Structurally modified firefly luciferase. Effects of amino acid substitution at position 286

Article abstract of DOI:10.1021/ja971927a

Ser was replaced at position 286 of firefly luciferase (Luciola mingrelica) by a series of naturally occurring and unnatural amino acids. The effect of these substitutions on the properties of luciferase, such as thermostability, pH dependence, and color

Full text of DOI:10.1021/ja971927a

VOLUME 119, NUMBER 45
NOVEMBER 12, 1997
© Copyright 1997 by the
American Chemical Society
Structurally Modified Firefly Luciferase. Effects of Amino
Acid Substitution at Position 286
Tuncer Arslan, Sergey V. Mamaev, Natalia V. Mamaeva, and Sidney M. Hecht*
Contribution from the Departments of Chemistry and Biology, UniVersity of Virginia,
CharlottesVille, Virginia 22901
ReceiVed June 11, 1997X
Abstract: Ser was replaced at position 286 of firefly luciferase (Luciola mingrelica) by a series of naturally occurring
and unnatural amino acids. The effect of these substitutions on the properties of luciferase, such as thermostability,
pH dependence, and color of light emitted, was investigated. For these purposes, the Ser286 codon (AGT) was
replaced by an amber stop codon (TAG) within the luciferase gene and transformed into Escherichia coli strains
producing specific amber suppressor tRNA’s to express luciferase with different substitutions at this position. The
incorporation of Leu, Lys, Tyr, or Gln at this position reduced the thermostability of mutated luciferases. The color
of emitted light changed upon substitution from yellow-green (λmax 582 nm) for the wild-type enzyme having Ser286
to, for example, red (λmax 622 nm) for luciferase having Leu286. For further evaluation of the structural relationship
between the amino acid position at 286 and the wavelength of emitted light, we used the method of in Vitro
incorporation of unnatural amino acids, which involves readthrough of a nonsense (UAG) codon by a misacylated
suppressor tRNA. The amino acids incorporated at position 286 in this fashion included O-glucosylated serine,
serine phosphonate, tyrosine phosphate, and tyrosine methylenephosphonate. The wavelength of light emitted by
the luciferase analogues was measured. While the introduction of serine phosphonate and glucosylated serine did
not change the λmax of light produced by luciferase, the incorporation of tyrosine phosphate and tyrosine methylene-
phosphonate into position 286 altered the spectra of emitted light compared with those of Ser286 and Tyr286. The
pH dependence of the wavelength of light emitted by the luciferases containing the negatively charged phosphorylated
Tyr analogues was demonstrated and could be rationalized in terms of the pKa’s of the phosph(on)ate oxygens.
Introduction
substrate, luciferases isolated from different firefly species, or
even from different anatomical parts of a single firefly, can emit
light having different colors.4 Moreover, the λmax of light
emitted by luciferase can be affected by temperature, pH, and
metal ions.4 Light production has been suggested to involve
two excited species of oxyluciferin responsible for the color of
emitted light (Figure 1).5 The presently accepted chemical
mechanism of the light reaction postulates the formation of an
enzyme-bound oxyluciferin intermediate in an electronically
Firefly luciferase catalyzes the conversion of luciferin to
oxyluciferin in the presence of ATP, Mg2+, and molecular
oxygen.1 Due to its high sensitivity and narrow specificity for
ATP, luciferase is used to determine the amount of ATP in
biological samples.2 The firefly luciferase gene is also widely
used as reporter gene in many molecular biological systems.3
Although firefly luciferases all use the same firefly luciferin
X Abstract published in AdVance ACS Abstracts, October 15, 1997.
(1) DeLuca, M.; McElroy, W. D. Methods Enzymol. 1978, 57, 3.
(2) Ludin, A. In Bioluminescence and Chemiluminescence; DeLuca, M.,
McElroy, W. D., Eds.; Academic Press: New York, 1981; p 187.
(3) Szalay, A. A., Kricka, L. J., Stanley, P., Eds. Bioluminescence and
Cheimiluminescence; John Wiley & Sons Ltd.: Chichester, 1993.
(4) (a) Seliger, H. H.; McElroy, W. D. Proc. Natl. Acad. Sci. U.S.A.
1964, 52, 75. (b) McElroy, W. D.; Seliger, H. H.; White, E. H. Photochem.
Photobiol. 1969, 10, 153. (c) Wood, K. V.; Lam, Y. A.; Seliger, H. H.;
McElroy, W. D. Science 1989, 244, 700. (d) Wood, K. V.; Lam, Y. A.;
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S0002-7863(97)01927-6 CCC: $14.00 © 1997 American Chemical Society

10878 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Arslan et al.
Figure 1. Partial reactions leading to light production by firefly luciferase.
excited state. Under physiological conditions, a proton is
abstracted from C-5 of oxyluciferin, resulting in the formation
of an enolate dianion and subsequent yellow-green biolumi-
nescence upon its relaxation to the ground state. Alternatively,
at lower pH, the oxyluciferin monoanion is thought to emit red
light.5 It has not been clear, however, whether alteration of
the wavelength of emitted light must involve changes in
properties of a particular amino acid(s) that interacts directly
with the luciferin or can result from more global changes in
the structure of luciferase. It seems likely, though, that recent
advances in the structural characterization of firefly luciferase
at high resolution6 will permit this issue to be addressed
experimentally.
proteins by suppression of a TAG stop codon was employed.11
Using misacylated suppressor tRNA’s12 a series of natural amino
acids and unnatural amino acids, such as glycosyl and meth-
ylenephosphonate derivatives of Ser and both phosphate and
methylenephosphonate derivatives of Tyr, have been incorpo-
rated at position 286 of L. mingrelica luciferase, both in ViVo
and in Vitro.13 Presently, we provide a complete account of
our studies involving replacement of Ser286 in L. mingrelica
luciferase, including the syntheses of the requisite amino acid
analogues, their attachment to a suitable suppressor tRNA, and
incorporation into protein to afford luciferase analogues. Also
reported is the purification of a number of the derived lu-
ciferases, their light emission properties, and the effects of pH
on light emission, as well as the thermostability of representative
analogues.
Kajiyama and Nakano have identified five sites in Luciola
cruciata luciferase at which alteration of a single amino acid
effected significant alteration of the wavelength at which light
was emitted.7 These five sites are widely separated throughout
primary sequence of the protein, which provides at least
superficial support for the conclusion that numerous alterations
of protein structure can influence the emission wavelength. The
same authors have found that substitutions of amino acids at
position 217, of both L. cruciata and Luciola lateralis lu-
ciferases, by hydrophobic amino acids such as Leu, Ile, or Val
leads to thermostability.8 It may be noted that some researchers
argue against the ability of a single amino acid residue to
contribute to color determination and suggest that oligo- or
polypeptide fragments are actually responsible sites for the
observed changes.9
Results
Synthesis of Amino Acid Derivatives. Although synthesis
of bis(2-nitrobenzyl)-N,N-diisopropylphosphoramidite (2) in
analogy with the general method of Bannwarth and Trzeciak14
proved to be problematic, treatment of dichloro(diisopropyl-
amino)phosphine (1) with 2-nitrobenzyl alcohol in THF gave
2 in 72% yield as a colorless solid.
The preparation of tyrosine phosphate derivative 5 began from
tyrosine (Scheme 1). The amino group was protected with
2-nitroveratryl chloroformate (NVOCCl) in the presence of
Na2CO3, giving 3 in 93% yield. Conversion to the respective
In order to evaluate the relationship between the structure of
amino acid residues at one of the positions important for color
determination and consequent alterations of the wavelength of
light produced by firefly luciferase, we have studied Luciola
mingrelica luciferase, a thermolabile species.10 A methodology
that allows site-specific incorporation of amino acids into
(11) (a) Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P.
G. Science 1989, 244, 182. (b) Ellman, J. A.; Mendel, D.; Anthony-Cahill,
S. J.; Noren, C. J.; Schultz, P. G. Methods Enzymol. 1992, 202, 301. (c)
Bain, J. D.; Wacker, D. A.; Kuo, E. E.; Chamberlin, A. R. Tetrahedron
1991, 47, 2389. (d) Bain, J. D.; Diala, E. S.; Glabe, C. G.; Wacker, D. A.;
Lyttle, M. J. D.; Dix, T. A.; Chamberlin, A. R. Biochemistry 1991, 30,
5411. (e) Bain, J. D.; Switzer, C.; Benner, S. A.; Chamberlin, A. R. Nature
1992, 356, 537. (f) Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.
Chem., Int. Ed. Engl. 1995, 34, 621. (g) Karginov, V. A.; Mamaev, S. V.;
An, H.; Van Cleve, M. D.; Hecht, S. M.; Komatsoulis, G. A.; Abelson, J.
N. J. Am. Chem. Soc. 1997, 119, 8166.
(12) (a) Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. J. J. Biol.
Chem. 1978, 253, 4517. (b) Baldini, G.; Martoglio, B.; Zugliani, C.; Brunner,
J. Biochemistry 1988, 27, 7951. (c) Bain, J. D.; Glabe, C. G.; Dix, T. A.;
Chamberlin, A. R.; Diala, E. S. J. Am. Chem. Soc. 1989, 111, 8013. (d)
Roesser, J. R.; Xu, C.; Payne, R. C.; Surratt, C. K.; Hecht, S. M.
Biochemistry 1989, 28, 5185. (e) Robertson, S. A.; Noren, C. J.; Anthony-
Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Nucleic Acids Res. 1989, 17,
9649. (f) Hecht, S. M. Acc. Chem. Res. 1992, 25, 545. (g) Lodder, M.;
Golovine, S.; Hecht, S. M. J. Org. Chem. 1997, 62, 778.
(5) (a) DeLuca, M.; Leonard, N. J.; Gates, B. J.; McElroy, W. D. Proc.
Natl. Acad. Sci. U.S.A. 1973, 70, 1664. (b) White, E. H.; Steinmetz, M. G.;
Miano, J. D.; Wides, P. D.; Morland, R. J. J. Am. Chem. Soc. 1980, 102,
3199. (c) Rosendahl, M. S.; Leonard, N. J.; DeLuca, M. Photochem.
Photobiol. 1982, 35, 857. (d) Wood, K. V. Photochem. Photobiol. 1985,
62, 662.
(6) (a) Baldwin, T. O. Structure 1996, 4, 233. (b) Conti, E.; Franks, N.
P.; Brick, P. Structure 1996, 4, 287.
(7) Kajiyama, N.; Nakano, E. Protein Eng. 1991, 4, 691.
(8) (a) Kajiyama, N.; Nakano, E. Biochemistry 1993, 32, 13795. (b)
Kajiyama, N.; Nakano, E. Biosci., Biotech. Biochem. 1994, 58, 1170.
(9) Ohmiya, Y.; Hirano, T.; Ohashi, M. FEBS Lett. 1996, 384, 83.
(10) Devine, J. H.; Kutuzova, G. D.; Green, V. A.; Ugarova, N. N.;
Baldwin, T. O. Biochim. Biophys. Acta 1990, 1173, 121.
(13) Mamaev, S. V.; Laikhter, A. L.; Arslan, T.; Hecht, S. M. J. Am.
Chem. Soc. 1996, 118, 7243.
(14) Bannwarth, B; Trzeciak, A. HelV. Chim. Acta 1987, 70, 175.

Structurally Modified Firefly Luciferase
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10879
Scheme 1a
(6) as a thick oil. This intermediate was used immediately for
the next step to preclude oxidation. Compound 6 was mixed
with dibromo-p-xylene and stirred at 100 °C for 20 min to give
phosphonate 7 in 30% yield (for two steps) as a colorless
powder. The coupling reaction between 7 and the protected
(N-diphenylmethylene)glycine using potassium bis(trimethyl-
silyl)amide as a base afforded tyrosine methylenephosphonate
8 in 58% yield.18 The amino group of 8 was deprotected in
ethereal HCl; subsequent treatment with 2-nitroveratryl chloro-
formate (NVOCCl) afforded fully protected tyrosine methyl-
enephosphonate analogue 9 in 66% overall yield. The ethyl
ester moiety of 9 was removed selectively by treatment with
0.5 N NaOH in THF in the presence of pyridine; free
carboxylate derivative 10 was obtained as a colorless solid in
75% yield. Conversion to the respective cyanomethyl ester 11
was accomplished in 92% yield.
a (a) 2-NO2C6H5CH2OH, EtN(iPr)2, 70%; (b) 2, tetrazole; (c)
Bu4NIO4.
Preparation of Aminoacyl pdCpA Esters. The coupling
reaction between several activated amino acids and the tris-
(tetrabutylammonium) salt of pdCpA was investigated under
conditions that included variation of solvents, reaction times,
activated ester:dinucleotide ratios and the addition of a base as
catalyst. In general, the coupling reactions proceeded to the
maximal extent within one hour; longer reaction times did not
result in higher yields. Bases such as Et3N or KF failed to
increase the coupling yields, but the addition of an excess of
the activated ester did promote complete consumption of pdCpA
to form the desired esters. Cyanomethyl esters 5 and 11 were
coupled with the tris(tetrabutylammonium) salt of the pdCpA
in DMF at 25 °C to give 12 and 13, respectively (Scheme 3).
The progress of the reactions was followed by HPLC; both were
complete within 1 h. The products were purified by preparative
HPLC. The photochemical deprotection reaction was studied
using aminoacylated dinucleotides 12 and 13; the reactions were
complete within 8 min, as judged by HPLC analysis and the
UV spectra of the derived products (Supporting Information,
Figures 1-9).
Scheme 2a
Preparation of Misacylated tRNA’s. The misacylated
suppressor tRNA’s required for the protein synthesis experi-
ments were prepared by T4 RNA ligase-mediated attachment
of N-protected 2′(3′)-O-aminoacyl-pdCpA derivatives to tRNA’s
or tRNA transcripts lacking the last two nucleotides.12 The
transformation is exemplified in Scheme 3 for the attachment
of tyrosyl-pdCpA derivatives 12 and 13 to a truncated yeast
a (a) 2-NO2C6H5CH2OH, tetrazole; (b) dibromo-p-xylene, 100 °C,
20 min; (c) Ph2CdNCH2COOEt, KHMDS, -78 °C, 3 h; (d) 1 N HCl;
(e) NVOCCl, Na2CO3; (f) 0.1 N NaOH; (g) ICH2CN, EtN(iPr)2.
cyanomethyl ester15 4 was achieved in 83% yield using
iodoacetonitrile. The coupling reaction between 2 and 4 was
then accomplished by activation of the phosphoramidite reagent
with tetrazole to give the corresponding phosphite. This
intermediate was not isolated, but was oxidized directly with
tetrabutylammonium periodate to afford tyrosine phosphate
derivative 5 in 61% overall yield for the two steps.
Also prepared for incorporation into luciferase was the
respective methylenephosphonate derivative of tyrosine. Since
ribosomal protein synthesizing systems exhibit good selectivity
for (S)-amino acids, even when present in a racemic mixture,16
racemic tyrosine methylenephosphonate was prepared rather
than the (S)-isomer. The strategy employed for the synthesis
of the methylenetyrosine phosphonate derivative employed the
well-established Arbuzov reaction,17 but under modified condi-
tions compatible with the stability of the benzylic bromide
functionality.
suppressor tRNAPhe
transcript lacking the 3′-terminal CA
CUA
sequence.11a,12
A number of misacylated tRNA’s were prepared in this
fashion, notably tRNA’s I-III and IV-VI, involving deriva-
tives of serine and tyrosine, respectively. The ligation yields
were determined by polyacrylamide gel electrophoresis, carried
out in 0.1 M NaOAc, pH 5.0, by modification of the method of
Varshney et al.19 (Figure 1, Supporting Information).
As shown in Scheme 2, phosphoramidite 2 was employed as
the starting material and was treated with 2-nitrobenzyl alcohol
in the presence of tetrazole to give tris(2-nitrobenzyl) phosphite
In ViWo Substitutions of Ser286 in Luciferase by Other
Naturally Occurring Amino Acids. The luciferase gene of
(15) Bodanszky, M.; Bodanszky, A. In The Practice of Peptide Synthesis,
2nd revised ed.; Springer-Verlag: Berlin, 1994; p 96.
(16) Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.-I.; Hecht, S. M.
Biochemistry 1988, 27, 7254.
(18) Hamilton, R.; Shute, R. E.; Travers, J.; Walker, B.; Walker, B. J.
Tetrahedron Lett. 1994, 35, 3597.
(19) Varshney, U.; Lee, C.-P.; RajBhandary, U. L. J. Biol. Chem. 1991,
266, 24712.
(17) Kosolapoff, G. M. J. Am. Chem. Soc. 1944, 66, 109.

10880 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Arslan et al.
Scheme 3a
a (a) DMF, 25 °C; (b) tRNA-COH, T4 RNA ligase, pH 7.5; (c) hυ.
Table 1. Emission Characteristics of Luciferase Analogues as a
Function of pH
wavelength of emitted light (nm)
amino acid at
position 286
pH 5.2
pH 6.1
pH 6.6
pH 7.0
pH 8.0
Ser
618
623
a
622
a
611
623
619
622
614
590
622
615
621
582
619
613
620
612
581
613
608
619
609
Tyr
Lys
Leu
Gln
a Not detected.
Figure 2. Plasmid constructs used for expression of the luciferase gene
(L. mingrelica).
light from 581 nm at pH 8.0 to 618 nm at pH 5.2, with a
significant change apparent at pH 6.6. This pH dependence is
very similar to that observed for the luciferases from other
sources.4a In comparison, the luciferase containing Leu286 did
not exhibit significant alteration in the wavelength of emitted
light at any pH from 8.0 to 5.2, consistent with the interpretation
that this analogue may be able to support only the oxyluciferin
monoanion species under both physiological and more acidic
conditions. The luciferases containing Gln, Lys, and Tyr at
position 286 were intermediate in their behavior, although it is
interesting that the pH values at which the greatest changes in
emission wavelength occurred were not the same for each of
these luciferase analogues.
L. mingrelica was cloned into expression vector pTrc-99A under
control of a strong trc promoter. Site-specific mutagenesis was
used to change Ser codon AGT at position 286 to an amber
stop codon TAG (Figure 2). For in ViVo incorporation of natural
amino acids, Escherichia coli strains, each containing a specific
suppressor tRNA recognized by a different endogenous amino-
acyl-tRNA synthetase, were used.20 Following the transforma-
tion of these strains with plasmid pTrcLuc-St286, the spectrum
of light emitted by each of the elaborated luciferases was
measured. It was found that luciferases with Leu, Lys, Tyr, or
Gln at position 286 produced light with λmax shifted toward
longer wavelengths than luciferase having Ser286 (λmax 582).13
Effect of pH on the Bioluminescence of Luciferases
Altered at Position 286. The cell extracts containing the
luciferases with substitutions at position 286 were prepared as
described in the Experimental Section and used directly in
luciferase assays that employed 0.1 M MOPS-MES buffer, 5
mM MgSO4, 0.5 mM luciferin, 1.5 mM ATP, and 1 mM EDTA.
Spectra were measured at pH values from pH 5.2 to 8.0. The
results of this experiment are presented in Table 1.
Comparison of the Thermostability of Wild-Type Lu-
ciferase with Luciferase Analogues Altered at Position 286.
Comparison of the thermostabilities of individual luciferases
was carried out in cell free extracts after expression in ViVo
and also after purification of individual enzymes on Ni-NTA
agarose. The purified luciferases were incubated at 22 or 37
°C (pH 7.8). The residual luciferase activity was assayed at
pH 7.8 and the spectrum of emitted light was measured at
various times. As shown in Figure 3, wild-type luciferase
(Ser286) exhibited the greatest thermostability when incubated
at 22 °C. The luciferase analogue containing Tyr286 had the
next greatest thermostability at 22 °C and was actually more
stable than the wild-type enzyme when both were incubated at
37 °C. In parallel with its apparent inability to support the
formation of the excited state oxyluciferin dianion (Vide supra),
As is clear from Table 1, the five luciferases studied exhibited
somewhat different behavior as a function of pH. Wild-type
luciferase (Ser286) exhibited a change in the λmax of emitted
(20) Promega Corporation Interchange in ViVo system; see: (a) Kleina,
L. G.; Masson, J.-M.; Normanly, J.; Abelson, J.; Miller, J. H. J. Mol. Biol.
1990, 212, 705. (b) Normanly, J.; Kleina, L. G.; Masson, J.-M.; Abelson,
J.; Miller, J. H. J. Mol. Biol. 1990, 212, 719.

Structurally Modified Firefly Luciferase
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10881
effect readthrough of the UAG codon at codon position 286.
Aliquots of the translation mixture were used directly to measure
the spectrum and intensity of the light produced (Figure 4).
SDS-PAGE analysis of [35S]methionine-labeled luciferase was
used for estimation of suppression efficiency. It was found that
luciferases obtained after in Vitro translation produced light with
the same spectral characteristics as luciferases obtained from
E. coli cells (cf. Tables 1-3).13 The suppression efficiency is
defined as the ratio of the amount of luciferase produced by
suppressing a gene containing a stop codon relative to the
amount produced from wild-type plasmid pTrcLuc.
Purification of a number of the luciferase analogues was
accomplished by translation of a fusion protein that had a
dodecapeptide at the C-terminus which included a hexahistidine
sequence. These fusion proteins could be purified on Ni-NTA
agarose columns11g and were shown to have unaltered properties
of light emission as a consequence of introduction of the
dodecapeptide.
Incorporation of Unnatural Amino Acids into Position 286
of Luciferase. The several protected pdCpA derivatives of Ser
and Tyr were ligated to yeast tRNAPheCUA(-CA) in typical yields
of about 60-70%, as evaluated by PAGE analysis at acid pH
(Supporting Information, Figure 1). After photochemical depro-
tection (5 min for GlcSer, 2 min for Val, and 8 min for Tyr
derivatives), the deprotected aminoacyl tRNA’s were introduced
into an E. coli S30 translation system containing plasmid
pTrcLuc-St286 DNA and [35S]methionine. Protein synthesis
was carried out at 22 °C for 2 h. Aliquots of the reaction
mixtures were used for assay of light production and for SDS-
PAGE analysis.
Representative results from the electrophoretic analysis of
[35S]methionine-labeled luciferases are shown in Figure 5. Lane
2 illustrates luciferase production from wild-type luciferase
mRNA. Lane 3 shows that E. coli tRNATyrCUA can efficiently
suppress the nonsense codon at position 286 (cf. lane 1, which
uses the same mRNA but lacks an activated suppressor tRNA).
Lanes 4 and 5 illustrate the production of luciferase analogues
containing tyrosine phosphonate and tyrosine phosphate, re-
spectively. Protein production levels were determined by
phosphorimager analysis of the band corresponding to luciferase;
this permitted a calculation of the specific activities of the
individual luciferase analogues. A summary of the enzymatic
activities and wavelengths of emitted light for several luciferase
analogues is given in Table 2.
Figure 3. Thermostability of the Ni-NTA-purified luciferase analogues.
Luciferases were incubated at 22 °C (upper panel) and 37 °C (lower
panel) in 100 mM K phosphate buffer, pH 7.8, containing 1 mM
MgSO4, 1 mM EDTA, and 15% glycerol. The luciferase activities were
determined after 1, 2, 3, and 4 h, and shown as a percentage of the
activity measured after maintenance of the enzyme at 0 °C.
As shown in the table, wild-type luciferase (elaborated from
pTrcLuc in the absence of a suppressor tRNA) had the same
specific activity and emission wavelength as luciferase prepared
by suppression of the stop codon at position 286 with seryl-
the luciferase analogue containing Leu286 was also the least
thermostable of those species studied, both at 22 and 37 °C.
None of the luciferase analogues studied was noted to alter
the wavelength of emitted light at any time during the course
of the thermostability study (Supporting Information, Table 1).
In Vitro Synthesis of Luciferase Analogues. The general
strategy employed for the synthesis of luciferase analogues in
Vitro is illustrated in Scheme 4. In common with the approach
employed in ViVo, this scheme uses misacylated suppressor
tRNA’s for readthrough of nonsense codons placed at pre-
determined sites in the mRNA of interest. However, because
the misacylated tRNA’s prepared in Vitro can include amino
acid analogues for which there is no aminoacyl-tRNA syn-
thetase, the derived proteins can have a much greater variety
of amino acids at the position of interest.
tRNAPhe
. Luciferase analogues containing Phe286 and
CUA
Val286 exhibited lower specific activities and emission maxima
at 608 and 621 nm, respectively. Interestingly, substitution of
Ser286 with O-glucosylated serine or serine methylenephos-
phonate had no effect on the emission wavelength, although
the efficiency of light production was diminished somewhat
(Figure 6 and Table 2).
Substitution of position 286 with tyrosine, tyrosine phosphate
and tyrosine phosphonate afforded luciferases all of which
emitted light at different wavelengths. In the belief that the
differences in emission wavelength for the latter two might
reflect differences in the pKa’s for tyrosine phosphate (pKa2 6.22)
and tyrosine methylenephosphonate (pKa2 7.72),21 the light
Each N-protected misacylated tRNA was deprotected by hυ
irradiation and the misacylated suppressor tRNA having a
deblocked amino acid was included in an E. coli S30 coupled
transcription-translation system containing [35S]methionine to
(21) (a) Smyth, M. S.; Ford, H., Jr.; Burke, T. R., Jr. Tetrahedron Lett.
1992, 33, 4137. (b) Burke, T. R., Jr.; Smyth, M. S.; Nomizu, M.; Otaka,
A.; Roller, P. P. J. Org. Chem. 1993, 58, 1336.

10882 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Arslan et al.
Scheme 4. General Strategy Employed for the Synthesis of Proteins Containing Synthetic Amino Acids at Single, Predetermined
Sites
Figure 5. Phosphorimager analysis of a 10% polyacrylamide gel
containing crude in Vitro suppression products labeled with [35S]-
methionine and synthesized using pTrcLuc-St286 plasmid DNA and
tRNAPheCUA activated with the following amino acids: lane 1, plasmid
pTrcLuc-St286 + unactivated tRNAPheCUA; lane 2, plasmid pTrcLuc
Figure 4. Emission spectra of L. mingrelica luciferase in which Ser286
has been replaced by tyrosine (curve 1), lysine (curve 2), and valine
(curve 3).
+ no suppressor tRNA; lane 3, plasmid pTrcLuc-St286 + E. coli
tRNATyrCUA; lane 4, plasmid pTrcLuc-St286 + phosphonotyrosyl-
tRNAPheCUA; lane 5, pTrcLuc-St286 + phosphorotyrosyl-tRNAPhe
.
CUA
The asterisk indicates the bands corresponding to full length luciferases.
emission characteristics of these luciferases were measured at
pH 8.5, as well as pH 7.8. As shown in Table 3, light emission
from luciferases having Ser286, Tyr286, and PhTyr286 was not
altered at the higher pH. However, the luciferase containing
PhnTyr286 emitted light at shorter wavelength at pH 8.5 than
at pH 7.8, undoubtedly reflecting deprotonation of this amino
acid at the higher pH value. This strongly suggests a real
dependence of the wavelength of emitted light on the extent of
negative charge on the tyrosine moiety at this site.
Discussion
The luciferases from L. mingrelica and L. cruciata are about
80% identical in amino acid sequence, although they do exhibit
differences in thermostability and in the spectrum of emitted
light.10 On the basis of the finding of Kajiyama and Nakano7
that a single amino acid change at any of five sites in L. cruciata
luciferase could alter the wavelength of light produced, we
sought to alter the structure of luciferase systematically at single

Structurally Modified Firefly Luciferase
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10883
Table 2. Emission Characteristics of Luciferases Synthesized in a Cell Free Systema
(misacylated)
suppressor tRNA
suppression
(%)
specific
emission
wavelength (nm)
DNA
pTrcLuc
activityb (%)
color
c
d
11
7.5
6
27
25
85
6
100
d
583
d
yellow-green
pTrcLuc-St286
pTrcLuc-St286
pTrcLuc-St286
pTrcLuc-St286
pTrcLuc-St286
pTrcLuc-St286
pTrcLuc-St286
pTrcLuc-St286
pTrcLuc-St286
tRNACUA
tRNACUA-Ser (I)
tRNACUA-PhnSer (II)
tRNACUA-GlcSer (III)
tRNACUA-Phe
109
29
36
27
21
14
20
17
584
584
584
608
621
613
593
603
yellow-green
yellow-green
yellow-green
orange
red-orange
orange
tRNACUA-Val
tRNATyrCUA-Tyr (IV)
tRNACUA-PhTyr (V)
tRNACUA-PhnTyr (VI)
yellow
orange
11
a Transcription and translation were carried out in an E. coli S30 system, as described in the Experimental Section. b Relative to that of wild
type. c Not applicable. The amount of luciferase protein was determined by phosphorimager analysis of the appropriate band on a SDS-PAGE gel
and calibrated by reference to a purified luciferase standard of known specific activity. d No detectable light production.
enzyme may be altered structurally in a fashion that precludes
the generation of an excited state oxyluciferin dianion even at
higher pH values. In this context it is important to note that
the introduction of the polar amino acids Gln and Tyr, or
positively charged Lys at position 286, did not decrease
thermostability dramatically, although they all resulted in
changes in the wavelength of emitted light (Figure 3 and
Supporting Information, Table 1). Thus the factors that control
the wavelength of light emission may well include, but need
not be limited to, alterations of protein structure linked to
conformational stability.
It is interesting to consider the foregoing results in the context
Figure 6. Emission spectra of L. mingrelica luciferases produced by
readthrough of a UAG codon at position 286: curve 1, Val286; curve
of observations made for other luciferases. All known firefly
luciferases are hydrophobic proteins.22 Although 60% of their
amino acid residues are hydrophobic, the overall hydrophobicity
of the Photinus pyralis luciferase was judged to be only
moderate, suggesting that a significant portion of the hydro-
phobic residues are buried within the protein matrix.22 Unlike
the results obtained by substitution of Leu or Val at position
286, Kajiyama and Nakano8 have shown that substitution of
hydrophobic amino acids at position 217 of L. cruciata and L.
lateralis luciferases (for the threonine normally present) actually
increased the thermostability of these enzymes. Thus the
presence of hydrophobic interactions in luciferases is well-
documented and need not have a deleterious effect on protein
function.
In addition to the data obtained in the present study, the results
obtained from analysis of five click beetle Pyrophorus pla-
giophthalamus luciferases4c and five L. cruciata luciferases
obtained after random mutagenesis7 indicate that alteration of
the color of light produced could be induced by changing amino
acid residues widely distributed throughout the sequence of the
proteins. This is in sharp contrast to the reports from the
laboratories which posit the involvement of larger structural
elements in luciferase as responsible for determining the color
of emitted light. These were defined as the fragments 223-
24723 within click beetle luciferase and 208-318 in Hataria
parVula and Pyrocoelia miyako luciferases.9 To some extent
this may be regarded as a semantic issue since changes at a
single site can effect larger structural elements within a protein.
However, the present results, as well as earlier studies employing
other luciferases,4c,7 clearly indicate that alteration of a single
amino acid is sufficient to change the spectrum of emitted light,
even if the global change to protein structure resulting from
that substitution extends well beyond the specific amino acid
altered.
2, Ser286; curve 3, GlcSer286; curve 4, PhnSer286. Curve 5 was
recorded for luciferase produced in the presence of deacylated sup-
pressor tRNA; curve 6 in the absence of a suppressor tRNA.
Table 3. pH Dependence of Light Emission by Luciferases with
Tyrosine Derivatives at Position 286
wavelength of emitted light (nm)
amino acid at
position 286
pH 7.8
pH 8.5
serine
tyrosine
tyrosine phosphate
tyrosine phosphonate
581
611
590
601
582
612
590
596
sites to define the basis for alterations in the wavelength of
emitted light. The luciferases from L. cruciata and L. mingrelica
are homologous at four of the five sites shown to be capable of
controlling the wavelength of light emitted by the former
species. One of these sites, Ser286, was chosen for initial
modification in L. mingrelica luciferase.13
As shown in Tables 1 and 2 and Figure 4, alteration of Ser286
in L. mingrelica luciferase can also result in a change in the
wavelength of emitted light. Interestingly, the largest changes
were noted for the luciferase analogues containing the lipophilic
amino acids Leu and Val at position 286. Amino acids such
as Leu and Val might be thought to promote conformational
changes through the introduction of new hydrophobic inter-
actions, inducing alterations in luciferase structure by changing
the protein environment around the bound luciferase sub-
strate. Consistent with this thesis, it was found that luciferase
[Val286]-13 and [Leu286]luciferase (Figure 3) exhibited the poorest
thermostability of any of the analogues studied. Measurement
of the pH dependence of the emission wavelength for several
luciferase analogues differing only at position 286 revealed that
most of these species emitted light at longer wavelengths as
the pH decreased. This is consistent with light emission by
the putative excited stated oxyluciferin monoanion at lower pH.
In contrast, luciferase Leu286 emitted light at virtually the same
wavelength (619-622 nm) at all pH values, suggesting that this
(22) Hlady, V.; Yeh, P.; Andrade, J. D. J. Fluorescence 1991, 1, 47.
(23) Morozov, V. M. In Bioluminescence and Chemiluminiscence:
Fundamental and Applied Aspects; Campbell, A. K., Krica, L. J., Stanley,
P. E., Eds.; Wiley Interscience: Chichester, 1994; p 536.

10884 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Arslan et al.
Among the most interesting modifications of luciferase
structure in the present study are those involving the introduction
of a glucosylated amino acid, as well as a phosphorylated amino
acid (and two methylenephosphonate analogues). Such modi-
fications are widespread in nature and are believed to be
introduced post-translationally. They are strongly associated
with the biological properties of the modified proteins; for
example the carbohydrates in glycoproteins are thought to be
essential for elements of biological recognition.24 Likewise, the
phosphorylation of proteins is involved in biological processes
such as receptor activation, carcinogenesis, and protein synthe-
sis.25 Phosphorylation is also involved in regulation of the
activity of certain enzymes, including DNA topoisomerase I,26
and can play a role as a recognition element in peptides.27
Clearly, the ability to introduce such modifications at single
sites in proteins can have an enormous facilitating effect on
the study of normal modification processes and on the mech-
anisms by which the modified species function, as well as
providing a vehicle for the elaboration of fundamentally new
species for investigation. It is particularly encouraging that
modifications, such as the introduction of glucosylserine or
serine phosphonate into a specific site in luciferase, could be
made without alteration of the primary function of the enzyme
(Table 2).
pocket. In any case, the availability of a crystal structure for a
firefly luciferase should prove extraordinarily valuable in guiding
the dissection of those structure elements that contribute to
luciferase function.
Experimental Section
General Methods. Oligonucleotides were obtained from Cruachem,
Inc. Restriction enzymes and T4 RNA ligase were purchased from
New England Biolabs. E. coli S30 coupled transcription-translation
system, Interchange in ViVo amber suppression mutagenesis system,
luciferase assay system, beetle luciferin, and pGEM-5Zf(+) vector were
purchased from Promega Corporation; pTrc 99A vector was from
Pharmacia Biotech. Acrylamide, N,N-methylenebisacrylamide, urea,
Tris base, and dNTPs were obtained from Sigma Chemicals. Yeast
extract and bacto tryptone were from Difco.
Plasmid DNAs were isolated using a Wizard Minipreps purification
system (Promega) or QIAGEN tip 100 columns (QIAGEN) according
to the protocols provided. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis was carried out using the standard Laemmli
procedure.30 Gels were visualized and quantified utilizing a Molecular
Dynamics 400E phosphorimager with ImageQuant version 3.2 software.
Sequence analysis was performed using a Sequenase version 2.0 DNA
sequencing kit (USB). All procedures involving water employed
distilled, deionized water from a Milli-Q system.
Experiments requiring anhydrous conditions were performed under
an argon atmosphere. Reactions were done at 25 °C unless otherwise
indicated. Solvents were J.T Baker p.a. and were used without further
purification unless mentioned otherwise. THF was distilled from
potassium/benzophenone. Melting points were measured on a Thomas
Hoover apparatus and are uncorrected. 1H NMR and 13C NMR were
recorded on a General Electric QE-300; all δ values are given in ppm
relative to tetramethylsilane and J values are in Hz. Thin layer
chromatography (TLC) was run on Merck silica gel F245 precoated
plates; spots were visualized by dipping the plates in a Ce-Mo staining
reagent. For column chromatography, Fluka silica gel 60, mesh size
230-400, was used.
A more sobering perspective is provided by introduction of
tyrosine phosphate and tyrosine phosphonate into position 286
of luciferase. Nonhydrolyzable analogues of phosphorylated
amino acids28 have been widely employed due both to the
greater ease of handling such species and to the potential for
probing biochemical events that normally result in dephos-
phorylation. It is frequently assumed in such studies that the
phosphorylated amino acids and their phosphonate analogues
are essentially equivalent since they involve only a small
structural change in a large molecule. The results shown in
Tables 2 and 3 indicate clearly that this need not be true. While
the differences noted for luciferases with PhTyr286 and
PhnTyr286 very likely are due to differences in pKa values, as
described above, these results nonetheless argue for caution in
the interpretation of results obtained with structural analogues
of the phosphorylated amino acids.
Recently the crystal structure of firefly luciferase from
Photinus pyralis has been determined.29 According to this
structure Ser284 in Photinus pyralis luciferase (corresponding
to Ser286 in Luciola mingrelica luciferase) is located in one of
the â strands inside the large domain of luciferase. As argued
previously,13 it seems likely that this residue is not part of the
active site of luciferase but may well play an important role in
maintaining the native structure of luciferase. In the context
of the changes in emission wavelength resulting from the
introduction of hydrophobic amino acids into position 286 in
L. mingelica luciferase (Vide supra), it is interesting to note that
several amio acids (notably Phe286, Phe273, Leu287, Leu291,
Phe295, and Leu309) within reasonable proximity to Ser284 in
P. pyralis luciferase form what appears to be a hydrophobic
Synthesis of the Unnatural Amino Acids. Bis(2-nitrobenzyl) N,N-
Diisopropylphosphoramidite (2). Dichloro(diisopropylamino)phos-
phine (1) (5.0 g, 24.7 mmol) was dissolved in 20 mL of THF and the
resulting solution was added slowly to a solution containing 12.8 mL
(73 mmol) of N,N-diisopropylethylamine and 7.56 g (49.4 mmol) of
2-nitrobenzyl alcohol in 50 mL of THF at 0 °C. The reaction mixture
was stirred at 0 °C for 30 min and then at 25 °C for another 30 min.
The colorless precipitate was isolated by filtration and the solid was
washed with 100 mL of ethyl acetate. The organic phase was washed
successively with 50-mL portions of saturated NaHCO3 and saturated
NaCl and then dried (MgSO4) and concentrated under diminished
pressure at 25 °C. The residue was precipitated from ethyl acetate-
hexane, affording bis(2-nitrobenzyl) N,N-diisopropylphosphoramidite
1
(2) as a colorless solid: yield 7.6 g (72%); mp 71-73 °C; H NMR
(CDCl3) δ 1.24 (d, 12 H, J ) 7 Hz), 3.72 (m, 2 H), 5.10 (dd, 2 H, J
) 7.5, 16 Hz), 5.11 (dd, 2 H, J ) 7, 16 Hz), 7.43 (m, 2 H), 7.65 (m,
2 H), 7.86 (d, 2 H, J ) 8 Hz), and 8.10 (d, 2 H, J ) 8.5 Hz); 13C NMR
(CDCl3) δ 25.1, 25.2, 43.8, 43.9, 62.9, 63.0, 125.1, 128.2, 129.0, 134.2,
136.5, 136.6, and 147.3. Anal. Calcd for C20H26O6N3P: C, 55.14; H,
4.74. Found: C, 54.94; H, 4.91.
N-[[(2-Nitroveratryl)oxy]carbonyl]-(S)-tyrosine (3). Tyrosine (181
mg, 1.0 mmol) was dissolved in 3 mL of water and treated with 210
mg (2.0 mmol) of Na2CO3. To this solution was added 303 mg (1.1
mmol) of 2-nitroveratryl chloroformate in 5 mL of dioxane at 0 °C.
The mixture was stirred at 25 °C for 1 h. The reaction mixture was
then treated with 30 mL of 1 N NaHSO4 and the aqueous phase was
extracted with ethyl acetate. The combined organic phase was dried
(MgSO4) and concentrated under diminished pressure. The residue was
purified by flash chromatography on a silica gel column (15 × 3 cm);
elution with 10:10:1 ethyl acetate-hexane-acetic acid afforded NVOC
tyrosine derivative 3 as a yellow powder: yield 390 mg (93%); silica
gel TLC Rf 0.25 (10:10:1 ethyl acetate-hexane-acetic acid); mp 133-
(24) (a) Baenziger, J. U. FASEB J. 1994, 8, 1019. (b) Lasky, L. A. Annu.
ReV. Biochem. 1995, 64, 113.
(25) (a) Pawson, T.; Schlessinger, J. Current Biol. 1993, 3, 434. (b)
Panayotou, G.; Waterfield, M. D. Bioassays 1993, 15, 171. (c) Pawson, T.
FASEB J. 1994, 8, 1113. (d) Crabtree, G. R.; Clipstone, N. A. Annu. ReV.
Biochem. 1994, 63, 1045. (e) Hunter, T. Cell 1995, 80, 225.
(26) Pommier, Y.; Kerrigan, D.; Hartman, K. D.; Glazer, R. I. J. Biol.
Chem. 1990, 265, 9418.
(27) (a) Escobedo, J. A.; Kaplan, D. R.; Kavanaugh, W. M.; Turck, C.
W.; Williams, L. T. Mol. Cell Biol. 1991, 11, 1125. (b) Thatcher, G. R. J.;
Campbell, S. A. J. Org. Chem. 1993, 58, 2272.
(28) Burke, Jr., T. R.; Smyth, M. S.; Otaka, A.; Nomizu, M.; Roller, P.
P.; Wolf, G.; Case, R.; Shoelson, S. E. Biochemistry 1994, 33, 6490.
(29) Conti, E.; Franks, N. P.; Brick, P. Structure 1996, 4, 287.
(30) Laemmli, U. K. Nature 1970, 227, 680.

Structurally Modified Firefly Luciferase
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10885
140 °C; 1H NMR (CDCl3) δ 2.82 (dd, 1 H, J ) 9.5, 14 Hz), 3.11 (dd,
1 H, J ) 4.5, 14 Hz,), 3.84 (s, 3 H), 3.87 (s, 3 H), 4.33 (m, 1 H), 5.37
(d, 1 H, J ) 15.5 Hz), 5.42 (d, 1 H, J ) 15.5 Hz), 6.67 (d, 2 H, J )
8.5 Hz), 7.03 (d, 2 H, J ) 8.5 Hz), 7.04 (s, 1 H), 7.50 (d, 1 H, J ) 8.5
Hz), and 7.71 (s, 1 H). Anal. Calcd for C19H20O9N2: C, 54.28; H,
4.79. Found: C, 54.47; H, 4.97.
N-(Diphenylmethylene)-4-[[bis(2-nitrobenzyl)phosphono]methyl]-
(R,S)-phenylalanine Ethyl Ester (8). Phosphonate 7 (140 mg, 0.262
mmol) was dissolved in 5 mL of THF and the solution was added slowly
to a solution containing 84 mg (0.315 mmol) of ethyl N-(diphenyl-
methylene)glycine and 60 mg (0.315 mmol) of potassium bis-
(trimethylsilyl)amide in 10 mL of THF at -78 °C. The combined
solution was stirred at -78 °C for an additional 3 h and then permitted
to warm to 25 °C. Ethyl acetate (100 mL) was added. The organic
phase was washed successively with 30-mL portions of saturated
NaHCO3 and saturated NaCl and then dried (MgSO4) and concentrated
under diminished pressure. The residue was purified by flash chro-
matography on a silica gel column (40 × 3 cm). Elution with 5:2:1
CH2Cl2-hexane-ethyl acetate afforded N-(diphenylmethylene)-4-[[bis-
(2-nitrobenzyl)phosphono]methyl]-(R,S)-phenylalanine ethyl ester (8)
as a yellowish oil: yield 110 mg (58%); silica gel TLC Rf 0.30 (5:2:1
CH2Cl2-hexane-ethyl acetate); 1H NMR (CDCl3) δ 1.24 (t, 3 H, J )
7 Hz), 3.22 (m, 3 H), 3.34 (s, 1 H), 4.09-4.25 (m, 3 H), 5.22-5.43
(m, 4 H), 6.99-7.56 (m, 20 H), and 8.04 (m, 2 H); 13C NMR (CDCl3)
δ 14.6, 32.7, 34.5, 39.7, 61.5, 64.8, 64.9, 67.6, 125.4, 125.6, 128.4,
128.7, 128.9, 129.0, 129.1, 129.3, 129.4, 129.6, 130.0, 130.1, 130.7,
132.8, 132.9, 133.0, 134.4, 134.6, 136.5, 137.7, 137.8, 139.7, 147.2,
171.3, and 172.1; mass spectrum (FAB) m/z 722 (M + H)+; (FAB)
m/z 722.226 (M + H)+ (C39H36O9N3P requires 722.226).
N-[[(2-Nitroveratryl)oxy]carbonyl]-(S)-tyrosine Cyanomethyl Es-
ter (4). Tyrosine derivative 3 (80 mg, 0.190 mmol) was dissolved in
5 mL of CH3CN and treated with 132 µL (0.76 mmol) of N,N-
diisopropylethylamine followed by 27.5 µL (0.38 mmol) of iodo-
acetonitrile. The reaction mixture was stirred at 25 °C for 12 h and
then treated with 75 mL of ethyl acetate. The organic phase was washed
successively with 25-mL portions of saturated NaHCO3 and saturated
NaCl and then dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by flash chromatography on a silica
gel column (20 × 3 cm). Elution with ethyl acetate afforded
cyanomethyl ester 4 as a colorless powder: yield 72 mg (83%); silica
gel TLC Rf 0.70 (ethyl acetate); mp 148-152 °C; 1H NMR (CDCl3) δ
3.07 (m, 2 H), 3.95 (s, 6 H), 4.55 (m, 1 H), 4.68 (d, 1 H, J ) 15.5 Hz),
4.82 (d, 1 H, J ) 15.5 Hz), 4.90 (s, 1 H), 5.22 (d, 1 H, J ) 8 Hz), 5.46
(d, 1 H, J ) 15 Hz), 5.56 (d, 1 H, J ) 15 Hz), 6.77 (d, 2 H, J ) 8.5
Hz), 6.92 (s, 1 H), 7.01 (d, 2 H, J ) 8.5 Hz), and 7.70 (s, 1 H); 13C
NMR (CDCl3) δ 37.3, 49.5, 55.4, 56.8, 56.9, 64.6, 108.7, 110.5, 114.3,
116.3, 126.8, 128.0, 130.8, 140.0, 148.6, 154.1, 155.9, and 171.0.
Anal. Calcd for C21H21O9N3: C, 54.88; H, 4.60. Found: C, 55.05;
H, 4.66.
N-[[(2-Nitroveratryl)oxy]carbonyl]-O-[bis[(2-nitrobenzyl)oxy]-
phosphoryl]-(S)-tyrosine Cyanomethyl Ester (5). Tyrosine cyano-
methyl ester 4 (72 mg, 0.156 mmol) was dissolved in 10 mL of THF
and treated with 105 mg (0.241 mmol) of bis(2-nitrobenzyl) N,N-
diisopropylphosphoramidite (2) and 22 mg (0.285 mmol) of tetrazole.
The solution was stirred at 25 °C for 30 min under an argon atmosphere.
Tetrabutylammonium periodide (100 mg, 0.23 mmol) was added in 3
mL of THF. The combined solution was stirred for 10 min at 25 °C
and then treated with 75 mL of ethyl acetate. The organic phase was
washed successively with 25-mL portions of saturated NaHCO3 and
saturated NaCl and then dried (MgSO4) and concentrated under
diminished pressure. The residue was purified by flash chromatography
on a silica gel column (30 × 3 cm). Elution with 1:1 ethyl acetate-
hexane afforded cyanomethyl ester 5 as a colorless powder: yield 77
mg (61%); silica gel TLC Rf 0.40 (1:1 ethyl acetate-hexane); mp 83-
87 °C; 1H NMR (CDCl3) δ 3.14 (m, 2 H), 3.94 (s, 3 H), 3.96 (s, 3 H),
4.69 (d, 1 H, J ) 15.5 Hz), 4.73 (m, 1 H), 4.82 (d, 1 H, J ) 15.5 Hz),
5.28 (d, 1 H, J ) 8.5 Hz), 5.45 (d, 1 H, J ) 14.5 Hz), 5.57 (d, 1 H, J
) 14.5 Hz), 5.63 (d, 4 H, J ) 7.5 Hz), 6.95 (s, 1 H), 7.12 (d, 2 H, J
) 8.5 Hz), 7.19 (d, 2 H, J ) 8.5 Hz), 7.50 (m, 2 H), 7.70 (m, 5 H),
and 8.12 (d, 2 H, J ) 8.5 Hz); 13C NMR (CDCl3) δ 37.4, 49.5, 55.1,
56.9, 57.0, 64.6, 67.3, 67.4, 108.7, 110.6, 114.2, 120.8, 120.9, 125.6,
129.0, 129.6, 131.2, 132.2, 132.8, 134.7, 147.2, 150.2, 154.1, 155.7,
and 170.7. Anal. Calcd for C35H32O16N5P: C, 51.91; H, 3.98.
Found: C, 52.27; H, 4.11.
N-[[(2-Nitroveratryl)oxy]carbonyl]-4-[[bis(2-nitrobenzyl)phospho-
no]methyl]-(R,S)-phenylalanine Ethyl Ester (9). Amino acid 8 (110
mg, 0.153 mmol) was dissolved in 10 mL of ether and the solution
was treated with 2 mL of 1 N HCl. The reaction mixture was stirred
at 25 °C for 30 min. The pH of the aqueous phase was then adjusted
to 7 by addition of 1 N NaOH. The reaction mixture was treated with
10 mL of hexane and the organic phase was separated. The aqueous
phase containing the amino acid analogue was treated with 25.8 mg
(0.25 mmol) of Na2CO3, then with 51 mg (0.19 mmol) of NVOCCl in
5 mL of dioxane at 0 °C. The mixture was stirred at 25 °C for 2 h and
then treated with 100 mL of ethyl acetate. The organic phase was
washed with 30-mL portions of saturated NaHCO3 and then with
saturated NaCl. The organic phase was dried (MgSO4) and concentrated
under diminished pressure. The residue was purified by flash chro-
matography on a silica gel column (25 × 3 cm); elution with 3:1 ethyl
acetate-hexane afforded ethyl ester (9) as a yellowish powder: yield
80 mg (66% for two steps); silica gel TLC Rf 0.50 (3:1 ethyl acetate-
1
hexanes); mp 139-142 °C; H NMR (CDCl3) δ 1.24 (t, 3 H, J ) 7
Hz), 3.13-3.48 (m, 4 H), 3.92 (s, 3 H), 3.93 (s, 3 H), 4.17 (q, 2 H, J
) 7 Hz), 4.68 (m, 1 H), 5.20-5.59 (m, 7 H), 6.99 (s, 1 H), 7.14 (d, 2
H, J ) 8 Hz), 7.21 (d, 2 H, J ) 8 Hz), 7.46-7.69 (m, 6 H), 7.68 (s,
1 H), and 8.04 (d, 2 H, J ) 8 Hz); 13C NMR (CDCl3) δ 14.6, 32.8,
34.7, 38.1, 55.3, 56.8, 56.9, 62.1, 64.2, 65.0, 108.8, 110.3, 125.4, 128.5,
129.1, 130.2, 130.5, 132.8, 134.4, 147.3, 148.5, 154.1, 155.7, and 171.8.
N-[[(2-Nitroveratryl)oxy]carbonyl]-4-[[bis(2-nitrobenzyl)phospho-
no]methyl]-(R,S)-phenylalanine (10). Tyrosine phosphonate deriva-
tive 9 (80 mg, 0.10 mmol) was dissolved in 3 mL of THF and treated
with 200 µL of pyridine and 515 µL of 0.5 N NaOH. The reaction
mixture was stirred at 25 °C for 30 min and the pH was adjusted to 4
by the addition of 20 mL of 1 N NaHSO4. The reaction mixture was
extracted twice with 50-mL portions of ethyl acetate, and the combined
organic extract was dried (MgSO4) and concentrated. The residue was
precipitated from ethyl acetate-hexane, affording N-[[(2-nitroveratryl)-
oxy]carbonyl]-4-[[bis(2-nitrobenzyl)phosphono]methyl]-(R,S)-
phenylalanine (10) as a colorless solid: yield 58 mg (75%); silica gel
Bis(2-nitrobenzyl) 4-(Bromomethyl)benzylphosphonate (7). Phos-
phoramidite reagent 2 (435 mg, 1 mmol) in 3 mL of CH2Cl2 was added
to a solution containing 140 mg (2 mmol) of tetrazole and 153 mg (1
mmol) of 2-nitrobenzyl alcohol in 10 mL of CH2Cl2 under an argon
atmosphere. The reaction mixture was stirred at 25 °C for 50 min and
then treated with 25 mL of CH2Cl2 and washed with 10 mL of saturated
NaCl. The organic phase was dried (Na2SO4) and concentrated under
diminished pressure at 25 °C. Crude product 6, isolated as a thick oil,
was used directly in the next step to preclude oxidation. Crude 6 and
dibromo-p-xylene (263 mg, 1 mmol) were mixed and stirred together
under an argon atmosphere at 100 °C for 20 min. The solution was
permited to cool to room temperature and the product was purified by
flash chromatography on a silica gel column (35 × 3 cm). Elution
with 5:2:1 ether-hexanes-ethyl acetate gave phosphonate 7 as a
colorless powder: yield 160 mg (30% for two steps); silica gel TLC
1
TLC Rf 0.10 (ethyl acetate); mp 96-98 °C; H NMR (CDCl3) δ 3.12
(m, 2 H), 3.30 (s, 1 H), 3.37 (s, 1 H), 3.94 (s, 3 H), 3.96 (s, 3 H), 4.78
(d, 1 H, J ) 16 Hz), 4.74 (m, 1 H), 4.80 (d, 1 H, J ) 15.5 Hz), 5.30-
5.59 (m, 7 H), 6.96 (s, 1 H), 7.11 (d, 2 H, J ) 8 Hz), 7.27-7.64 (m,
8 H), 7.69 (s, 1 H), and 8.06 (d, 2 H, J ) 8 Hz). Anal. Calcd for
C34H33O15N4P: C, 53.13; H, 4.32. Found: C, 53.02; H, 4.39.
N-[[(2-Nitroveratryl)oxy]carbonyl]-4-[[bis(2-nitrobenzyl)phospho-
no]methyl]-(R,S)-phenylalanine Cyanomethyl Ester (11). Tyrosine
phosphonate derivative 10 (58 mg, 0.075 mmol) was dissolved in 5
mL of CH3CN and treated with 70 µL (0.376 mmol) of N,N-
diisopropylethylamine followed by 50 µL (0.19 mmol) of iodoaceto-
nitrile. The reaction mixture was stirred at 25 °C for 12 h and then
treated with 25 mL of ethyl acetate. The organic phase was washed
successively with 10-mL portions of saturated NaHCO3 and saturated
1
Rf 0.60 (5:2:1 ether-hexanes-ethyl acetate); mp 110 °C; H NMR
(CDCl3) δ 3.31 (s, 1 H), 3.38 (s, 1 H), 4.480 (s, 1 H), 4.483 (s, 1 H),
5.39 (s, 2 H), 5.41 (s, 2 H), 7.28-7.68 (m, 10 H), and 8.07 (m, 2 H);
13C NMR (CDCl3) δ 32.9, 33.5, 34.7, 65.0, 65.1, 125.4, 129.1, 129.4,
129.9, 130.7, 130.8, 132.8, 132.9, 134.5, 137.4, and 147.3. Anal. Calcd
for C22H20O7N2PBr: C, 49.36; H, 3.76. Found: C, 49.50; H, 3.68.

10886 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Arslan et al.
NaCl and then dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by flash chromatography on a silica
gel column (15 × 3 cm). Elution with ethyl acetate afforded
cyanomethyl ester 12 as a colorless powder: yield 56 mg (92%); silica
digestion by NcoI-PstI and ligated with the pTrc 99A vector that had
been digested by the same restriction enzymes. The cloning of the
luciferase gene into the pTrc 99A expression vector was confirmed by
restriction analysis and the luciferase assay. A TAG codon at position
286 was introduced into pTrcLuc using a T7-Gen in Vitro mutagenesis
kit (USB). The mutation was confirmed by sequencing by the dideoxy
chain termination method of Sanger.32
Expression of pTrcLuc-St286 in E. coli Strains Containing
Suppressor tRNAs. E. coli strains carrying suppressor tRNA’s for
Ser, Leu, and Gln and a control strain without any suppressor tRNA
were transformed by the DNA of plasmid pTrcLuc-St286. Individual
colonies obtained after transformation were cultured overnight at 22
°C in 100 mL of Luria-Bertani broth containing 50 µg/mL ampicillin
and 1 mM isopropyl â-D-thiogalactopyranoside (IPTG). The cells were
harvested by centrifugation, resuspended in 1 mL of lysis buffer (100
mM potassium phosphate, pH 7.8, containing 1 mM EDTA and 1 mg/
mL lysozyme), incubated on ice for 30 min, and then frozen on dry
ice. The frozen pellets were allowed to thaw at 25 °C and the DNA
was disrupted by sonication. Debris was removed by centrifugation
for 15 min at 14 000 rpm using a microcentrifuge. Glycerol was added
to the extracts to 17% v/v concentration, and the extracts were stored
at -20 °C.
Incorporation of a Fusion Dodecapeptide Containing Hexa-
histidine onto the C Terminus of Luciferase. A 36-base pair (bp)
synthetic oligonucleotide was ligated into SpeI-PstI sites of plasmids
pTrcLuc and pTrcLuc-St286, introducing a sequence of Tyr codon
(instead of the TAG stop codon) and the codons encoding hexahistidine
at the 3′-end of the luciferase gene, followed by the TAA stop codon
and a XhoI site. Incorporation of the hexahistidine sequence was
confirmed by the appearance of the XhoI site and by dideoxy sequencing
analysis. The resulting plasmids [pTrcluc(H) and pTrcLuc-St286(H)]
were used for expression in E. coli strains of the Interchange system
and for in Vitro translation. Luciferase assays carried out using
luciferases substituted at position 286 (obtained both in ViVo and in
Vitro) have demonstrated that the incorporation of the hexahistidine
fusion peptide at the C-terminus of the Luciola mingrelica luciferase
did not change properties of the enzyme such as emission wavelength
or specific activity.
Construction of a Plasmid for Runoff Transcription of Yeast
tRNAPheCUA(-CA). The DNA fragment containing the T7 promoter, a
sequence corresponding to yeast tRNAPheCUA, KpnI, HindIII, BstNI, and
FokI cleavage sites was constructed using two overlapping synthetic
oligonucleotides. The oligonucleotides were purified on a 8% denatur-
ing polyacrylamide gel; 5 µg of each oligonucleotide was combined
and annealed in 100 µL of 50 mM Tris-HCl, pH 7.5, containing 10
mM MgCl2, 1 mM DTT, and 50 mg/mL of bovine serum albumin.
Transcription was carried out in the presence of 1 mM each dNTP and
5 units of DNA polymerase I (Klenow) at 37 °C for 30 min. The
double-stranded DNA was purified on a 6% polyacrylamide gel,
digested with restriction endonucleases KpnI and HindIII, and ligated
to plasmid pUC19 that had been digested with the same restriction
endonucleases. The structure of the resulting plasmid (pYRNA) was
confirmed by restriction digest analysis and dideoxy sequencing.
Runoff Transcription of Suppressor tRNA(-CA). Plasmid
pYRNA DNA was digested with FokI and then transcribed using an
AmpliScribe T7 transcription kit (Epicentre Technologies) in a 500-
µL reaction mixture at 37 °C for 4 h according to the protocol supplied
with the kit. After incubation, 500 µL of 80% formamide containing
0.02% xylene cyanol and bromophenol blue was added and mixture
was applied to an 8% denaturing polyacrylamide gel (400 × 200 × 2
mm). After electrophoresis for 3 h at 800 V, the RNA band was
visualized by UV shadowing,33 excised from the gel, and cut into small
pieces. The RNA was eluted from the gel by treatment with 5 mL of
100 mM sodium acetate, pH 4.6, containing 1 mM EDTA and 0.01%
SDS at 4 °C for 12 h. The tRNA was precipitated with two volumes
of cold ethanol, dried, and then dissolved in RNase-free water and
divided into aliquots and stored at -80 °C.
1
gel TLC Rf 0.80 (ethyl acetate); mp 83-85 °C; H NMR (CDCl3) δ
3.12 (m, 2 H), 3.30 (s, 1 H), 3.37 (s, 1 H), 3.94 (s, 3 H), 3.96 (s, 3 H),
4.68 (d, 1 H, J ) 16 Hz), 4.74 (m, 1 H), 4.80 (d, 1 H, J ) 15.5 Hz),
5.50-5.59 (m, 7 H), 6.96 (s, 1 H), 7.11 (d, 2 H, J ) 8 Hz), 7.27-7.64
(m, 8 H), 7.69 (s, 1 H), and 8.06 (d, 2 H, J ) 8 Hz). Anal. Calcd for
C36H34O15N5P: C, 53.53; H, 4.24. Found: C, 53.84; H, 4.40.
N-[[(2-Nitroveratryl)oxy]carbonyl]-O-[bis[(2-nitrobenzyl)oxy]-
phosphoryl]-(S)-tyrosine pdCpA Ester (12). A solution of the tris-
(tetrabutylammonium) salt of pdCpA (4.1 mg, 3 µmol) dissolved in
50 µL of freshly distilled DMF was added to a flame-dried conical
vial containing 20 mg (25 µmol) of cyanomethyl ester 5. The reaction
mixture was stirred at 25 °C and monitored by HPLC. Four-microliter
aliquots were removed periodically and diluted with 40 µL of 50 mM
NH4OAc, pH 4.5; 10 µL of each diluted aliquot was analyzed on a 3
µm C18 reverse phase HPLC column (100 × 4.6 mm). The column
was washed with 1% f 65% CH3CN-50 mM NH4OAc, pH 4.5, over
45 min at a flow rate of 1.0 mL/min [detection at 260 nm; the desired
product had a retention time of 30.2 min (Supporting Information,
Figures 2 and 3)]. The remaining reaction mixture was diluted with
600 µL of 1:1 CH3CN-50 mM NH4OAc, pH 4.5, and purified by C18
reverse phase HPLC on a semipreparative column (250 × 10 mm) using
the same gradient described above at a flow rate of 4.0 mL/min.
Dinucleotide derivative 12 was recovered from the appropriate fractions
by lyophilization as a colorless solid: yield (3.2 mg, 77%); mass
spectrum (FAB) m/z 1389 (M + H)+; (FAB) m/z 1389.255 (M + H)+
(C52H56O28N12P3 requires 1389.254).
N-[[(2-Nitroveratryl)oxy]carbonyl]-4-[[bis[(2-nitrobenzyl)oxy]-
phosphono]methyl]-(R,S)-phenylalanine pdCpA Ester (13). A solu-
tion of the tris(tetrabutylammonium) salt of pdCpA (4.1 mg, 3 µmol)
dissolved in 50 µL of freshly distilled DMF was added to a flame-
dried conical vial containing 20 mg (25 µmol) of cyanomethyl ester
11. The reaction mixture was stirred at 25 °C and monitored by HPLC.
Four-microliter aliquots were removed periodically and diluted with
40 µL of 50 mM NH4OAc, pH 4.5; 10 µL of each diluted aliquot was
analyzed on a 3 µm C18 reverse phase HPLC column (100 × 4.6 mm).
The column was washed with 1% f 65% CH3CN in 50 mM NH4OAc,
pH 4.5, over a period of 45 min at a flow rate of 1.0 mL/min [detection
at 260 nm; the desired product had a retention time of 28.7 min
(Supporting Information, Figures 4 and 5)]. The remaining reaction
mixture was diluted with 600 µL of 1:1 CH3CN-50 mM NH4OAc,
pH 4.5, and purified by C18 reverse phase HPLC on a semipreparative
column (250 × 10 mm) using the same gradient described above at a
flow rate of 4.0 mL/min. Dinucleotide derivative 13 was recovered
from the appropriate fractions by lyophilization as a colorless solid:
yield (3.4 mg, 82%); mass spectrum (FAB) m/z 1387 (M + H)+; (FAB)
m/z 1387.273 (M + H)+ (C53H58O27N12P3 requires 1387.275).
Photochemical Deprotection of pdCpA Esters 12 and 13. The
photochemical deprotection of aminoacyl-pdCpA derivatives 12 and
13 was carried out following literature precedent31 by use of a 500 W
mercury-xenon lamp using Pyrex and water filters. The samples were
irradiated for 8 min. The deblocking of 12 and 13 was monitored by
C18 reverse phase HPLC as described above. Retention time (tR) of
12 was 30.2 min (Supporting Information, Figures 2 and 3) and the tR
of the corresponding fully deprotected aminoacyl pdCpA was 9.7 min
(Supporting Information, Figures 6 and 7). The tR of 13 was 28.7 min
(Supporting Information, Figures 4 and 5) and the tR of the corre-
sponding fully deprotected aminoacyl pdCpA was 6.9 min (Supporting
Information, Figures 8 and 9) under the same conditions. The UV
spectra reflected complete deprotection.
Construction of the Plasmid for Expression of Luciferase Gene.
pGK3 plasmid10 was kindly provided by Dr. G. Kutuzova. The
luciferase gene was subcloned into the pGEM-5Zf(+) vector (Promega)
using the NcoI-MamI fragment of pGK3 containing the luciferase gene
and NcoI-EcoRV-digested pGEM-5Zf(+) plasmid DNA. The lu-
ciferase gene was isolated from intermediate plasmid pGEM5-luc after
Synthesis of Misacylated tRNAs. Ligation reactions were carried
out in reaction mixtures containing per 50 µL: 0.5 A260 unit of
(32) Sanger, F.: Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A.
1977, 74, 5463.
(33) Hassur, S. M.; Whitlock, H. W. Anal. Biochem. 1974, 59, 162.
(31) Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc.
1991, 113, 2722.

Structurally Modified Firefly Luciferase
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10887
aminoacyl pdCpA, 10 µg tRNAPheCUA(-CA), 50 mM Hepes buffer, pH
7.5, 15 mM MgCl2, 10-20% DMSO and 100 units of T4 RNA ligase.
Reactions were incubated at 37 °C for 25 min and then quenched by
addition of 0.1 volume of 3 M sodium acetate, pH 4.5. The tRNA
was precipitated by the addition of two volumes of cold ethanol and
collected by centrifugation. The tRNA was washed with 70% ethanol,
dried, and dissolved in 1 mM potassium acetate solution to a
concentration of 1 mg/mL. Photodeprotection was carried out using a
500 W mercury-xenon lamp with Pyrex and water filters. The
aminoacyl-tRNA solution in a transparent microfuge tube was placed
into a glass container with cooling water circulated at 2 °C. Irradiation
was carried out for 2 min for Val and Phe, 5 min for Ser and
glucosylated Ser, and 8 min for Ser phosphonate, as well as Tyr
phosphate and phosphonate derivatives. Deprotected aminoacyl-tRNAs
were used in in Vitro suppression experiments immediately after
irradiation.
In Vitro Suppression. In Vitro synthesis of luciferase were carried
out in an E. coli S30 translation system for circular DNA. Four
micrograms of plasmid DNA and 5 µg of misacylated tRNA were
typically used in a 50-µL reaction mixture (total volume), which also
contained amino acids (100 µM final conc) 20 µL of S30 premix,34 10
µCi of [35S]methionine (1200 Ci/mmol), and 15 µL of S30 extract.
The reaction mixture was maintained at 22 °C for 1-2 h. The reaction
was stopped by immersion in an ice bath for 5 min. Ten microliters
of this translation mixture was used for luciferase assay and 5-µL
aliquots were used for 10% SDS-PAGE analysis after acetone
precipitation.
columns were washed successively with 5 mL of 100 mM K phosphate
buffer, pH 7.8, and then with 5 mL of the same buffer containing 60
mM imidazole. The luciferase fusion proteins were eluted from the
column with 4 mL of 100 mM K phosphate buffer, pH 7.8, containing
120 mM imidazole; 1-mL fractions were collected. The fractions
containing luciferase were determined using an assay for light produc-
tion. The fractions containing highest luciferase activity (usually 2
and 3) were pooled and dialyzed against 100 mM K phosphate, pH
7.8, containing 1 mM MgSO4, 1 mM EDTA, and 15% glycerol v/v.
The concentrations of the purified luciferases were estimated using
SDS-PAGE followed by Coomassie Brillant Blue staining.
Luciferase Assay. Luciferase assays were typically carried out using
a commercial luciferase assay system (Promega). After translation,
10 µL of the translation reaction mixture was added to 100 µL of the
luciferase assay reagent and light emission was measured immediately
using an Hitachi F2000 fluorescence spetrophotometer. Experiments
monitoring pH dependence of the spectra of light emitted were carried
out using 0.1 M MOPS-MES buffer, 5 mM MgCl2, 0.5 mM luciferin,
1.5 mM ATP, and 1 mM EDTA.
Acknowledgment. We thank Dr. Galina Kutuzova for the
plasmid encoding L. mingrelica luciferase and Drs. Kenneth
Rothschild, Boston University, and Uttam RajBhandary, Mas-
sachusetts Institute of Technology, for the strain of E. coli from
which mature tRNATyr
was isolated. This work was sup-
CUA
ported by NIH Research Grant GM43328 and Grant B10-94-
015 from Virginia’s Center for Innovative Technology.
Purification of Luciferase Containing a Hexahistidine Fusion
Peptide. The luciferases were expressed in Interchange E. coli strains
and cell free extracts were prepared as described previously.13 The
extracts were applied to 0.5-mL Ni-NTA (QIAGEN) columns. The
Supporting Information Available: A table outlining the
thermostability of the luciferase analogues and PAGE, UV, and
HPLC analysis of some analogues (11 pages). See any current
masthead page for ordering and Internet access instructions.
(34) Lesley, S. A.; Brow, M. A. D.; Burgess, R. R. J. Biol. Chem. 1991,
266, 2632.
JA971927A