Design and synthesis of photochemically controllable restriction endonuclease BamHI by manipulating the salt-bridge network in the dimer interface

Article abstract of DOI:10.1021/jo035774n

The strategy for the design of photochemically controllable enzymes by manipulating the dimer interface is described. Employing a restriction endonuclease BamHI, the selective incorporation of amino acids having a photoremovable 6-nitroveratryl group into

Full text of DOI:10.1021/jo035774n

Design a n d Syn th esis of P h otoch em ica lly Con tr olla ble Restr iction
En d on u clea se Ba m HI by Ma n ip u la tin g th e Sa lt-Br id ge Netw or k in
th e Dim er In ter fa ce
Masayuki Endo, Koji Nakayama, and Tetsuro Majima*
Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka,
Ibaraki, Osaka 567-0047, J apan
majima@sanken.osaka-u.ac.jp
Received December 4, 2003
The strategy for the design of photochemically controllable enzymes by manipulating the dimer
interface is described. Employing a restriction endonuclease BamHI, the selective incorporation of
amino acids having a photoremovable 6-nitroveratryl group into the specific position (Lys132) in
the dimer interface of the BamHI mutant (H133A) was performed. The activity of the photofunc-
tionalized BamHI mutant was significantly suppressed, and the following photoirradiation induced
the recovery of the activity. In addition, uncaging of the 6-nitroveratryl group introduced to Lys132
did not seriously reduce the catalytic activity and affinity for the substrate. These results indicate
that the activity of the enzyme can be effectively regulated by caging and uncaging of the specific
amino acid in the dimer interface using the photoremovable group.
In tr od u ction
a photoremovable group for inactivation followed by
uncaging it with photoirradiation for activation. These
studies have revealed that the photoregulation using a
caged protein is practically advantageous for the initia-
tion of the protein activity in vitro and in vivo.3
To control the activity of the enzyme, we planned to
manipulate the amino acids involved in a salt-bridge
network in a protein-protein interface by introduction
of photoreactive molecules. We employed a restriction
endonuclease BamHI which has a dimer interface includ-
ing hydrogen bondings (H-bondings) with salt bridges
and requires dimerization to exhibit the activity.4 The
salt-bridge network in the dimer interface of BamHI
consists of four amino acids: two basic amino acids,
Lys132 and His133, in the R helix specifically interact
with two acidic amino acids, Glu167 and Glu170, in the
counterpart R helix (Figure 1).4
Complex formation of proteins is a key phenomenon
for expressing specific activities and functions in vitro
and in vivo. The higher order structures of proteins to
form active complexes need precise assembly through
several key amino acids in the protein-protein interface.1
A salt-bridge network involving these amino acids often
seen in the dimer interface stabilizes the formation of
the complex and contributes to the precise alignment of
the dimer. Substitution of these amino acids, for example,
alanine scanning, results in a significant decrease or
inactivation of the functions and formation of the com-
plex.2 This means that if chemically controllable func-
tional groups, especially photoreactive residues, can be
selectively introduced into these amino acids, the as-
sociation of the proteins and the enzymatic activity can
be regulated by photoirradiation from the outside without
the addition of reagents for activation. Numbers of
studies have been made by caging of a target protein with
For labeling of proteins, a promising method for site-
selective incorporation of unnatural amino acids has been
developed using an in vitro translation system with
special codons and the corresponding aminoacyl-tRNA.5-7
The incorporation of a photoremovable group has been
achieved by these methods.8 To regulate the enzymatic
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10.1021/jo035774n CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/29/2004
4292
J . Org. Chem. 2004, 69, 4292-4298

Synthesis of Photofunctionalized BamHI
CHART 1. P h otofu n ction a lized Am in o Acid s
Em p loyed in Th is Exp er im en t: NVOC-Lys (KNVOC
)
a n d NV-Glu (ENV
)
SCHEME 1. Syn th etic Sch em e for Site-Selective
In cor p or a tion of P h otofu n ction a lized Am in o Acid s
in to Ba m HIa
F IGURE 1. Crystal structure of endonuclease BamHI (PDB,
1BHM). (A) The complex of BamHI dimer and DNA (red rope).
(B) The salt-bridge network with the amino acid side chains
in the BamHI dimer interface. Dashed lines indicate the
H-bondings with salt-bridges. Asterisks denote the counterpart
BamHI monomer in the dimer interface. Figures were created
by a Cn3D program distributed by the National Center for
Biotechnology Information.
a
In the first step, a synthetic aminoacyl-pdCpA was coupled
with tRNACCCG(-CA) to give a photofunctionalized aminoacyl-
tRNA. In the second step, mRNA with CGGG codon at the specific
position was translated with the aminoacyl-tRNA using the in
vitro translation system.
reduction of electrostatic interaction by introduction of
the photoremovable groups to the side chains of the basic
and acidic amino acids; (3) steric effect caused by the
bulkiness of the 6-nitroveratryl groups to prevent correct
positioning of amino acid side chains in the dimer
interface. Based on this strategy, we designed and
synthesized the photofunctionalized BamHI and inves-
tigated for photochemical control of the correct dimer
formation of BamHI and subsequent recovery of the
enzymatic activity.
activity by photoirradiation, we individually introduced
a photoremovable 6-nitroveratryloxycarbonyl (NVOC)
group to Lys132 and a 6-nitroveratryl (NV) group to
Glu167 and Glu170 (Chart 1). We expected to control the
following three important changes in the salt-bridge
network of the dimer interface by protection of the amino
and carboxylate groups with 6-nitroveratryl deriva-
tives: (1) inhibition of H-bond formations between the
amino acid side chains in the salt-bridge network; (2)
Resu lts a n d Discu ssion
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1995, 34, 1629-1630. (c) Nowak, M. W.; Kearney, P. C.; Sampson, J .
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Arslan, T.; Hecht, S. M. J . Am. Chem. Soc. 1999, 121, 478-479.
The synthesis of the photofunctionalized BamHI has
been performed by the in vitro translation system. The
biochemical method using four-base codon-anticodon
interaction was employed for site-selective incorporation
of photofunctionalized amino acids.7 As shown in Scheme
1, first, a photofunctionalized aminoacyl-tRNA with a
four-base anticodon was obtained from the coupling of a
photofunctionalized aminoacyl-pdCpA and tRNACCCG lack-
ing a 3′-terminal CA nucleotide [tRNACCCG(-CA)]. Sec-
ond, site-selective incorporation of photofunctionalized
amino acids was performed by in vitro translation with
mRNA containing the four-base codon (CGGG) and the
photofunctionalized aminoacyl-tRNACCCG
.
Syn th esis of P h otofu n ction a lized Am in o Acid s
a n d Am in oa cyl-tRNACCCG. The synthesis of active
esters of lysine and glutamic acid with a 6-nitroveratryl
group was carried out according to Scheme 2. The
6-nitroveratryl group to the Nꢀ position of lysine (1) was
introduced by a reaction of NR-(tert-butoxycarbonyl)-L-
lysine with 6-nitroveratryl chloroformate. Esterification
of 1 with chloroacetonitrile gave NR-Boc-Nꢀ-NVOC-Lys
cyanomethyl ester 2. Introduction of the 6-nitroveratryl
J . Org. Chem, Vol. 69, No. 13, 2004 4293

Endo et al.
SCHEME 2. Syn th etic Sch em e for F u n ction a lized
Am in oa cyl-p d Cp A 3 a n d 7a
SCHEME 3. P h otoch em ica l Rea ction s of
Nr-Boc-NE-NVOC-Lys 1 a n d Nr-Boc- γ-NV-Glu 5
w ith P h otoir r a d ia tion a t 365 n m
and proteins previously studied,3,9 the removal of the
NVOC and NV gave NR-Boc-Lys and NR-Boc-Glu during
the photoirradiation of NR-Boc-Nꢀ-NVOC-Lys 1 and
NR-Boc-γ-NV-Glu 5, respectively. The half-lives for deg-
radation of the Boc-NVOC-Lys 1 and Boc-NV-Glu 5 were
1.2 and 3.5 min, respectively, under these experimental
conditions.
a
(a) 6-Nitroveratryl chloroformate; (b) chloroacetonitrile; (c)
F IGURE 2. In vitro translation of BamHI mutants with
pdCpA Bu4N+ salt; (d) 6-nitroveratryl alcohol, DCC; (e) (i) TFA,
(ii) 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile; (f) chlo-
roacetonitrile; (g) pdCpA Bu4N+ salt.
unnatural aminoacyl-tRNACCCG
: lane 1, translation with
mRNA of wild-type BamHI; lanes 2 and 3, translation with
K132KNVOC mutant mRNA without and with NVOC-Lys-
tRNACCCG, respectively; lanes 4 and 5, translation with
E167ENV mutant mRNA without and with NV-Glu-tRNACCCG
,
group to the γ-carboxylate of glutamic acid was performed
by the coupling of NR-tert-butoxycarbonylglutamic acid-
R-tert-butyl ester and 6-nitroveratyl alcohol with dicy-
clohexyl carbodiimide (DCC) to give 4. tert-Butoxycarbo-
nyl and tert-butyl groups of 4 were removed by treatment
with trifluoroacetic acid (TFA), and then the NR-amino
group was protected with the Boc group using 2-(tert-
butoxycarbonyloxyimino)-2-phenylacetonitrile to afford 5.
Esterification of 5 with chloroacetonitrile gave NR-Boc-
γ-NV-Glu cyanomethyl ester 6.
respectively; lanes 6 and 7, translation with E170ENV mutant
mRNA without and with NV-Glu-tRNACCCG, respectively.
Syn th esis of Site-Selectively P h otofu n ction a lized
Ba m HI Mu ta n ts. The photofunctionalized amino acids
previously synthesized were incorporated into the specific
positions of BamHI. The introduction of the modified
amino acids into the specific positions of BamHI was
carried out by employing the in vitro translation system
with mRNA containing a four-base codon and the syn-
thesized aminoacyl-tRNACCCG. In vitro translation was
carried out in a mixture containing mRNA, unnatural
aminoacyl-tRNACCCG, E. coli S30 extract, and amino acid
with [35S]-methionine at 30 °C for 3 h. After the reaction,
the mixtures were loaded onto an 18% SDS-PAGE, and
the gel was visualized by an imaging analyzer (Figure
2). Wild-type BamHI was expressed as a full-length form
at 27 kDa (lane 1). In the cases of translation with mRNA
containing a four-base codon mutation, incomplete length
of BamHI was observed in the absence of aminoacyl-
tRNA (lanes 2, 4, and 6). In contrast, full-length BamHI
was obtained in the presence of the specific aminoacyl-
tRNA (lanes 3, 5, and 7). The efficiency of incorporation
of NVOC-Lys at position 132 (K132KNVOC) was 60%
quantified by the radioactivity of [35S]-methionine. On the
other hand, the incorporation efficiencies of the NV-Glu
The incorporation of the modified amino acids was
performed using a four-base codon-anticodon method
according to the previously reported method.7b At first,
aminoacyl-tRNAs were obtained by a reaction of the
NR-Boc-Nꢀ-NVOC-Lys cyanomethyl ester (2) or NR-Boc-
γ-NV-Glu cyanomethyl ester (6) with a nucleoside dimer
5′-phospho-2′-deoxycytidylyl-(3′-5′)-adenosine (pdCpA) to
give NR-Boc-Nꢀ-NVOC-Lys-pdCpA 3 (75% yield) and
NR-Boc-γ-NV-Glu-pdCpA 7 (93% yield). The Boc-protected
aminoacyl-pdCpA was treated with TFA followed by
ligation onto a tRNACCCG(-CA) using T4 RNA ligase to
afford Nꢀ-NVOC-Lys-tRNACCCG and γ-NV-Glu-tRNACCCG
.
The photoreactivities of the NR-Boc-Nꢀ-NVOC-Lys (1)
and NR-Boc-γ-NV-Glu (5) were examined (Scheme 3). The
photoirradiation was carried out in a 1 mM methanoic
solution of the nitroveratryl derivatives at 0 °C by a
transilluminator at 365 nm. The products were analyzed
by a reversed-phase HPLC. The nitroveratryl groups
were easily deprotected, and no other byproduct was
detected. Like the photodeprotection for caged peptides
(9) (a) Tatsu, Y.; Shigeri, Y.; Sogabe, S.; Yumoto, N.; Yoshikawa, S.
Biochem. Biophys. Res. Commun. 1996, 227, 688-693. (b) Curley, K.;
Lawrence, D. S. Curr. Opin. Chem. Biol. 1999, 3, 84-88.
4294 J . Org. Chem., Vol. 69, No. 13, 2004

Synthesis of Photofunctionalized BamHI
F IGURE 4. In vitro translation of BamHI H133A mutants
with unnatural aminoacyl-tRNACCCG: lane 1, translation with
an mRNA of H133A mutant; lanes 2 and 3, translation with
an H133A/K132KNVOC mutant mRNA without and with NVOC-
Lys-tRNACCCG, respectively; lanes 4 and 5, translation with
an H133A/E167ENV mutant mRNA without and with NV-Glu-
tRNACCCG, respectively; lanes 6 and 7, translation with an
H133A/E170ENV mutant mRNA without and with NV-Glu-
tRNACCCG, respectively.
F IGURE 3. Cleavage of a substrate DNA (pBR322 restriction
fragment) with a wild-type and mutant BamHI without and
with photoirradiation: lane 1, substrate DNA only; lane 2,
DNA cleavage with a commercially available BamHI (20
units); lane 3, a wild type BamHI prepared by the in vitro
translation system; lanes 4 and 5, DNA cleavage with a
K132KNVOC BamHI mutant without (-) and with (+) photoir-
radiation, respectively; lanes 6 and 7, DNA cleavage with an
E167ENV BamHI mutant without (-) and with (+) photoirra-
diation, respectively; lanes 8 and 9, DNA cleavage with an
E170ENV BamHI mutant without (-) and with (+) photoirra-
diation, respectively. The photoirradiation was carried out at
0 °C for 20 min, and then the cleavage of substrate DNA was
performed at 30 °C for 2 h. The retardation of cleavage bands
in lanes 3-9 may originate from the binding of proteins in
the in vitro translation mixtures.
introduced to the amino acid side chains cannot prevent
the dimer formation for the BamHI activity. Because of
the long methylene chains of Lys and Glu, these would
facilitate the exclusion of the bulky 6-nitroveratryl groups
outside of the dimer interface. In addition, one or two
H-bondings in the salt-bridge network are still preserved
in the BamHI mutants even when the photoremovable
groups are introduced. These results suggest that one
H-bonding in the dimer interface would be sufficient to
exhibit the activity and can maintain the correct dimer
formation. The NV-Glu BamHI mutants showed poor
activities (40-60% of DNA cleavage), which may indicate
some distortion or failure of the interaction with other
amino acids by some misfolding. Further manipulation
of the salt-bridge network in the dimer interface is
required to prevent the dimer formation using the
photoremovable groups.
To suppress the BamHI activity before photoirradia-
tion, we replaced histidine at position 133 to alanine
(H133A mutant) to reduce the number of H-bondings
(Figure 1b). When the NVOC group is introduced to the
Lys132 of the H133A mutant (H133A/K132KNVOC), all the
H-bondings are excluded from the salt-bridge network,
meaning that the dimer formation should be completely
prohibited. In the cases of Glu167 and Glu170 of the
H133A mutants with NV groups (H133A/E167ENV and
H133A/E170ENV, respectively), one H-bonding is still
preserved between the Lys132 and glutamates. If the
activity of the H133A/K132KNVOC mutant is compared
with those of the H133A/E167ENV and H133A/E170ENV
mutants, the effect of the H-bonding for the dimer
formation could be estimated.
Su p p r ession a n d Recover y of th e Activity of
P h otofu n ction a lized Ba m HI Mu ta n ts w ith P h otoir -
r a d ia tion . We prepared the mRNA of the H133A mutant
and those with a four-base codon in positions 132, 167,
and 170 individually. Incorporation of the NVOC-Lys into
position 132 and NV-Glu into positions 167 and 170 was
carried out by the same method as previously described.
As shown in Figure 4, when the NVOC group was
incorporated into position 132 and the NV groups into
positions 167 and 170, the full-length proteins were
obtained in the presence of the specific aminoacyl-tRNA
(compare lanes 3, 5, and 7 with the corresponding lanes
2, 4, and 6, respectively in Figure 4). The efficiency of
incorporation of NVOC-Lys at position 132 was 55% and
at positions 167 and 170 (E167ENV and E170ENV, respec-
tively) were 11 and 10%, respectively. Incorporation effi-
ciencies for NV-Glu were obviously lower than that of
NVOC-Lys. Since the efficiencies of incorporation depend
on the shape and bulkiness of the side chains of the
amino acids,7b steric hindrance would exist to some extent
in the 6-nitroveratryl ester on glutamate as compared
to the longer methylene chain of NVOC-Lys when in-
corporated into the proteins. We employed these BamHI
mutants for the investigation of the photochemical con-
trol of enzymatic activities without further purification.
Activity of P h otofu n ction a lized Ba mHI with P h o-
toir r a d ia tion . Enzymatic activities of the wild-type and
mutant BamHI were identified by double-strand cleavage
of the pBR322 restriction fragment digested by EcoRI as
a substrate (Figure 3). The wild-type BamHI prepared
by in vitro translation showed sufficient activity and
sequence selectivity (lane 3). Photoreaction for the func-
tionalized BamHI was carried out in a solution contain-
ing the previous BamHI. For uncaging of the photore-
movable groups, photoirradiation was performed by a
transilluminator (365 nm) at 0 °C for 20 min. On the
basis of the results of deprotection of the photoremovable
groups of the amino acid monomers (NVOC-Lys and NV-
Glu), over 90% of the protecting groups of photofunction-
alized BamHI could be removed under these experimen-
tal conditions. After the photoirradiation, the mixtures
were diluted with a buffer containing 10 mM tris-
(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)
(pH 8.0), 10 mM MgCl2, 100 mM NaCl, and 1 mM DTT,
and the solutions were incubated with the substrate DNA
fragment at 30 °C for 2 h for examining enzymatic
activities. Unexpectedly, all the mutants (K132KNVOC
,
E167ENV, and E170ENV) exhibited the activities without
photoirradiation (lanes 4, 6, and 8) (Figure 3). After
photoirradiation and subsequent enzymatic reaction, the
mutants exhibited the same activities as those of the
mutants without photoirradiation (lanes 5, 7, and 9).
These results suggest that the 6-nitroveratryl groups
J . Org. Chem, Vol. 69, No. 13, 2004 4295

Endo et al.
F IGURE 6. DMS chemical cross-linking of the wild-type
BamHI, K132KNVOC, and H133A/K132KNVOC mutants without
and with photoirradiation: lanes 1 and 2, wild-type BamHI
without (-) and with (+) photoirradiation, respectively; lanes
3 and 4, DMS-treated wild-type BamHI without (-) and with
(+) photoirradiation, respectively; lane 5, K132KNVOC mutant;
lanes 6 and 7, DMS-treated K132KNVOC mutants without (-)
and with (+) photoirradiation, respectively; lane 8, H133A/
K132KNVOC mutant; lanes 9 and 10, DMS-treated H133A/
K132KNVOC mutants without (-) and with (+) photoirradiation,
respectively. For the negative control of DMS cross-linking,
wild-type BamHI treated with 0.1% SDS for inhibition of the
dimer formation was detected as a monomer.
F IGURE 5. Cleavage of a substrate DNA (λDNA) with
BamHI H133A mutants without and with photoirradiation:
lane 1, cleavage with a BamHI H133A mutant; lanes 2 and 3,
cleavage with an H133A/K132KNVOC BamHI mutant without
(-) and with (+) photoirradiation, respectively; lanes 4 and 5,
cleavage with an H133A/E167ENV BamHI mutant without (-)
and with (+) photoirradiation, respectively; lanes 6 and 7,
cleavage with an H133A/E170ENV BamHI mutant without (-)
and with (+) photoirradiation, respectively. Photoirradiation
was carried out at 0 °C for 20 min, and then the cleavage of
substrate DNA was performed at 30 °C for 2 h.
those of the NV-Glu at positions 167 and 170 were 23
and 17%, respectively.
level of the affinity and activity as compared to the
H133A BamHI mutant. These results indicate that
caging and following uncaging of the NVOC group
attached to the K132 of the H133A mutant do not
seriously affect the activity and affinity for the substrate.
The effect of the H133A mutation is critical for the
activity (KM ) 1.7 nM and Vmax ) 8.8 × 10-1 nM min-1
for the wild-type BamHI), suggesting that the H-bonding
between H133 and E167 would largely contribute to
stabilizing the dimer structure. Because of the decrease
of the number of the H-bondings involved in the salt-
bridge network, elimination of the H133 would weaken
the interaction of the two BamHI monomers, which may
facilitate to control the dimer formation by protection and
deprotection using the photoremovable group.
Dir ect Obser va tion of th e Dim er for m a tion by
Ch em ica l Cr oss-Lin k in g. To directly examine the
dimer formation of the photofunctionalized BamHI mu-
tants, the wild-type, K132KNVOC, and H133A/K132KNVOC
mutants were investigated by a chemical cross-linking
method using dimethyl suberimidate dihydrochloride
(DMS) (Figure 6).11 In this experiment, we used the
purified BamHI for cross-linking the proteins. In the case
of the wild-type BamHI, both proteins before and after
photoirradiation were detected as the dimer on SDS-
PAGE (lanes 3 and 4). The K132KNVOC and H133A/
K132KNVOC mutants before photoirradiation were de-
tected as the dimer (lanes 6 and 9), indicating that the
NVOC does not prevent the dimer formation. After
photoirradiation, both mutants were also detected as the
dimer. In the case of the K132KNVOC mutant, both
mutants without and with photoirradiation had the
activities, and the cross-linking results are consistent
with the cleavage results (see Figure 3). In contrast, the
To detect the activities and specificities of these mutant
proteins, we performed the cleavage of λDNA as a
substrate (Figure 5). The H133A mutant of BamHI
prepared by in vitro translation showed sufficient activity
and sequence selectivity (lane 1); however, the catalytic
activity of the H133A mutant was significantly decreased
as compared to that of the wild-type BamHI (vide
infra). Photoirradiation to the BamHI mutants (H133A/
K132KNVOC, H133A/E167ENV, and H133A/E170ENV) was
carried out at 0 °C for 20 min. In the case of H133A/
K132KNVOC (lanes 2 and 3), the activity of the H133A/
K132KNVOC mutant was almost suppressed without pho-
toirradiation (2.7% of cleavage product), and after
photoirradiation and incubation, the mutant recovered
the activity and specificity, and 23% of the DNA was
cleaved (lane 3). This suggests that the reconstitution of
the salt-bridge network in the dimer interface by removal
of the NVOC group. In contrast, the H133A/E167ENV and
H133A/E170ENV mutants did not show the activities even
after photoirradiation, suggesting that serious misfolding
would abolish the enzymatic activities by introduction
of the NV groups into these glutamates.
The activity of H133A/K132KNVOC mutant after pho-
toirradiation was evaluated by kinetic parameters ac-
cording to the previously reported method.10 Using the
pBR322 restriction fragment as a substrate, kinetic
parameters were obtained as KM ) 2.9 nM and Vmax
)
5.4 × 10-2 nM min-1 for the photoirradiated H133A/
K132KNVOC mutant and KM ) 2.3 nM and Vmax ) 6.5 ×
10-2 nM min-1 for the H133A mutant. The caging and
uncaging of H133A/K132KNVOC mutant lowered both
affinity and catalytic activity, but still preserved the same
(10) (a) Hinsch, B.; Mayer, H.; Kula, M. Nucleic Acids Res. 1980, 8,
2547-2559. (b) Hinsch, B.; Kula, M. Nucleic Acids Res. 1981, 9, 3159-
3174.
(11) Dubey, A. K.; Bisaria, V. S.; Mukhopadhyay, S. N.; Ghose, T.
K. Biotech. Bioeng. 1989, 33, 1311-1316.
4296 J . Org. Chem., Vol. 69, No. 13, 2004

Synthesis of Photofunctionalized BamHI
H133A/K132KNVOC mutant only showed the activity after
photoirradiation (Figure 5), meaning that the H133A/
K132KNVOC forms an inactive dimer before photoirradia-
tion. This result is similar to the case of photofunction-
alized HIV-1 protease, which forms a dimer without
photoirradiation as an inactive form.8f The results we
obtained may suggest that the photoremovable NVOC
group in the dimer interface would prevent the correct
dimer formation, and the removal of the NVOC group
from the dimer interface would reconstitute the salt-
bridge network and rearrange the BamHI dimer as an
active form.
positions 132, 167, and 170 were prepared by the QuikChange
site-directed mutagenensis method (Stratagene, LaJ olla, CA)
with Pfu DNA polymerase. Mutant BamHI gene substituted
by alanine at position 133 was also prepared by the same
mutagenesis method. The sequences for all the plasmids
prepared here were confirmed by automated dideoxy DNA
sequencing.
In Vitr o Tr a n sla tion of Wild -Typ e a n d Mu ta n t Ba m HI.
Preparation of mRNA was carried out in a solution containing
template DNA (5 µg), T7 RNA polymease (110 units), 2.5 mM
NTP, 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2, and 5 mM DTT
at 37 °C for 6 h. Prepared mRNA was purified by an RNeasy
Mini Kit (Qiagen, Hilden, Germany), and approximately 50-
150 µg was obtained. E. coli S30 Extract System (Promega,
Madison, WI) was employed for in vitro protein synthesis
following the manufacturer’s protocol. The translation reaction
was carried out in 10 µL of reaction mixture containing mRNA
Con clu sion s
We have demonstrated the direct photochemical con-
trol of the enzymatic activity and function by precise
manipulation of the salt-bridge network in the dimer
interface employing the photofunctionalized BamHI mu-
tants. In our experiment, the key amino acid in BamHI
for control of the enzymatic activity is the Lys132, and
this residue can be available for the potential control of
the BamHI activity. Using the method as we have
demonstrated here, the photochemical activation of the
enzymes and proteins can be achieved by introducing
photoreactive molecules into the key amino acids in the
dimer interface based on precise and rational modifica-
tion of the proteins with respect to the structure. The
strategy of the photochemical regulation of the enzy-
matic activities by the control of protein-protein inter-
actions can be applicable to various enzymes and pro-
teins possessing the salt-bridge network in the dimer
interface.
(2-3 µg), aminoacyl-tRNA
(1 µg), 0.10 mM amino acid
CCCG
mixture (lacking methionine and arginine), 0.01 mM arginine,
4 µL of premix, 3 µL of E. coli S30 extract, and L-[35S]
methionine (3 µCi) at 30 °C for 3 h. Wild-type and H133A
BamHI were prepared by the same method without aminoacyl-
tRNA. The mixtures of proteins were denatured in a solution
containing 50 mM Tris-HCl (pH 6.8), 0.1 M DTT, 2% SDS,
and 10% glycerol, and loaded onto an 18% SDS-polyacrylamide
gel for electrophoresis. The SDS-PAGE gels were visualized
and quantified by an imaging analyzer (Fujix BAS1000
analyzer, Tokyo, J apan). For cold samples, the same procedure
previously described was employed by just replacing the [35S]
methionine to 0.1 mM methionine, and the concentration was
quantified by Western blotting using a hexahistidine antibody
as a primary antibody. Generation of the full-length proteins
was about 10 ng from a 10 µL scale synthesis of the in vitro
translation system.
P h ot oir r a d ia t ion a n d Mea su r em en t s of t h e Ba m H I
Activities. Photoirradiation for the BamHI mutants was
carried out with a UV transilluminator (LMS-20E, 40 W; UVP,
Inc., Upland, CA) with 365 nm light on ice for 20 min. The
reaction mixtures of BamHI (3 µL) with or without photoir-
radiation were diluted by a solution containing 10 mM Tris-
HCl (pH 8.0), 10 mM MgCl2, 100 mM NaCl, and 1 mM DTT,
and then the pBR322 EcoRI restriction fragment (2.0 µg) or
λDNA (1.7 µg) was added. The reaction mixtures were incu-
bated at 30 °C for 2 h. The digested mixtures were loaded onto
a 0.7% agarose gel and electrophoresed in TBE (Tris-Borate-
EDTA) buffer. The gels were visualized by ethidium bromide
staining. The gels were photographed, and the bands were
quantified by the ImageJ program distributed by NIH. The
kinetic parameters of the enzymes were calculated according
to the previously reported method.10 The reactions were carried
out at 30 °C in the same reaction solutions containing the in
vitro translation mixture of H133A or photoirradiated H133A/
K132KNVOC mutant (1 ng) and pBR322 restriction fragment
(3-18 nM).
Exp er im en ta l Section
Syn t h esis of Am in oa cyl-t R NACCCG
. Deprotection of
NR-Boc-Nꢀ-NVOC-Lys-pdCpA 3 and NR-Boc-γ-NV-Glu-pdCpA
7 was performed with trifluoroacetic acid according to a
previous method.7b The deprotected aminoacyl-pdCpA was
dissolved in DMSO to a concentration of 5 mM and stored at
-20 °C. A tRNACCCG (-CA) was obtained by in vitro transcrip-
tion from a FokI treated pUC19 plasmid containing a T7
promoter and a synthetic yeast tRNAPhe sequence.12 Ligation
of aminoacyl-pdCpA to tRNA CCCG(-CA) was carried out in a
30 µL solution containing 5 µg of tRNA CCCG(-CA), 0.5 mM
aminoacyl-pdCpA, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
10 mM DTT, 1 mM ATP, 0.01% BSA, 10% DMSO, and T4 RNA
ligase (40 units; Takara Shuzo, Kyoto, J apan). The reaction
mixture was incubated at 10 °C for 2 h and then quenched by
the addition of 3 µL of 3 M sodium acetate (pH 5.2). The
aminoacyl-tRNACCCG was desalted by ethanol precipitation,
dried, and dissolved in RNase-free water to a final concentra-
tion of 1 µg/µL.
Ch em ica l Cr oss-Lin k in g of Ba m HI. The BamHI wild-
type, K132KNVOC, and H133A/K132KNVOC mutants were puri-
fied by a Ni-NTA spin column (Qiagen) for chemical cross-
linking. A reaction mixture of [35S]-labeled BamHI (100 µL)
Con str u ction of Exp r ession P la sm id s for Wild -Typ e
a n d Mu ta n t Ba m HI. Wild-type BamHI gene (642 bp) was
prepared by assembling six short fragments of double-strand
DNA (about 110 base pairs each) by T4 DNA ligase. Synthetic
wild-type BamHI gene was subcloned into a pUC19 plasmid,
and the sequence was confirmed by dideoxy DNA sequencing.
The full length BamHI gene amplified by PCR with primers
having NdeI and XhoI restriction sites was then inserted into
a pET26b expression plasmid (Novagen, Madison, WI). Mutant
BamHI genes substituted by a CGGG four base codon at
was centrifuged at 10000g for 5 min to obtain
a clear
supernatant. To the supernatant was added 500 µL of a buffer
[10 mM HEPES (pH 8.0), 10 mM MgCl2, 0.3 M NaCl, 1 mM
EDTA, and 2 mM 2-mercaptoethanol] with 10 mM imidazole.
The mixture was loaded onto a Ni-NTA spin column equili-
brated with the same buffer as above, and the column was
washed twice with 500 µL of wash buffer (the same buffer with
20 mM imidazole). The purified protein was eluted with 150
µL of an elution buffer (the same buffer with 250 mM
imidazole). Photoirradiation for the purified BamHI was
carried out on ice for 20 min at 365 nm. For chemical cross-
linking, the BamHI was treated in a solution containing 3.7
mM DMS, 10 mM HEPES (pH 8.0), 0.3 M NaCl, 10 mM MgCl2,
(12) Nowak, M. W.; Gallivan, J . P.; Silverman, S. K.; Labarca, C.
G.; Dougherty, D. A.; Lester, H. A. Methods Enzymol. 1998, 293, 504-
529.
J . Org. Chem, Vol. 69, No. 13, 2004 4297

Endo et al.
1 mM EDTA, and 2 mM 2-mercaptoethanol. After incubation
at 30 °C for 30 min, the reaction was quenched by the addition
of 1/10 volume of 1 M Tris-HCl (pH 7.6). The reaction mixtures
were analyzed by an 18% SDS-PAGE, and the gel was
visualized by an imaging analyzer.
Science and Technology (MEXT) of the J apanese Gov-
ernment.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the synthesis of the compounds 1-7, 1H NMR and
mass spectra of selected compounds, and the sequences of
BamHI and tRNACCCG. This material is available free of charge
via the Internet at http://pubs.acs.org.
Ack n ow led gm en t. This work has been partly sup-
ported by a Grant-in-Aid for Scientific Research on
Priority Area (417), 21st Century COE Research, and
others from the Ministry of Education, Culture, Sports,
J O035774N
4298 J . Org. Chem., Vol. 69, No. 13, 2004