in bioluminescence emission spectra among firefly

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The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than ...
www.biochemj.org Biochem. J. (2008) 412, 27–33 (Printed in Great Britain)

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doi:10.1042/BJ20070733

Site-directed mutagenesis of firefly luciferase: implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases Narges Kh. TAFRESHI*, Majid SADEGHIZADEH*1 , Rahman EMAMZADEH†, Bijan RANJBAR‡, Hossein NADERI-MANESH‡ and Saman HOSSEINKHANI†1

The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the

bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as K m and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging.

INTRODUCTION

tissue, whereas longer wavelengths (red light) are less affected [8,9]. Furthermore, haemoglobin is the main absorber of light in the body, with strong absorption peaks in the blue and green part of the visible spectrum, but absorption is reduced for wavelengths longer than 600 nm (red region) [10]. Therefore red light can be transmitted through several centimetres of tissue and be detected externally more easily than absorbed blue or green light. This property is an important consideration in the selection of appropriate luciferases for use in bioluminescence imaging [3]. Moreover, red-emitting variants of luciferase are also desirable for multiple labelling systems in whole cells as well as for dual-reporter systems [4,5,11]. Sample matrix or environmental conditions create high variability in the response, which is the main drawback encountered in an assay using luciferase as a reporter. To overcome this problem, a dual-reporter system can be used in which, rather than a main reporter (e.g. a greenemitting luciferase), a second control reporter (e.g. a red-emitting luciferase) to improve the analytical signal and distinguish the main signal from non-specific interference signals is used [5]. Consequently, red-emitting luciferase is also appropriate for multiplex analysis and dual-reporter systems in biosensor application and for minimizing light absorption by tissue in whole animals. Most investigations on light emission changes to red wavelengths have been focused on the North American firefly Photinus pyralis [12–14]. The red-emitting mutants of luciferase have also been isolated in the Japanese firefly (Luciola cruciata) luciferases by random mutagenesis [15]. Differences in bioluminescence colour are attributed to the luciferase structure and assay conditions [16,17]. The construction of chimaeric luciferases [18,19] and site-directed and random mutagenesis

Bioluminescence is the emission of visible light by living organisms. In this process, chemical energy is converted into light [1,2]. The reaction is the luciferase-catalysed adenylation and oxidation of benzothiazolyl-thiazole luciferin. In fact, light emission is a consequence of formation of an intermediate in an electronically excited state; relaxation to the ground state results in the emission of light [1,2]. Since even a few photons can be detected using available lightmeasuring technology (such as a luminometer or charged-coupled devices), bioluminescence has been exploited as a powerful technology for numerous applications over the last decade. It is already being used for monitoring gene expression, protein localization and protein–protein interactions, as well as detection of infections, tumour growth and metastasis in whole animals [3,4], reporter gene assays [5], protein trafficking [3] and detection of environmental contamination [6,7]. The major advantages of techniques based on bioluminescence are the versatility, noninvasiveness, reproducibility, high rate and ease of assay performance with high sensitivity and low cost [3–5]. Bioluminescence imaging enables and facilitates real-time analysis of disease processes at the molecular level in living organisms, monitoring throughout the course of disease and progression and tracking of infection. In addition, serial quantification of biological processes in intact animals is possible by bioluminescence imaging [3,8]. Lack of intrinsic bioluminescence in mammalian tissues makes bioluminescence imaging a tool for in vivo imaging [3]. Photons are both scattered and absorbed as they pass through mammalian tissue. Shorter wavelengths of light (blue and green) are largely absorbed by

Key words: bioluminescence, firefly luciferase, Lampyris turkestanicus (Iranian firefly), mutagenesis, red shift.

Abbreviations used: IPTG, isopropyl β-D-thiogalactoside; Ni-NTA, Ni2+ -nitrilotriacetate; rLuc(Arg), recombinant mutant luciferase containing an arginine insertion; SOE-PCR, splicing overlap extension PCR. 1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).  c The Authors Journal compilation  c 2008 Biochemical Society

Biochemical Journal

*Department of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran, 14115-175, †Department of Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran, 14115-175, and ‡Department of Biophysics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran, 14115-175

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Scheme 1 Mechanism of adenylation and oxidation steps in the firefly luciferase-catalysed bioluminescence

studies have revealed certain important residues and regions involved in bioluminescence colour [16,18–20]. On the basis of these results, four mechanisms have been proposed to explain colour variations in beetle luciferases. (i) Solvent effect: interaction between solvent and emitter molecules that influences the difference of the energies between the ground and excited states and therefore luminescence intensity [21]. (ii) The interaction of basic residues of luciferase with oxyluciferin involved in enol– keto tautomerization of oxyluciferin by which bioluminescence spectra of beetle luciferases are determined by the ratios between enol (green–yellow emitter) and keto (red emitter) forms of excited oxyluciferin (Scheme 1) [22,23]. (iii) The active-site conformation, which could have an effect on the freedom of rotation of the oxyluciferin thiazolinic rings; on the other hand, it affects the rotation of excited oxyluciferin along the C2 –C2  bond [24,25]. (iv) Resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state under control of luciferase modulation [26]. To date, the crystal structure for P. pyralis without substrates and in complex with a high-energy intermediate from Luciola cruciata has been resolved [27,28]. However, the detailed mechanism for the bioluminescence colour change is still unclear, and generalization in explaining the mechanism of colour emission among different luciferases is lacking. In order to better understand the relationship between luciferase structure and bioluminescence colour in firefly luciferases, we carried out an investigation on the effect of site-directed mutagenesis in residues potentially involved in bioluminescence colour. In the present study, we made a set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase [29] by sitedirected mutagenesis on the basis of sequence homology. The kinetic and structural characterizations of mutants are reported. EXPERIMENTAL Reagents

IPTG (isopropyl β-D-thiogalactoside), kanamycin and ATP were from Roche, D-luciferin potassium salt was from Sigma, restriction enzymes were from Fermentas, Pfu polymerase, plasmid extraction kit, gel purification kit and PCR purification kit were from Bioneer Co., Ni-NTA (Ni2+ -nitrilotriacetate) spin kit was from Qiagen, and pET28a vector was from Novagen.

of primers were used for cloning: F-Cloning, containing a BamHI restriction site (5 -CGTTGGATCCATGGAAGATGCAAAAAATATTATG-3 ), and R-Cloning, containing a HindIII restriction site (5 -CAGCAAGCTTTTACAATTTAGATTTTTTTCCCATC-3 ); the sequence of the restriction enzyme site is underlined. The overlapping mutagenesis primers were for S284T (5 -ATTATAAAATTCAAACTGCGTTGCTGGTACCTAC-3 and 5 -CAACGCAGTTTGAATTTTATAATCTTGAAG-3 ), H245N (5 -ATGAAATGGTATAACCGTTAAAATCGCAG-3 and 5 -CGGTTATACCATTTCATAATGGTTTTGGAATGTTTAC-3 ) and H431Y (5 -ACCATCTTTGTCGTAGTAAGCTATGTCACCAGAGTG-3 and 5 -CTTACTACGACAAAGATGGTTACTTCTTCATAGTAGATCG-3 ); the mutated residues are indicated in bold. The plasmid carrying the native luciferase was used as template. Two PCRs to carry out primary amplification of the two DNA fragments to be spliced were conducted using forward mutant primer and R-Cloning, F-Cloning and reverse mutant primer, and Pfu polymerase under the following conditions: initial denaturation at 94 ◦C for 5 min, 25 cycles of 94 ◦C for 1 min, 68 ◦C for 1 min and 72 ◦C for 1.5 min, and a final extension for 5 min at 72 ◦C. Subsequently, the primary PCR products were purified using a clean-up kit to remove redundant primers, and the resulting fragments from the primary PCR were mixed in a 1:1 molar ratio so that the amount of DNA added to the second PCR was approx. 100 ng. PCR conditions were essentially identical with those of the primary PCR, except that it was carried out for ten cycles without primers. Finally, F-Cloning and R-Cloning primers were added to each tube and the next step of PCR was carried out. The mutagenesis products, digested with BamHI/HindIII, were inserted into the BamHI/HindIII restriction sites of digested/dephosphorylated pET28a high expression vector, and ligated mixtures were transformed into the competent cells of Escherichia coli BL21 by electroporation. Protein expression and purification

A 10 ml aliquot of 2XYT medium containing 50 µg/ml kanamycin with a fresh bacterial colony harbouring the expression plasmid was inoculated and grown at 37 ◦C overnight. Subsequently, 300 ml of medium was inoculated with 500 µl of overnight culture and grown at 37 ◦C with vigorous shaking until the D600 reached 0.6, after which IPTG was added to a final concentration of 1.5 mM to the solution and incubated at 22 ◦C overnight with vigorous shaking. The cells were harvested by centrifugation at 5000 g for 15 min. The pellet from the cells was resuspended in lysis buffer (50 mM NaH2 PO4 , 300 mM NaCl and 10 mM imidazole, pH 8) and was stored at − 70 ◦C until use in the next step. Purification of His6 -tagged fusion protein was carried out using Ni-NTA spin columns as described by the manufacture (Qiagen). The colour of emitted light of the firefly luciferase reaction was evident by the addition of luciferin to the purified luciferases. Photographs of glowing colonies on an LB (Luria–Bertani) agar plate and cocktails containing luciferase molecules were taken using a Konika 400 ASA film. Sequencing

pET28a carrying native and mutant luciferases were sequenced using an automatic sequencer (MWG) by T7 promoter and T7 terminator universal primers.

Site-directed mutagenesis

Determination of kinetic parameters

The mutants, including S284T, H245N and H431Y, were prepared by SOE-PCR (splicing overlap extension PCR) [30]. Two pairs

ATP and luciferin kinetic parameters were measured at 25 ◦C. To estimate the K m for luciferin, 100 µl of the assay reagent

 c The Authors Journal compilation  c 2008 Biochemical Society

Site-directed mutagenesis of firefly luciferase

containing 20 mM MgSO4 and 2 mM ATP in 50 mM Tricine (pH 7.8) was mixed with 50 µl of the various concentrations of luciferin (from 0.01 to 2 mM) in a well of a 96-well plate. The reaction was initiated with injection of 50 µl of diluted enzyme, and light emission was recorded over 5 s (Orion microplate luminometer, Berthold Detection Systems). The calculation of the Michaelis–Menten parameters for ATP was carried out. The estimation of the ATP kinetic constant was performed in a similar way, but various concentrations of ATP were mixed with the assay reagent including 20 mM MgSO4 and 2 mM luciferin in 50 mM Tricine (pH 7.8). Apparent kinetic parameters were calculated from Lineweaver–Burk plots. The decay times of the native and mutant luciferases were measured in 20 min. The residual activity for each enzyme was reported as a percentage of the original activity. Approximate protein concentrations were calculated using a Bradford assay [31], and relative specific activities (enzyme activity/protein concentration) were also calculated. Measurement of bioluminescence emission spectra

The bioluminescence spectra were recorded using a Cary Eclipse luminescence spectrophotometer (Varian) from 400 to 700 nm wavelengths. A volume of 300 µl of Tricine buffer (pH 7.8 or 5.5) including 2 mM ATP, 5 mM MgSO4 and 1 mM luciferin was added to 100 µl of each purified luciferase solution (approx. 50 pg) in a quartz cell. The spectra were automatically corrected for photosensitivity of the equipment. Thermostability, optimum pH and temperature

The purified luciferase solutions (10 µg/ml) were incubated in the range of 10–40 ◦C for 10 min. Enzyme activities were measured at room temperature (25 ◦C), and the remaining activities were recorded as a percentage of the original activity. In order to obtain the optimum temperature of activity for the native and mutant luciferases, activities were measured in the temperature range 20–37 ◦C. Moreover, optimum pH of activity for the enzymes was measured by incubation of the enzyme in a mixed buffer at the pH range 5.5–9.5. Structural studies Intrinsic fluorescence

The purified luciferases were dialysed in dialysis buffer containing 50 mM Tris/HCl (pH 7.8), 1 % (v/v) glycerol, 1 mM EDTA, 50 mM NaCl and 0.05 % 2-mercaptoethanol at 4 ◦C. The intrinsic fluorescence was recorded using 0.01 µg/ml purified and dialysed enzyme. The fluorescence emission spectrum of the enzyme was performed in a Cary Eclipse luminescence spectrophotometer. The fluorescence emission was scanned between 290 and 440 nm, with an excitation wavelength of 296 nm. CD measurements

The far-UV CD spectra were recorded on a JASCO J-715 spectropolarimeter using 0.2 mg/ml solution with the purified and dialysed proteins. The results were expressed as molar ellipticity, [θ ] (deg · cm2 · dmol−1 ), based on a mean amino acid residue weight (m.r.w.) assuming its average molecular mass for firefly luciferase. The molar ellipticity was determined as [θ ] = (θ m.r.w. × 100)/cl, where c is the protein concentration in mg/ml, l is the light path length in centimetres, and θ is the measured ellipticity in degrees at wavelength λ. The instrument was calibrated with ammonium (+)-10-camphorsulfonic acid, assuming [θ ]291 =

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7820 deg · cm2 · dmol−1 [32], and with JASCO standard nonhydroscopic ammonium (+)-10-camphorsulfonate, assuming [θ ]290.5 = 7910 deg · cm2 · dmol−1 [33]. Noise in the data was smoothed using the JASCO J-715 software, including the fast Fourier-transform noise-reduction routine which allows enhancement of noisy spectra without distorting their peak shapes [34]. Homology-based modelling studies

The homology-based model of Lampyris turkestanicus luciferases were constructed by satisfaction of spatial restraints using the crystal structure of Luciola cruciata luciferase complexed with a transient structure as a template with the Modeler program. The models were visualized with Swiss-Pdb Viewer version 3.7 [35]. RESULTS AND DISCUSSION

Site-directed mutagenesis using the SOE-PCR method was used to make variants of Lampyris turkestanicus luciferase displaying different colours and functional properties. Similar mutations have been reported in other species of firefly luciferases: H245N and S284T in P. pyralis [5,36], and H433Y in Luciola cruciata (corresponding to a mutation of His431 in Lampyris turkestanicus) [15]. Three bright variants emitting in the red regions of the spectrum were obtained, and further analysis and characterization was performed. Construction, expression and purification of the native and mutant luciferases

Three red-emitting luciferases from Lampyris turkestanicus were made according to sequence similarity by SOE-PCR, and their kinetic and structural properties were compared. After cloning and transformation, mutations at specific residues were confirmed by sequencing. Upon addition of D-luciferin, the photographs of the IPTG-induced bright bacterial cells containing the native and mutant luciferases were taken in a darkroom. In order to improve further the purification and characterization of the native and mutant forms, overexpression of luciferases containing a His6 tag was carried out in an E. coli BL21 host. The purification of His6 -tag fusion luciferases was also performed by affinity (Ni-NTA–Sepharose) chromatography. The purified native and mutant luciferases had purities of more than 95 % according to analyses by SDS/PAGE. The colour of reaction upon addition of substrate to the purified luciferase could be seen with the naked eye in the darkroom (Figure 1, inset). Bioluminescence spectra

The in vitro bioluminescence spectra of the native and mutant luciferases are depicted in Figure 1. Among the mutants, only S284T exhibits a single peak in the red region of the spectrum. A similar spectrum was also reported for a similar mutant of P. pyralis luciferase [5], suggesting that a single substitution at this position (284) is sufficient to cause a complete shift to the red region of the spectrum in firefly luciferases. The crystal structure of a red-emitting mutant (S286N) form of Luciola cruciata luciferase complexed with a high-energy intermediate analogue DLSA {5 -O-[N-(dehydroluciferyl)-sulfamoyl]adenosine} has revealed transient movement of the Ile288 side chain towards the excited state of oxyluciferin [28]. Therefore it is postulated that a conformational displacement of the Leu286 (corresponding to Ile288 in Luciola cruciata) side chain towards the excited state of oxyluciferin may be involved in red-light emission by the mutant form (S284T) of Lampyris turkestanicus luciferase.  c The Authors Journal compilation  c 2008 Biochemical Society

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Phe268 , a conserved residue in firefly luciferases, which has been changed to cysteine in Lampyris turkestanicus (results not shown). It is important to note that the residues Asn229 and Gly247 that have been identified as critical residues for pH-sensitivity in firefly luciferases are conserved in Lampyris turkestanicus [38]. Influence of the mutations on the kinetic constants, decay and thermostability

Figure 1 Bioluminescence emission spectra produced by the wild-type and mutant luciferase-catalysed oxidation of luciferin at pH 7.8 (A) and 5.5 (B). Insets show bioluminescence of the different luciferases at the same pHs used for emissions spectra.

As indicated in Figure 1(A), H245N and H431Y exhibit a bimodal spectrum with a maximum in the red region (at 615 nm) and a smaller shoulder at 560 nm in the green region, whereas the native luciferase exhibits a spectrum with only a peak at 555 nm. According to the proposed residues of the enzyme active site (H244 HGF247 ), the imidazole group of His245 is located at the cavity entrance of the putative luciferin-binding site close to its carboxy group and to the γ -phosphate group of ATP [12,37]. In fact, His245 participates as a base (nucleophile) in enol–keto tautomerization. This implies that a red shift in the bioluminescence spectra of the mutant luciferases is determined by increasing the keto tautomer (red emitter) to the enol (green–yellow emitter) form of the excited oxyluciferin [12,37]. Moreover, the bioluminescence spectra of H245N and H431Y mutants at lower pH (pH 5.5) have a shorter shoulder in the green region, as indicated in Figure 1(B). It is worthwhile to note that the shape of the emission spectrum for the native luciferase was only slightly changed under acidic conditions. In contrast with most firefly (Lampyridae) luciferases, the luciferase from Lampyris turkestanicus does not show any significant shift in the emission peak under this condition and is very similar to that of pHinsensitive luciferases (click beetles and railroad worm). Although firefly luciferases have significant amino acid identity, multiple sequence alignment of Lampyris turkestanicus luciferase with other firefly luciferases revealed certain amino acid substitutions in Lampyris turkestanicus which are probably necessary for the pH-independent luminescence spectra profile. Among them is  c The Authors Journal compilation  c 2008 Biochemical Society

Lineweaver–Burk plots were used to estimate the apparent K m . The kinetic parameters, including relative activity, quantum yield, optimum temperature, optimum pH, and luciferin and ATP K m , are shown in Table 1. The values shown in Table 1 demonstrate that the mutations have adversely affected the performance of the enzyme activity in S284T and H431Y mutants (24 and 16.6 % of wild-type respectively). However, the specific activity of the H245N mutant luciferase is higher than other known mutants (76.6 % of wild-type). This may indicate that the imidazole ring of His245 is not necessary to maintain an environment for efficient decay of the oxyluciferin excited state. A similar interpretation has been deduced for the H245A mutant in P. pyralis firefly luciferase [12]. Table 1 also compares the specific activity and quantum efficiency of the mutant luciferases with those of the wild-type enzyme. As indicated in Table 1, the quantum yield comparison with specific activity was less affected by S284T and H245N mutations, owing to an increase in decay rate. The enzyme’s K m values are elevated in comparison with those in the wild-type: approx. 1.8-, 1.4- and 1.6-fold for luciferin and 1.84-, 1.2- and 1.4-fold for ATP in S284T, H245N and H431Y respectively. Times of light decay for the native and mutant forms are shown in Figure 2. The time of light emission for the H431Y mutant luciferase was shorter and for the S284T and H245N mutants were relatively longer than for the native form. As indicated in Figure 2, upon mutation of the Ser284 into threonine, we observed slower light emission decay. At the same time, the bioluminescence spectra of the S284T mutant have only a peak at 620 nm without a shoulder in the green region. These results confirm the idea that the rate of light decay depends on formation of a specific intermediate. That is to say, formation of the keto tautomer for the S284T mutant has led to a lower decay rate than in the native form. Change in the rate of decay, upon a single mutation, has also been reported for a mutant of bacterial luciferase [39]. On the other hand, H245N mutation brought about an increase in the thermostability of luciferase (Figure 3), suggesting therefore that this mutant luciferase could be considered as a good reporters for in vivo imaging, since most native luciferases are unstable at higher temperatures. The activity of the native enzyme fell suddenly after 25 ◦C, but mutant luciferases were inactivated more gradually. As indicated in Figure 3, the H245N mutant kept approx. 80 % of its original activity at 35 ◦C, whereas the native enzyme was completely inactive at this temperature. Structural characterization of the native and mutant luciferases

The native and mutant forms of Lampyris turkestanicus luciferase were compared by CD and fluorescence spectroscopy. The CD spectra of the native and mutant forms of luciferase obtained in PBS, pH 7.0, are shown in Figure 4. The far-UV CD spectra of the native and mutant forms of luciferase show only slight differences in the secondary structure of the S284T mutant. On the other hand, the CD spectra of the H431Y and H245N mutants have been altered from that of the wild-type more noticeably by mutation. An apparent increase in the helical structure of the H431Y mutant has occurred in luciferase. However, change of the helical structure is accompanied by a small decrease in

Site-directed mutagenesis of firefly luciferase Table 1

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Kinetic constants and spectral properties of the wild-type and mutant enzymes

Asterisks identify minor peaks. The errors associated with K m values fall within + − 10 % of the value. λmax (nm) Enzyme

Luciferin K m (µM)

ATP K m (µM)

Quantum yield ( × 1014 )

Specific activity (1013 × RLU/s per mg)

Relative activity (%)

pH 7.8

pH 5.5

Optimum temperature ( ◦C)

Optimum pH

Wild-type S284T H245N H431Y rLuc(Arg)†

16.12 30 23 26 24

135 248.4 168 187.2 142

1 0.5 0.9 0.14 0.3

1.5 + − 0.15 0.36 + − 0.04 1.15 + − 0.14 0.25 + − 0.07 1.21 + − 0.14

100 24 76.6 16.6 81

555 618 572∗ , 617 564∗ , 612 558∗ , 616

560 619 617 619, 564∗ 618, 560∗

24 30 24 24 34

8 8 8.5 8.5

†Data taken from [42].

Figure 2

Comparison of decay time of the wild-type and mutant luciferases

The residual activity for each enzyme was reported as a percentage of the original activity.

Figure 4 Far-UV CD spectra for the wild-type and mutant forms of luciferases The concentration of protein used for the far-UV CD spectrum (200–250 nm) was 0.2 mg/ml. Each enzyme was equilibrated in 0.05 M Tris/HCl buffer (pH 7.8) at 25 ◦C. Each spectrum represents the average of five scans. The content of secondary-structure elements of the native and mutants forms are also indicated in the inset.

Figure 3

Comparison of thermostability of wild-type and mutant luciferases

The remaining activity was expressed as a percentage of the original activity. Time courses for the inactivation of the recombinant wild-type (䉬), S284T (䉱), H245N (䊉) and H431Y (䊐) luciferases are shown.

disordered structures. A decrease in fluorescence intensity was also observed for the mutant luciferases (Figure 5). Therefore the above substitutions may bring about alteration of the microenvironment of a tryptophan residue, resulting in a fluorescence intensity change which indicates displacement of the only tryptophan residue (417) to a more hydrophilic environment. Even small changes in the enzyme conformation have been proven to affect the tryptophan fluorescence of proteins, as reported previously for similar cases [40]. It is noteworthy that the H433Y (corresponding to His431 in Lampyris turkestanicus) mutant has earlier been obtained by random mutagenesis in the Japanese firefly (Luciola cruciata)

Figure 5 Intrinsic fluorescence spectra for the wild-type and mutant luciferases The spectra were measured at 25 ◦C in 0.05 M Tris/HCl buffer (pH 7.8). The protein concentration was 0.01 µg/ml. The excitation wavelength was 296 nm.

and also by site-directed mutagenesis in Luciola mingrelica [41]. It seems that the mutation of His431 which is 12 Å (1 Å = 0.1 nm) from the active site has a strong effect on the catalytic activity of the enzyme. The X-ray data for luciferase showed that His431 is located in a region containing a flexible loop Tyr425 –Phe433 [27]. The imidazole ring of His431 forms a hydrogen bond with the  c The Authors Journal compilation  c 2008 Biochemical Society

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carboxy group of Asp429 . This hydrogen bond fixes the position of the imidazole ring and increases the rigidity of the flexible loop. Calculation of the geometry of the hydrogen bonds revealed that upon replacement of the histidine residue, the hydrogen bond disappears. Therefore it is suggested that the H431Y mutation increases the flexibility of the polypeptide loop between the Nterminal and C-terminal domain of luciferase and exposes the active site to water, resulting in red light emission [41]. The crystal structure of P. pyralis firefly luciferase was used as a template to elucidate the structure of the mutant Lampyris turkestanicus luciferase. The sequence identity and the sequence similarity were 84 and 91 % respectively. Superposition of the three-dimensional structures of native and mutant luciferase revealed structures with distinct similarities, thus indicating that the mutation did not change the three-dimensional structure of luciferase as verified by molecular homology modelling, and similar interpretations to P. pyralis luciferase can be deduced for the mechanism of colour change in the mutants of Lampyris turkestanicus luciferase.

The properties of the mutants for bioluminescence applications

In a recent publication, a red-emitting form of P. pyralis (S284T) with 26 % specific activity compared with the native form has been introduced as the best candidate for bioluminescence imaging [5]. However, as the present findings testify, the H245N mutant could be another suitable red-emitting luciferase based on the composite properties including λmax = 617 nm and an astonishing high relative activity (76.6 % of that of the wildtype). Furthermore, the K m value of neither ATP nor luciferin of this mutant is significantly different from that of the wildtype. Therefore it could be considered as a possible candidate for monitoring of gene expression in in vivo conditions. In addition, another new mutant [42] with an additional arginine residue in position 354, rLuc(Arg) (recombinant mutant luciferase containing an arginine insertion), may have great potential as a bioluminescence reporter and in imaging applications owing to the fact that kinetic activity, such as substrate K m value and relative specific activity, is roughly similar to that of the wild-type. Moreover, the optimum temperature of activity for rLuc(Arg) (34 ◦C) is much higher than for the native form (24 ◦C). This is very intriguing, since the mutant luciferase emits a brighter and more stable signal at physiological temperature and makes a case for using the red-emitting rLuc(Arg) as a reporter for bioluminescence imaging and also in in vivo diagnostic applications. It should be noted that increased light emission and sensitivity have been observed in the tumours bearing thermostable luciferase [43].

Conclusions

In the present paper, we have described the red-emitting variants of luciferase from Lampyris turkestanicus, designed and produced based on sequence alignment and homology analysis of conserved residues among different beetle luciferases. Our results emphasize the importance of certain specific residues and regional structure in the determination of bioluminescence colour among firefly luciferases. The bioluminescence colours of these mutants change from yellow–green to red at pH 7.8. Owing to lower absorption of the longer wavelengths, and therefore easier diffusion through tissues, the mutant luciferases hold a promising future for in vivo imaging. Moreover, the availability of multicoloured luciferases (from green to red) could lead to the construction of multiplex biosensors and microchips which  c The Authors Journal compilation  c 2008 Biochemical Society

could allow the monitoring of multiple and spontaneous biological events. This work was supported by a grant (TMU 85-04-13) from the Research Council of Tarbiat Modares University and Department of High-Tech Industries, Ministry of Mines and Industries. We thank Mr M. R. Ganjalikhani, Mr M. Ebrahimi, Dr Maryam Nikkhah and Dr Sara Gharavi for their helpful co-operation.

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Received 31 May 2007/8 January 2008; accepted 6 February 2008 Published as BJ Immediate Publication 6 February 2008, doi:10.1042/BJ20070733

 c The Authors Journal compilation  c 2008 Biochemical Society