Il

Ma

b

a

ARRA

KLFDSB

1

dIadqiitl

L

L

L

om[

(

0h

Enzyme and Microbial Technology 51 (2012) 186– 192

Contents lists available at SciVerse ScienceDirect

Enzyme and Microbial Technology

j our na l ho me p age: www.elsev ier .com/ locate /emt

mplication of an unfavorable residue (Thr346) in intrinsic flexibility of fireflyuciferase

aryam Moradia, Saman Hosseinkhania,∗, Rahman Emamzadehb

Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IranDepartment of Biology, Faculty of Sciences, Isfahan University, Iran

r t i c l e i n f o

rticle history:eceived 13 March 2012eceived in revised form 9 June 2012ccepted 12 June 2012

eywords:uciferase

a b s t r a c t

In order to better understand the functional role of an unusual residue (Thr346) of firefly luciferasemutagenesis at this residue was performed. Firefly luciferase, catalyzes the bioluminescence reaction andis an excellent tool as a reporter in nano-system biology studies. Nonetheless, the enzyme rapidly losesits activity at temperatures above 30 ◦C and this leads to reduced sensitivity and precision in analyticalapplications. Residue Thr346 in a connecting loop (341–348) of firefly luciferase is located in a disallowedregion of Ramachandran plot. In this study, we have substituted this residue (T346) with anomalous

lexibilityihedral anglestabilityioluminescence

dihedral angles with Val, Gly and Pro to clarify the role of this residue in structure and function of theenzyme using site-directed mutagenesis. Substitution of this unfavorable residue (T346) with atypicaldihedral angles ( , ϕ) with other residues brought about an increase of thermostability and decreaseof specific activity. Structural and functional properties of the mutants were analyzed using differentspectroscopic methods. It seems that this residue is a critically conserved residue to support the functionalflexibility for a fast kinetic bioluminescence reaction at the expense of lower stability.

. Introduction

The production and emission of light by a living organism isue to a type of chemical reaction called bioluminescence [1].

n Photinus pyralis, a beetle in the superfamily of Cantharidae, monomeric luciferase (EC.1.13.12.7) is responsible for the pro-uction of yellow–green light (�max ≈ 560 nm) with a remarkableuantum yield [2,3]. It is located in specialized peroxisomes [4]

n an evolutionarily developed organ known as the lantern of thensect [5]. As indicated in Eqs. (1)–(3), the luciferase enzyme func-ions as a monooxygenase by catalazing a two-step oxidation ofuciferin to produce light, oxyluciferin, CO2, and AMP [5,1]

uc + LH2 + ATP → Luc·LH2-AMP + PPi (1)

uc·LH2-AMP + O2 → Luc·AMP·Oxyluciferin ∗ + CO2 (2)

uc·AMP·Oxyluciferin∗ → Luc + Oxyluciferin + AMP + h� (3)

Firefly luciferase has been extensively utilized for a number

f applications, particularly for analyzing of gene expression [6],onitoring of in vivo protein folding and chaperonin activity

7], non-invasive imaging of tumor growth [8] and metastasis in

∗ Corresponding author. Tel.: +98 21 82884407; fax: +98 21 82884484.E-mail addresses: saman h@modares.ac.ir, shosseinkhani@yahoo.com

S. Hosseinkhani).

141-0229/$ – see front matter © 2012 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.enzmictec.2012.06.002

© 2012 Elsevier Inc. All rights reserved.

whole animals [9], measuring microbial contamination [10], andas a reporter for efficiency of gene and drug transfer and ther-apy [11,12]. In spite of wide range of luciferase applications, someinherent properties limit further application and development ofluciferase-based technologies, including the low stability of theenzyme, a low turnover number, and a high Km for the substrate ATP[13]. The crystal structure of P. pyralis luciferase without bound sub-strates was shown a unique molecular architecture consisting of alarge N-terminal domain (residues 1–436) and a small C-terminaldomain (residues 440–550), which are separated by a wide cleft[14]. Conformational analysis in backbone dihedral angles (ϕ, )space was revealed that there is one sterically disallowed residue(ϕ = 74◦, = −65◦) in the luciferase structure [14]. Thr346 in the341–348 loop is the only firefly luciferase residue located in a dis-allowed region of Ramachandran plot. Energetically unfavorableconformations are rare in proteins and are usually in regions of thestructures involved in function [15].

GN Ramachandran developed a method to visualize backbonedihedral angles against ϕ of amino acid residues in protein struc-ture known as the Ramachandran plot. The Ramachandran plot is auseful tool for the analysis of the stereochemistry of the polypep-tide chain backbone in protein structures.

The relative configuration of two linked peptide residues ateach alpha carbon atom in the main chain can be specified by givingthe orientation of the two dihedral angles ϕ and (called ϕ andϕ′ by Ramachandran) by which they are rotated about the bonds

crobia

N ipv

fiEsssbwrtsaedRhtctc

ctsc

2

2

�Ak(

2

dmp

(

i6wDuad

2

ap

2

b

M. Moradi et al. / Enzyme and Mi

-C� and C�-C′ respectively [16,17]. The range of accessible ϕ and values at each non-Gly amino acid residue in a protein structure

s sharply restricted by local sterically unfavorable interactions. Inrotein structures the majority of non-Gly residues adopt ϕ and alues in allowed regions of the Ramachandran map [15,18–21].

However, it is evident that individual residues are occasionallyound in forbidden areas [22], suggesting that unfavorable localnteractions may be offset by other compensating factors [23,24].xpeditions into the Ramachandran disallow regions may cause atrain of up to at least 5 kcal/mol [15]. An amino acid may toleratemall deviations from its ideal conformations in order to optimizetabilizing tertiary interactions in the protein, such as hydrogenonding or keeping hydrophobic residues buried or interactionsith the substrate or ligand at the active site [25]. Disallowed

esidues (0.4% of all non-Gly residues) cluster in five regions ofhe Ramachandran map and in most of the cases, the unusualtereochemistry was conserved in related protein structures. Prond the branched aliphatic residues (Val, Ile and Leu) do not gen-rally adopt such distortions [22,25]. An analysis of selected 66isallowed residues (non-Gly), clustered in distinct regions of theamachandran map, revealed that while Ser, Asn, Thr, and Cysave the highest propensities to exhibit unusual conformations,he branched aliphatic residues show the lowest tendency for suchonformations [22]. Disallowed residues are usually located closeo the surface of the molecule and in short loops, being in manyases at the junction of two secondary structural elements [25].

In this study, In order to better understand the importance of theonformational strain of Thr346 on luciferase structure and func-ion, we have replaced it by Gly, Val, and Pro. The changes in proteintability, the effects on the Kinetic properties, as well as structuralhanges of the mutants are evaluated.

. Experimental procedures

.1. Materials

The following materials were obtained from the indicated sources: isopropyl--d-thiogalactopyranoside (IPTG), ATP (Roche); d-luciferin potassium salt (Resem);NS, 8-anilino-aphthalene-1-sulfonic acid (Merck, Germany); plasmid extractionit, gel purification kit, PCR purification (Bioneer CO. South Korea), Ni-NTA spin kitQIAGEN Inc.).

.2. Site-directed mutagenesis

For the construction of single mutants including T346G, T346V, T346P, site-irected mutagenesis was performed using the Quick-change PCR. The desired pointutants for interesting position was generated by site-directed mutagenesis on

ET16b-luc using the following mutagenesis primers:

T346V 5′-GG CTC ACT GAG ACT GTT TCT GCT ATT CTG ATT AC-3′;T346P 5′-GG CTC ACT GAG ACT CCA TCT GCT ATT CTG ATT AC-3′;T346G 5′-GG CTC ACT GAG ACT GGT TCT GCT ATT GTG ATT AC-3′

where underline represents the mutated codon).PCR was carried out using Pfu DNA polymerase under the following condition:

nitial denaturation at 95 ◦C for 1 min, 18 cycles (95 ◦C for 1 min, 55 ◦C for 1 min and8 ◦C for 11 min), and a final extension for 10 min at 68 ◦C. PCR products were treatedith Dpn I (37 ◦C, overnight) restriction endonuclease to eliminate the methylatedNA templates. Subsequently, the mutagenesis products were purified using a cleanp kit to remove redundant primers. Thereafter, DH5� competent cells were useds cloning hosts for the transformation of the nicked vector DNA containing theesired mutations.

.3. Sequencing

pET16-b containing native and mutant luciferases were sequenced using anutomatic sequencer (MWG, Germany) by T7 promoter and T7 terminator universalrimers.

.4. Expression and purification of recombinant luciferase

Ten ml of terrific broth (TB) medium containing 50 �g/ml ampicilin with a freshacterial colony harboring the expression plasmid was inoculated and grown at

l Technology 51 (2012) 186– 192 187

37 ◦C overnight. Then 250 ml of medium with 500 �l overnight cultures was inocu-lated and grown at 37 ◦C with vigorous shaking until the OD600 reaches 0.8. Glycerolstocks of cells were prepared in 10% (v/v) glycerol and stored at −80 ◦C. After which,IPTG and lactose were added to a final concentration of 1 mM and 4 mM, respectively,to the solution and incubated at 22 ◦C overnight with vigorous shaking. Followingovernight induction, the cell pellets were collected by centrifugation (12,000 × gfor 15 min at 4 ◦C) and resuspended in 10 ml lysis buffer (5 mM imidazole, 50 mMTris–HCl, 300 mM NaCl, pH 7.8). The cells were sonicated to disrupt the bacterial cellson ice to prevent excessive heating of the lysate. The cell lysate was clarified by cen-trifugation (18,000 × g for 25 min at 4 ◦C). The native and mutant luciferases werepurified by Ni-Sepharose affinity chromatography as described by the manufac-turer (Novagen, USA). The purified enzymes had purities of greater than 95% basedon analysis by SDS-polyacrylamide gel electrophoresis. Concentrations of nativeand mutant forms of luciferase were determined by the Bradford method [26]. Thecolor of the emitted light of the purified firefly luciferase reaction was obvious afteraddition of luciferin and Mg+2-ATP to purified luciferases.

2.5. Specific activity measurements

Specific activity values were measured using a Sirius Single Tube luminome-ter (Berthold Detection Systems, Germany) by integration of average of total lightemitted in 10 s. Reactions were initiated by the injection of 2.5 �l purified enzymesto 25 �l standard solution [2 mM luciferin, 4 mM ATP, and 10 mM MgSO4 in 50 mMTris–HCl (pH 7.8)] and 22.5 �l Tris–HCl buffer at 25 ◦C, and total light units weremeasured in 1 s.

2.6. Measurement of bioluminescence emission spectra

Bioluminescence emission spectra of purified enzymes were obtained usingCary-Eclipse luminescence spectrophotometer (Varian) from 400 to 700 nm wave-lengths. Reactions were initiated by addition of 300 �l of 50 mM Tris–HCl buffer (pH7.8), including 2 mM ATP, 5 mM MgSO4, and 1 mM luciferin, to 100 �l of a purifiedluciferase solution in a quartz cell.

2.7. Characterization of kinetic parameters

Km value for ATP was determined by assaying 5 �l of diluted enzyme in 25 �l ofassay reagent, including 10 mM MgSO4 and 1 mM luciferin in 50 mM Tris–HCl (pH7.8) in addition of 20 �l of the various concentration of ATP (from 0.004 to 8 mM). Theestimation of LH2 kinetic constants was performed in a similar way, except that 25 �lthe assay reagent containing 10 mM MgSO4, 2 mM ATP in 50 mM Tris–HCl pH 7.8 inaddition of 20 �l of the various concentration of luciferin (from 0.01 to 2.5 mM) wereused. Light emission recorded over 1 s (Sirius tube luminometer, Berthold DetectionSystem, Germany) and kinetic constants were calculated according to the methodof Hanes–Woolf plot [27].

The time of light decay were measured by injection of 5 �l substrate solutions(10 mM MgSO4, 1 mM luciferin, 2 mM ATP, 50 mM Tris–HCl, pH 7.8) to 5 �l nativeand mutant purified enzymes in 2 min. The residual activity for each enzyme wasreported as a percentage of the original activity. To obtain the optimal temperatureof activity for native and mutant luciferases, activities were measured in the rangeof 10–45 ◦C. Moreover, the optimum pH of activity for these enzymes was measuredby incubation of enzyme in a mixed buffer in the pH range of 6–11.5.

2.8. Thermal inactivation and thermal stability studies

For the measurement of thermal stability, the purified luciferases (10 �g/ml)were incubated in the range of 20–45 ◦C (intervals 5 ◦C) for 5 min. Enzyme activi-ties were measured at room temperature (25 ◦C), and the remaining activity wasrecorded as percentage of the original activity after incubation for 5 min in ice.Thermal inactivation rates of the luciferases were studied by incubating enzymesolutions in a circulating water bath at 30 ◦C. At regular intervals (0–45 min), sampleswere removed and cooled on ice (5 min), and the remaining activity were recordedas a percentage of the original activity. The activity of the enzyme solution kept onice was considered as the control (100%).

2.9. Fluorescence measurements

The purified luciferases were dialyzed in dialysis buffer containing 50 mMTris–HCl, 1% glycerol, 1 mM EDTA, 150 mM NaCl, 2 mM �-mercaptoethanol, and0.8 mM ammonium sulfate (pH 7.8) at 4 ◦C.

Fluorescence studies were carried out on a Cary-Eclipse luminescence spec-trophotometer (Varian). Intrinsic fluorescence was determined using 20 �g/mlprotein and an excitation wavelength of 295 nm. Emission spectra were recordedbetween 300 and 400 nm [28]. Extrinsic fluorescence studies were carried out with8-anilino-1-naphthalenesulfonic acid (ANS) as a fluorescence probe. Measurements

were taken on the same spectrofluorometer that was used for intrinsic fluorescencestudies. All experiments were carried out at 25 ◦C. The final concentration of theANS in the enzyme solutions was 30 �M, and the molar ratio of protein to ANS was1:30. The ANS emission was scanned between 380 and 700 nm with an excitationwavelength of 350 nm [29,30].

188 M. Moradi et al. / Enzyme and Microbial Technology 51 (2012) 186– 192

F Protein Data Bank). Position 346 is shown in purple. (For interpretation of the referencest .)

2

qocSF

2

Jpweaumats

3

3m

forcamIoSfew

3

la

properties

Kinetic properties [relative specific activity, optimum pH andoptimum temperature of activity, in addition to Km for luciferin

ig. 1. Distribution of observed dihedral angles ϕ, in firefly luciferase (Brookhaveno color in this figure legend, the reader is referred to the web version of the article

.10. Dynamic quenching

Fluorescence quenching was carried out via the addition of KI (as an ionicuencher) to dialyzed protein solutions (20 �g/ml) at an excitation wavelengthf 295 nm and an emission wavelength of 300-450 nm in a Cary-Eclipse lumines-ence spectrophotometer (Varian). Quenching data were analyzed in terms of thetern–Volmer constant, KSV, which was computed from the Stern–Volmer equation0/F = 1+ KSV[Q], where [Q] is the molar concentration of the quencher [31].

.11. Circular dichroism (CD) measurements

The CD spectra of mutant and native enzymes at pH 7.8 were measured on aASCO J-715 spectropolarimeter (Japan) using solutions with purified and dialyzedrotein concentrations 0.2 mg ml−1 (far-UV) at room temperature. Protein bufferas the same as fluorescence measurements .The results were expressed as molar

llipticity, [�] (deg cm2 dmol−1), based on a mean amino acid residue weight (MW)ssuming its average weight for firefly luciferase. The molar ellipticity was calculatedsing the equation: [�] = (� × 100MW)/(cl), where c is the protein concentration ing ml−1, l is the light path length in cm, and � is the measured ellipticity in degrees

t wavelength �. The CD spectra were smoothed using the noise reduction rou-ines provided with the J-710 spectropolarimeter, as well as the contents of regularecondary structure were analyzed.

. Results

.1. Construction, expression, and purification of the native andutant luciferases

The crystal structure of firefly luciferase indicates a strained con-ormation in the presence of a threonine at position 346 (Fig. 1). Inrder to examine the effect of various amino acid substitutions atesidue 346, a set of studies using site-directed mutagenesis wasarried out. In this investigation, Thr346 was substituted with threemino acids: Glycin, Valine, and Proline. After PCR and transfor-ation, amino acid substitutions were confirmed by sequencing.

n order to purify and characterize native and mutant luciferases,verexpression was carried out in E. coli BL21. Affinity (Ni-NTA-epharose) chromatography was used to purify the His6-taggedusion luciferases. To estimate purity of the collected fractions, thelution fractions were analyzed by SDS-PAGE in which luciferasesere present as a band of 62 kDa (Fig. 2).

.2. Bioluminescence emission spectra

The in vitro bioluminescence spectra of native and mutantuciferases are shown in Fig. 3. The native and all mutants exhibit

similar spectrum with only a peak at 557 nm (Fig. 3).

Fig. 2. SDS–PAGE of purified wild-type (P. pyralis) and mutant luciferases afterpurification via affinity Ni-NTA column chromatography. Lane M is the molecularmarker.

3.3. Influence of the amino acids substitution on the kinetic

Fig. 3. Bioluminescence emission spectra produced by the native from P. pyralis(1) and mutant [(2) Val346, (3) Gly346 and (4) Pro346] luciferases by a luciferase-catalyzed reaction at pH 7.8.

M. Moradi et al. / Enzyme and Microbial Technology 51 (2012) 186– 192 189

Table 1Kinetic properties of the wild-type and mutant luciferases.

Enzyme Specific activity (×1010 RLU s−1 mg−1) Relative activity (%) Km (�M)a Optimum

ATP LH2 Temperature pH

Ppy 2.9 100 125 4 25 8T346G 1.7 60.77 175 2.5 25 8T346V 1.2 43.56

T346P 2.1 72.19

a The error associated with the Km values falls within ± 10% of the value.

Fa

(s

as(Aat

cTitatwcTWsl

The results are indicative of the conformational changes in

Fw

ig. 4. Comparison of decay times of native (1) and mutant [(2) T346P, (3) T346V,nd (4) T346G] luciferases. For further details, see Experimental procedures.

LH2 Km) and ATP (ATP Km)] of native and mutant luciferases areummarized in Table 1.

Km values for D-LH2 and ATP were determined for the mutantsnd WT by Hanes-Woolf plot. The T346G and T346P mutants havehown lower Km values for luciferin than the wild-type enzyme,0.62- and 0.7-fold, respectively). The Km value of Val346 for Mg+2-TP increased 2.3 times, while that for Pro346 and Gly346 arepproximately similar to each other and around 1.5 times higherhan native.

The time of light decay was measured by injection of 10 �locktail [2 mM luciferin, 4 mM ATP, and 10 mM MgSO4 in 50 mMris–HCl (pH 7.8)] to 10 �l of native and mutant enzymes. Accord-ng to the result, the decay time for mutant luciferases is faster thanhat for the native form (Fig. 4). While the optimum temperature ofctivity for native and mutants are identical with except to T346Phat decreased to 20 ◦C, a clear improvement in thermostabilityas observed for mutants T346V and T346G mutant luciferases

ompared to the native P. pyralis as indicated in Fig. 5A. Moreover,346P and T346V mutants exhibit a similar inactivation profile to

T luciferase, while T346G mutant was inactivated substantially

lower than WT luciferase (Fig. 5B). Optimum pH values for mutantuciferases did not exhibit any difference (Table 1).

ig. 5. Comparison of thermal stability (A) and thermal inactivation (B) of native ( ) anith each point falls within 5% of the value. For further details, see Experimental procedu

285 5 25 8175 2.8 20 8

Integrated specific activity values (RLU s−1 mg−1) were deter-mined from estimation of the total light output in the biolumines-cence assay conducted under optimum conditions for each enzyme.Specific activity for all mutants decreased compared with native(Table 1).

3.4. Structural characterization

In other to discriminate the conformational changes in mutantand native (P. pyralis) forms, intrinsic and extrinsic fluorescencestudies were carried out.

3.5. Intrinsic fluorescence

Fluorescence spectroscopy is widely used as an effective toolto monitor luciferase conformational changes because tryptophanfluorescence is highly sensitive to the environment polarity [32,33].Higher fluorescence intensity can be seen upon displacement ofTrp(s) to a hydrophobic environment (buried within the core of theprotein). In contrast, in a hydrophilic environment (Trp is exposedto solvent) their quantum yield decreases leading to low fluores-cence intensity. Furthermore, tryptophan fluorescence is stronglyinfluenced by nearby protonated groups such as Asp or Glu thatthey can cause quenching of Trp fluorescence. In this study, Trpwas excited selectively at 295 nm wavelength.

P. pyralis luciferase has two tryptophans, Trp417 and Trp426,which are located near the protein surface of N-terminal domain.As shown in Fig. 6, T346V, T346P and T346G mutants exhibitedincreases and decreases in intensity compared to the native form(P. pyralis), respectively.

mutant forms compared to the native form, presumably resulteda less hydrophobic environment for both Trps in T346P and a lesspolar region for those in T346V.

d mutant [T346G (*), T346V ( ), and T346P ( )] luciferases. The error associatedres.

190 M. Moradi et al. / Enzyme and Microbial Technology 51 (2012) 186– 192

Fig. 6. Intrinsic fluorescence spectra for wild-type and mutant luciferases. The spec-t ◦

wT

ie

3

atrErLw

3

irIiosfoaq[

F[iA

Fig. 8. Stern–Volmer plots of native ( ) and mutant [T346V ( ), T346P ( ), andT346G (*)] luciferases obtained by quenching with acrylamide. The excitation andemission wavelengths were 295 and 340 nm, respectively. The protein was dissolvedin 50 mM Tris–HCl buffer (pH 7.8), and the protein concentration was 30 �g/ml inall samples.

Fig. 9. Far-UV CD spectra for the wild type and mutant forms of luciferases [T346P

ra were taken at 25 C in Tris–HCl buffer (50 mM, pH 7.8). The protein concentrationas 20 �g/ml. The excitation wavelength was 295 nm (1: T346V, 2: P. pyralis, 3:

346G, 4: T346P).

Therefore, this result of intrinsic fluorescence may be usefuln illustrating the potential structural changes taking place in thenzyme induced by insertion of new residues (Fig. 6).

.6. Extrinsic fluorescence

It is known that ANS, a valuable probe for the detection andnalysis of conformational changes in proteins, bind preferentiallyo hydrophobic cavities on the protein surface with greater affinityesulting in a detectable blue shift in its fluorescence spectrum.nhancement of ANS fluorescence has been previously shown toeflect the level of hydrophobic patches on luciferase structure [34].oss of hydrophobic clusters on current mutant forms of Thr346as confirmed by fluorescence measurement using ANS (Fig. 7).

.7. Fluorescence quenching by acrylamide

The results of quenching of tryptophan fluorescence exper-ments allowed us to differentiate the location of tryptophanesidues and also to assess protein conformational flexibility [35].n order to reveal the differences in the accessibility and flexibil-ty of native and mutant luciferases, we carried out experimentn quenching of tryptophan fluorescence with acrylamide. Fig. 8hows the Stern–Volmer plots for quenching of native and mutantorms of firefly luciferase by acrylamide. It is shown that flu-rescence of Val346 mutant is quenched quite effectively by

crylamide, which indicates higher accessibility of tryptophan touencher molecules and exposure of fluorophores to the solvent36] (Fig. 8).

ig. 7. Fluorescence spectra of ANS (5) in the presence of native (1) and mutant(2) T346V, (3) T346P, and (4) T346G] luciferases. Spectra were recorded at 25 ◦Cn 50 mM Tris–HCl buffer (pH 7.8). The sample contained 1 �M protein and 30 �MNS. The excitation wavelength was 350 nm.

(1), T346V (2), P. pyralis (3) and T346G (4)]. The concentration of protein used for thefar-UV CD spectrum (200–250 nm) was 0.2 mg ml−1. Each enzyme was equilibratedin Tris–HCl buffer (50 mM, pH 7.8) at 25 ◦C.

3.8. CD spectra of native and mutant forms of luciferase

The CD spectra obtained in Tris–HCl buffer (0.05 M, pH 7.8) at25 ◦C (Fig. 9) show changes in the secondary structure content ofthe T346P and T346V mutants in comparison to native.

4. Discussion

The backbone dihedral angles (ϕ and �) have considerableimportance in protein tertiary structure. Although they vary from−180◦ to +180◦ [37], It is clear that all values of ϕ and may not beallowed due to the steric hindrance between the backbone atomsor between backbone and the C� carbon atom of side chains [22].The Ramachandran plot illustrates the sterically allowed regions ofthe dihedral angles [38].

In P. pyralis firefly luciferase, Thr346 is located in the loop con-necting the antiparallel strands 7 and 8 of �-sheet B. This residueis placed in a disallowed region of the Ramachandran plot (ϕ = 74◦, = −65◦) [14].

To assess the importance of the conformational strain in thisposition, we have replaced Thr346 by Gly, Val, and Pro. The effectson the kinetic parameters and protein stability of the three mutantsare studied.

Bioluminescence emission spectra of three mutants are similar

to the native luciferase without any shift in maximum wavelengthof light emission (Fig. 3). Therefore, substitution of these aminoacids do not affect the solvent accessibility to emitter site and thecritical residues involved in color determination are conserved [39].

crobia

T(pbcre

lfffdtet

TmtthmmdTbl

iwirspofc

Amp

mflmtwafAsf

tprmlalcTcAc

[

[

[

[

[

[

[

[

[

[

[

[

[

M. Moradi et al. / Enzyme and Mi

he rate of decay of native and mutant luciferases was measuredFig. 4), which shows higher rates of decay for mutant luciferasesresumably due to higher luciferase inhibition using by-products ofioluminescence reaction [40,41]. However, it should be noted thathange of decay rates may change quantum efficiency of luciferaseeaction. The direct relationship between color shift and quantumfficiency have been shown [42].

As shown in Table 1, the Km values of T346G and T346P foruciferin were decreased, indicating higher affinity of substratesor the mutant enzymes, but the Km values of mutant luciferasesor ATP were increased, indicating lower affinity of the substratesor the mutant enzymes. Optimum temperature of T346P wasecreased to 20 ◦C and for the other mutants was identical withhe native enzyme. The other basic kinetic properties of the mutantnzymes were relatively similar to those of native form. Moreover,he relative specific activity for all mutant forms was decreased.

As shown in Fig. 5A and B, the thermostability of T346G and346V mutant forms of P. pyralis luciferase were significantlyore than the wild-type enzyme. Several mechanisms in kinetic

hermostability of enzymes have been proposed [43]. Amonghem proteins aggregation, a process that is highly dependent onydrophobic clusters located on the protein surface, is a commonechanism of inactivation. Reducing surface hydrophobicity, uponutation at position 346 as confirmed by ANS fluorescence (Fig. 7),

ecreases sensitivity of P. pyralis luciferase to protein aggregation.herefore, it seems the thermal stability of luciferase is increasedy decreasing the aggregation-prone structure of P. pyralis

uciferase.To evaluate the effect of these substitutions on the local polar-

ty of the emitter (Trp) site, purified native and mutant luciferasesere compared by fluorescence spectroscopy. As can be seen

n Fig. 6, the fluorescence intensity is increased for Val346 andemarkably decreased for Pro346 mutant luciferase, suggesting aubstantial alteration in their conformations, which indicate dis-lacement of Trp (s) to more hydrophobic environment (Val346)r polar environment (Pro346). Alteration in the emission spectrarom tryptophan can be seen in response to the local environmenthanges surrounding the indole ring of Trp.

On the other hand, according to the extrinsic fluorescence ofNS, it may be concluded that hydrophobic patches on surface ofutant luciferases are displaced into the hydrophobic interior of

rotein which become less accessible to ANS binding (Fig. 7).Nevertheless, the distribution of hydrophobicity inside the

utants is not uniform. Therefore, comparison of the intrinsicuorescence spectrum (Fig. 6) indicates that the microenviron-ent around the Trp in T346V luciferase is more hydrophobic than

he other mutants. Furthermore, it seems that structural changesithin the protein make minor changes in spatial arrangement of

mino acid residues around the emitter site. Therefore, a brief dif-erence in kinetic properties of mutated luciferases is seen (Table 1).ccording to fluorescence spectroscopy results, however, it may beuggested higher thermostability of mutant luciferases may ariserom lower surface hydrophobicity upon mutation of T346 [44].

In order to determine the accessibility of the Trp residues inhe luciferases, quenching of the tryptophan fluorescence of P.yralis luciferases with acryl amide was carried out. The quenchingesults (Fig. 8) indicated the tryptophan fluorescence of the Val346utant was more effectively quenched with acrylamide. Although

ocated close to the surface of the protein, both tryptophans, Trp417nd Trp426, are not surface residues. Therefore, any change in theoop containing residue 346 can be associated with conformationalhanges and therefore, lead to quenching of these tryptophans.

rp417 is located inside the cavity surrounded by positivelyharged His419, Arg393, Lys372 and negatively charged Glu370,sp413, and Asp415 residues; therefore, it is not accessible forharged quencher and its fluorescence is quenched by acrylamide

[

l Technology 51 (2012) 186– 192 191

only. In contrast, Trp426 is located closer to the protein surface thanTrp417 and is accessible to acrylamide and iodide ions [45].

In conclusion, according to the result presented in thismanuscript, substitution of an unfavorable residue (T346) withanomalous dihedral angles ( , ϕ) with similar residues broughtabout with the increase of thermostability and decrease of specificactivity. However, it should be emphasized this residue with simi-lar odd dihedral angles is observed in L. crutiata and L. turkestanicus(unpublished data) luciferases. Therefore, it may be concluded, thisresidue is a critical conserved residue to support high flexibilityof firefly luciferase for a fast kinetic of bioluminescence reaction.Moreover, this unfavorable residue is kept at the expense of lowerthermostability.

Acknowledgments

This work was supported by a grant from the Iranian NationalScience Foundation (INSF). Research Council of Tarbiat ModaresUniversity is acknowledged for financial support of this work.

References

[1] DeLuca M. Firefly luciferase. Advances in Enzymology and Related Areas ofMolecular Biology 1976;44:37–68.

[2] Ugarova NN. Luciferase of Luciola mingrelica fireflies. Journal of Biolumines-cence and Chemiluminescence 1989;4:406–18.

[3] Ando Y, Niwa K, Yamada N, Enomoto T, Irie T, Kubota H, et al. Firefly biolu-minescence quantum yield and color change by pH-sensitive green emission.Nature Photonics 2008;2:44–7.

[4] McElroy WD. Methods in Enzymology 1955;2:851–6.[5] White EH, Rapaport E, Seliger HH, Hopkins TA. The chemi- and bioluminescence

of firefly luciferin:an efficient chemical production of electronically excitedstates. Bioorganic Chemistry 1971;1:92–122.

[6] Shapiro E, Lu C, Baneyx F. Protein Engineering, Design and Selection2005;18:581–7.

[7] Frydman J, Nimmesgern E, Erdjumentbromage H, Wall JS, Tempst P. EMBOJournal 1992;11:4767–78.

[8] Dickson PV, Hamner B, Ng CYC, Hall MM, Zhou J. In vivo bioluminescence imag-ing for early detection and monitoring of disease progression in amurine modelof neuroblastoma. Journal of Pediatric Surgery 2007;42:1172–9.

[9] Dothager RS, Flentie K, Moss B, Pan MH, Kesarwala A. Advances in biolumi-nescence imaging of live animal models. Current Opinion in Biotechnology2009;20:45–53.

10] Lundin A, Deluca M. Bioluminescence and Chemiluminescence. AcademicPress; 1981. pp. 187–196.

11] Shin JH, Yi JK, Kim AL, Park MA, Kim SH, Lee H, et al. Oncology Reports2003;10:2063–9.

12] Rehemtulla A, Hall DE, Stegman LD, Prasad U, Chen G, Bhojani MS, et al. Molec-ular Imaging 2002;1:43–55.

13] White PJ, Squirrell DJ, Arnaud P, Lowe CR, Murray JAH. Biochemical Journal1996;319:343–50.

14] Conti E, Franks NP, Brick P. Crystal structure of firefly luciferase throws light ona superfamily of adenylate-forming enzymes. Structure 1996;4:287–98.

15] Herzberg O, Moult J. Analysis of the steric strain in the polypeptide backbone ofprotein molecules. Proteins: Structure, Function and Genetics 1991;11:223–9.

16] Ramachandran GN, Ramakrishnan C, Sasisekharan V. Stereochemistry ofpolypeptide chain configurations. Journal of Molecular Biology 1963;7:95–9.

17] Ramakrishnan C, Ramachandran GN. Stereochemical criteria for polypeptidechain conformations. Allowed conformations for a pair of peptide units. Bio-physical Journal 1965;5:909–33.

18] MacArthur MW, Laskowski RA, Thornton JM. Knowledge-based validation ofprotein structure coordinates derived by X-ray crystallography and NMR spec-troscopy. Current Opinion in Structural Biology 1994;4:731–7.

19] Morris AL, MacArthur MW, Hutchinson EG, Thornton JM. Stereochemical qual-ity of protein structure coordinates. Proteins: Structure, Function and Genetics1992;12:345–64.

20] Thornton JM. Lessons from analyzing protein structures. Current Opinion inStructural Biology 1992;2:888–94.

21] Weaver LH, Tronrud DE, Nicholson H, Matthews BW. Some uses of theRamachandran (ϕ, ) diagram in the structural analysis of lysozymes. CurrentScience 1990;59:833–7.

22] Gunasekaran K, Ramakrishnan C, Balaram P. Disallowed Ramachandran con-

formations of amino acid residues in protein structures. Journal of MolecularBiology 1996;264:191–8.

23] Jia Z, Quail JW, Waygood EB, Delbaere LT. Active-centre torsion-angle strainrevealed in 1.6 A-resolution structure of histidine-containing phosphocarrierprotein. The Journal of Biological Chemistry 1993;268:22490–501.

1 crobia

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

92 M. Moradi et al. / Enzyme and Mi

24] Stites WE, Meeker AK, Shortle D. Evidence for strained interactions betweenside-chains and the polypeptide backbone. Journal of Molecular Biology1994;235:27–32.

25] Chakrabarti P, Pal D. On residues in the disallowed region of the Ramachandranmap. Biopolymers 2002;63:195–206.

26] Bradford MM. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. AnalyticalBiochemistry 1976;72:248–54.

27] Hanes CS. Studies on plant amylases. I. The effect of starch concentration uponthe velocity of hydrolysis by the amylase of germinated barley. BiochemicalJournal 1932;26:1406–21.

28] Mortavavi M, Hosseinkhani S. Design of thermostable luciferases through argi-nine saturation in solvent-exposed loops. Protein Engineering, Design andSelection 2011;24(12):893–903.

29] Riahi-Madvar A, Hosseinkhani S. Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis. Protein Engineering,Design and Selection 2009;22:655–63.

30] Semisotnov GV, Rodionova NA, Razgulyaev OI, Uversky VN, Gripas AF, Gilman-shin RI. Study of the molten globule intermediate state in protein folding by ahydrophobic fluorescent probe. Biopolymers 1991;31:119–28.

31] Eftink MR, Ghiron Ca. Exposure of tryptophanyl residues and protein dynamics.Biochemistry 1977;16:5546–51.

32] Nazari M, Hosseinkhani S. Design of disulfide bridge as an alternative mecha-nism for color shift in firefly luciferase and development of secreted luciferase.Photochemistry & Photobiological Sciences 2011;10(7):1203–15.

33] Amini-Bayat Z, Hosseinkhani S, Jafari R, Khajeh Kh. Relationship between sta-

bility and flexibility in the most flexible region of Photinus pyralis luciferase.Biochimica et Biophysica Acta 2012;1824:350–8.

34] Moradi A, Hosseinkhani S, Naderi-Manesh H, Sadeghizadeh M, Alipour BS.Effect of charge distribution in a flexible loop on the bioluminescence colorof firefly luciferases. Biochemistry 2009;48:575–82.

[

l Technology 51 (2012) 186– 192

35] Lakowicz JR. Topics in Fluorescence Spectroscopy – Biological Applications.New York: Plenum Press; 1992. p. 3.

36] D’Amico S, Marx JC, Gerday C, Feller G. Activity–stability relationshipsin extremophilic enzymes. Journal of Biological Chemistry 2003;278:7891–6.

37] Hirst DJ, Kountouris P. Prediction of backbone dihedral angles and pro-tein secondary structure using support vector machines. BMC Bioinformatics2009:2009.

38] Ramachandran GN, Sasisekharan V. Conformation of polypeptides and proteins.Advances in Protein Chemistry 1968;23:283–438.

39] Hosseinkhani S. Molecular enigma of multicolor bioluminescence of fireflyluciferase. Cellular and Molecular Life Sciences 2011;68:1167–82.

40] Emamzadeh R, Hosseinkhani S. RACE-based amplification of cDNA andexpression of a luciferin-regenerating enzyme (LRE): an attempt towards per-sistent bioluminescent signal. Enzyme and Microbial Technology 2010;47:159–65.

41] da Silva LP, da Silva JCGE. Kinetics of inhibition of firefly luciferase bydehydroluciferyl-coenzyme A, dehydroluciferin and l-luciferin. Photochem-istry & Photobiological Sciences 2011;10:1039–45.

42] Niwa K, Ichino Y, Kumata S, Nakajima Y, Hiraishi Y, Kato D, et al. Quantumyields and kinetics of the firefly bioluminescence reaction of beetle luciferases.Photochemistry and Photobiology 2010;86:1046–9.

43] Vieille C, Zeikus GJ. Hyperthermophilic enzymes:sources,uses,and molec-ular mechanisms for thrmodtability. Microbiology & Molecular Biology2001;65:1–43.

44] Law GH, Gandelman OA, Tisi LC, Lowe CR, Murray JA. Mutagenesis of solvent-

exposed amino acids in Photinus pyralis luciferase improves thermostabilityand pH-tolerance. Biochemical Journal 2006;397(2):305–12.

45] Dementieva EI, Fedorchuk EA, Brovko LYu, Savitskii AP, Ugarova NN. Fluo-rescent properties of firefly luciferases and their complexes with luciferin.Bioscience Reports 2000;20:21–30.