Biochem. J. (2008) 412, 27–33 (Printed in Great Britain)
27
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
*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
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
28
N. Kh. Tafreshi and others
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 =
29
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
30
N. Kh. Tafreshi and others
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
31
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
32
N. Kh. Tafreshi and others
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