRE-680 fusion proteins for similar purposes. Moreover, since the cited applications (14, 22) employ CCD cameras, the longer wavelength emission and superior BRET ratio of BXRE680 (e.g., 40- to 50-fold greater than the cLuc-containing material) may provide enhanced sensitivity in environments containing interfering visible light absorbing substances. However, additional experiments must be undertaken to determine whether the potential advantages of BXRE-680 can be realized. Factor Xa Assays with Agarose-SA:BXRE-680. We also explored the use of the agarose bound BXRE-680 material (Figure 3) as a substrate for assaying factor Xa activity. Briefly, various concentrations (1-20 µg/mL) of commercial factor Xa were incubated at 25 °C in citrated human blood containing the resin-bound substrate. The assay principle is that factor Xa cleavage of BXRE-680 releases AF680-labeled Ppy RE10 from the matrix into the blood medium. Physiologically significant levels of factor Xa activity were proportional to the light intensity produced by the solubilized BRET source (after brief centrifugation to separate unreacted matrix-bound substrate) upon addition of substrates LH2 and Mg-ATP (Figure 5). The advantage provided by the nIR source was that there was



Figure 6. Relative transmittance of bioluminescence through citrated human blood. The emission spectra shown in all panels were initiated by addition of solutions of Mg-ATP (2 mM) and LH2 (0.60 mM) in PBS to citrated human blood (dashed lines) or PBS (solid lines) containing (a) Ppy GR-TS (18); (b) Ppy RE10; (c) Ppy RE10-AF680; and (d) PpyRE10-AF750 as described in detail in the Experimental Procedures.

sufficient sensitivity to detect 1 µg/mL of factor Xa from aliquots of blood diluted only 1:1 with substrate solution. Emission spectra showing the attenuation of green (Ppy GR-TS), red (Ppy RE10), and nIR (Ppy RE10-AF680 and Ppy RE10-AF750) signals by whole human blood diluted 1:1 with an isotonic buffer are presented in Figure 6. Since the bioluminescence measurements were made with the proteins in direct contact with the blood components, attenuation may result not only from the absorption of light emission, but also from nonspecific effects, e.g., adsorption of the enzymes, and from quenching of bioluminescence and/or fluorescence of the nIR dyes. Strong hemoglobin absorption (30) is likely an important factor in the nearly complete loss (∼97%) of the green signal (Figure 6a) and the 550-600 nm component of the Ppy RE10 emission (Figure 6b). However, interference of Ppy RE10 bioluminescence also occurs as the total bioluminescence signal has been attenuated ∼90%. The loss of Ppy RE10 bioluminescence intensity no doubt contributes to the reduced intensity of the nIR signals produced by BRET (Figure 6c,d); however, the relative intensities produced by the AF-680 and AF-750 labeled enzymes in blood were ∼23% and ∼25%. This result very likely reflects the greater transmission of nIR light in a medium containing hemoglobin. Immobilized versions of the fusion protein with linkers containing other specific cleavage sites should provide new substrates for protease assays as demonstrated with factor Xa, possibly in media like blood where interference with visible light is a serious problem. Additional potential applications (31) include immunoassays and biosensors.

ACKNOWLEDGMENT The authors gratefully acknowledge the financial support provided by the Air Force Office of Scientific Research (FA9550-07-1-0043 and FA9550-10-1-0174), the National Science Foundation (MCB0842831), and the Hans & Ella McCollum ′21 Vahlteich Endowment. Supporting Information Available: Experimental Procedures including materials and general methods for cloning of Ppy RE8 variants, cloning of fusion protein BXRE10, expression

and purification of biotinylated fusion protein BXRE10 and relative quantum yield measurements; HPLC chromatograms of Ppy RE10-AF750. This material is available free of charge via the Internet at http://pubs.acs.org.

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