A Hydrophilic Azobenzene-Bearing Amino Acid for Photochemical

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Bioconjugate Chem. 2005, 16, 1360−1366

1360

ARTICLES A Hydrophilic Azobenzene-Bearing Amino Acid for Photochemical Control of a Restriction Enzyme BamHI Koji Nakayama, Masayuki Endo, and Tetsuro Majima* The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan. Received November 15, 2004; Revised Manuscript Received August 12, 2005

A novel hydrophilic and negatively charged azobenzene-bearing amino acid, 4′-carboxyphenylazophenylalanine (azoAla 1), has been designed and synthesized for investigation of the photochemical regulation of the enzyme activity. The properties of photoisomerization and thermal stability of the cis-isomer were similar to those of a commonly used phenylazophenylalanine (azoAla 2). For photochemical control of the enzyme, these two azobenzene-bearing amino acids were incorporated into the specific position at the dimer interface of a restriction enzyme BamHI. These trans-azobenzene derivatives in the BamHI suppressed the enzymatic activity, and the following photoirradiation at 366 nm induced the recovery of its activity. Although the activities of both azoAla-BamHI mutants were same level after a long time irradiation, the recovery of the activity of azoAla 1-BamHI was faster than that of azoAla 2-BamHI with a short time irradiation. This result suggests that the negatively charged carboxylate group introduced into an azobenzene moiety affects the behavior of azoAla in the protein scaffold during the trans-cis photoisomerization.

INTRODUCTION

The artificial and selective activation of an enzyme is one of the most important issues for the investigation of the specific biological phenomena in living cells. Caged compounds such as o-nitrobenzyl derivatives are widely used for regulation of the enzymatic activity by introduction of these compounds into an enzyme, which are then uncaged with photoirradiation for activation (1-7). These caged molecules unavoidably release the reactive nitroso derivatives after photoirradiation, which may cause a negative side-effect in the cells. In contrast, photochromic molecules, which change their conformations by photoirradiation, do not produce a side-product (8-15). When a photochromic moiety is incorporated into polymers and biomolecules, the mechanical movement of the photochromic molecules by irradiation at specific wavelengths can induce structural changes, which produces novel physical and biochemical properties (8-15). From this point of view, the use of photochromic molecules is a better choice for control of the activity of enzymes and the subsequent expression of biological phenomena. The most commonly used and reliable photochemical switches for the functionalization of polymers and biomolecules are azobenzene derivatives, which have excellent features; i.e., the simplest chemical structure, rapid photoisomerization in both the trans-cis directions, a large structural motion, and a thermally stable cis isomer (16, 17). However, for incorporation of phenylazophenylalanine into the proteins and enzymes, the hydrophobic property of the azobenzene moiety may cause the exclusion of water in the solvent-accessible surface of the * Corresponding author: [email protected]; Phone: +81-6-6879-8495; Fax: +81-6-6879-8499.

proteins, which may result in a negative effect such as an unexpected missfolding of the proteins around the incorporated sites. One of the simplest solutions for water-accessibility is the introduction of a hydrophilic property into an azobenzene derivative. In this report, we synthesized a negatively charged 4′carboxyphenylazophenylalanine moiety and incorporated it into the dimer interface of a restriction enzyme BamHI. In the previous study, we controlled the activity of BamHI, which is activated through the dimer formation (18-21), by arranging the specific amino acids involved in the dimer interface by introducing the photoremovable o-nitroveratryl groups (22). At this dimer interface, side chains of four amino acids, K132, H133, E167, and E170, consist of a salt bridge network to maintain the conformation of the BamHI dimer in the active form (Figure 1) (18-21). Especially, K132 in the dimer interface is the key position for the activity, since the K132 on the R-helix faces two glutamate side chains (E167 and E170) on the counterpart R-helix. We previously discovered that phenylazophenylalanine (azoAla 2) incorporated into the 132 position can regulate the activity of BamHI (23). For further investigation of relationship between the photochemical switching of azobenzene derivative and the expression of the enzymatic activity, we introduced a negatively charged 4′-carboxyphenylazophenylalanine (azoAla 1) into the 132 position for changing the solventaccessibility in the surface of BamHI. We examined whether the negative charge affects the behavior of the azobenzene moiety during the trans-cis photoisomerization in the dimer interface, which may accompany local structural change around the azoAla residue for effective inactivation and photochemical activation of BamHI.

10.1021/bc049724g CCC: $30.25 © 2005 American Chemical Society Published on Web 09/14/2005

Photochemical Control of a Restriction Enzyme BamHI

Figure 1. BamHI dimer structures. (A) Side view of the BamHI dimer-DNA complex (PDB 1BHM). (B) The detailed interaction of the amino acid side chains at the dimer interface. K132 and H133 are located on the same R-helix, and E167* and E170* are on the counterpart R-helix*. The asterisks represent the counterpart BamHI monomer. (C) Minimized structure of the trans and cis form of azoAla 2. (D) Photofunctionalized amino acid phenylazophenylalanine derivatives (azoAla 1 and 2) employed in this experiment. EXPERIMENTAL PROCEDURES

Materials. The reagents used in the experiment were purchased from the following companies: 4-nitrobenzoic acid methyl ester, NR-tert-butoxycarbonyl-4-nitro-L-phenylalanine, tert-butyl hypochlorite, chloroacetonitrile, Wako Chemicals (Osaka, Japan); 4-nitrobenzoic acid methyl ester, Tokyo Kasei (Tokyo, Japan); restriction endonucleases, New England BioLabs (Beverly, MA); pET26b, Novagen (Madison, WI); Pfu DNA polymerase, Stratagene (LaJolla, CA); nucleoside triphosphates and L-[35S]-methionine, Amersham Pharmacia Biotech (Piscataway, NJ); T4 RNA ligase, Takara Shuzo (Kyoto, Japan); T4 DNA ligase, T7 RNA polymerase, and RNase inhibitor, Toyobo (Osaka, Japan); RNeasy Project Mini

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Kit, Qiagen (Hilden, Germany); E. coli S30 Extract System, Promega (Madison, WI). Purification of the nucleotide derivatives was performed using a JASCO LC2000Plus series HPLC system (Tokyo, Japan). Photoirradiation was carried out using an ultrahigh-pressure mercury lamp (Ushio USH-500D; 500 W) equipped with a monochromator (Ritsuoyo Kogaku MC-10N) which can control the specific wavelength within a 4 nm full-width at half-maximum. Visualization and quantification of the gels were performed using a Fujix BAS1000 imaging analyzer (Tokyo, Japan). 4-Nitrobenzoic Acid tert-Butyl Ester (3). n-Butyllithium (0.10 mol, 65 mL of 1.58 M hexane solution) was slowly added to a mixture of the 4-nitrobenzoic acid methyl ester (9.05 g, 50 mmol) and distilled tert-butyl alcohol (9.5 mL, 0.10 mol) in anhydrous THF (30 mL) at 0 °C under an argon atmosphere (24). The reaction mixture was warmed to room temperature and stirred at room temperature. After 18 h, diethyl ether (200 mL) and a saturated ammonium chloride aqueous solution (200 mL) were added to the mixture, and the organic layer was extracted and washed twice with a saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. The resulting brown solid was purified by silica gel chromatography with toluene/ hexane (1:1) to give compound 3 (3.48 g, 31%) as a yellow solid; 1H NMR [270 MHz, CDCl3 (TMS)]: δ 8.15 (d, 2H, J ) 8.1 Hz), 7.96 (d, 2H, J ) 8.1 Hz), 1.63 (s, 9H). tert-Butyl 4-Hydroxylaminebenzoate (4). Zinc powder (90%, 205 mg, 3.2 mmol) was slowly added to a mixture of 3 (352 mg, 1.6 mmol) and ammonium chloride (84.4 mg, 1.6 mmol) in MeOH/water (10:1, 20 mL) over a period of 5 min (25). The reaction mixture was stirred at room temperature for 3 h under an argon atmosphere. The insoluble precipitate was removed by filtration, and the filtrate was dried under reduced pressure. The resulting orange solid was diluted with methylene chloride (200 mL) and a saturated aqueous sodium bicarbonate solution, and the organic layer was washed twice with saturated aqueous sodium chloride. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The orange solid was purified by silica gel chromatography with CH2Cl2/ MeOH (100:2) to give compound 4 (220 mg, 67%) as a yellow solid. 1H NMR [270 MHz, CDCl3 (TMS)]: δ7.91 (d, 2H, J ) 8.6 Hz), 6.97 (d, 2H, J ) 8.6 Hz), 5.21 (s, 1H), 1.63 (s, 1H), 1.58 (s, 9H). 4-tert-Butoxycarbonyl-Nr-Boc-phenylazophenylalanine (5). tert-Butyl hypochlorite (114 mg, 1.05 mmol) was added to a solution of 4 (220 mg, 1.05 mmol) in THF (5 mL) at -78 °C (26). The reaction mixture was stirred under argon at -78 °C for 10 min and then allowed to react at -20 °C for 1 h. The reaction proceeded more than 90% based on TLC monitoring. The solvent was removed under reduced pressure at 10 °C, and the brown solid was dried under reduced pressure. The crude product was dissolved in 10 mL of 10% AcOH/MeOH. This solution was added to a 10% AcOH/MeOH solution (10 mL) of NRBoc-4-aminophenylalanine (263 mg, 0.94 mmol) under argon at 0 °C. The reaction mixture was allowed to react at room temperature for 2 h. After removal of the solvent under reduce pressure, the brown solid was dissolved in CH2Cl2 (200 mL), and then a saturated aqueous sodium bicarbonate solution was added. The organic layer was washed twice with a saturated aqueous sodium chloride solution and then dried over anhydrous sodium sulfate. The resulting orange solid was purified by silica gel column chromatography with CH2Cl2/MeOH (100:8) to give compound 5 (335 mg, 76%) as an orange solid. 1H

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NMR [270 MHz, CDCl3(TMS)]: δ 8.15 (d, 2H, J ) 8.1 Hz), 7.88, (dd, 4H, J ) 8.1 Hz), 7.35 (d, 2H, J ) 8.1 Hz), 5.04 (s, 1H), 4.64 (s, 1H), 3.22 (m, 2H), 1.62 (s, 9H), 1.41 (s, 9H). 4-tert-Butoxycarbonyl-Nr-Boc-phenylazophenylalanine Cyanomethyl Ester (6). Chloroacetonitrile (24 mg, 0.32 mmol) was added to a mixture of compound 5 (77 mg, 0.16 mmol) and triethylamine (24 mg, 0.24 mmol) in dry acetonitrile (20 mL). The reaction mixture was stirred at room temperature for 12 h under argon. After removal of the solvent under reduced pressure, the product was purified by silica gel column chromatography with CH2Cl2/MeOH (100:2) to give compound 6 (46 mg, 57%) as an orange solid. 1H NMR [CDCl3 (TMS)]: δ 8.13 (d, 2H, J ) 8.1 Hz), 7.91, (dd, 4H, J ) 8.1 Hz), 7.33 (d, 2H, J ) 8.1 Hz), 5.00 (d, 2H, J ) 7.8 Hz), 4.77 (dd, 2H, J ) 15.8 Hz), 4.71 (s, 1H), 3.21 (m, 2H), 1.63 (s, 9H), 1.43 (s, 9H). FAB-MS (positive), m/z calcd for C27H33N4O6 509.2 [M+H]+, found 509.0. 4-tert-Butoxycarbonyl-NR-Boc-phenylazophenylalanyl pdCpA (7). Compound 6 (1.25 µmol) was added to a dry DMF solution of pdCpA tetra-n-butylammonium salt (27) (20 µL, 0.25 µmol) in a microtube. The reaction mixture was incubated at room temperature for 3 h, and the reaction was monitored by a reversed-phase HPLC [linear gradient; 0-80% acetonitrile-50 mM ammonium acetate buffer (pH 4.8) over 30 min, flow rate 2 mL/min, Chemcobond C18 column (7.5 × 150 mm), detection at 260 nm]. After the reaction, the mixture was diluted with 50 mM ammonium acetate solution (pH 4.5, 1 mL), and the product was purified by HPLC. The retention time of compound 7 was 22.8 min. The product was concentrated under reduced pressure and then lyophilized. 67% yield. ESI-MS (negative), m/z calcd for C40H46N11O18P2 1030.3 [M - H]-, found 1030.5. Preparation of 4′-Carboxyphenylazophenylalanyl-tRNACCCG (azoAla 1-tRNACCCG). Deprotection of compound 7 was performed in trifluoroacetic acid (20 µL) on ice for 10 min, and then the trifluoroacetic acid was removed by flowing nitrogen gas. The pellet was washed twice with ether and then dried under reduced pressure. The pellet was dissolved in DMSO to make a 0.5 mM solution and stored at -20 °C. The tRNACCCG (-CA) was obtained according to a previous method (22, 23). Ligation of aminoacyl-pdCpA to tRNACCCG(-CA) was carried out in a 20 µL solution containing 5 µg of tRNACCCG(-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). The reaction mixture was incubated at 4 °C for 2 h, and then the reaction was quenched by the addition of 2 µL of sodium acetate (3 M, pH 5.2). The aminoacyl-tRNACCCG was precipitated with ethanol, rinsed with 70% ethanol, and dried under reduced pressure. The aminoacyl-tRNACCCG was dissolved in RNase-free water to a final concentration of 1 µg/µL. In Vitro Translation of Wild-Type and azoAlaBamHI. Preparation of a plasmid containing the BamHI gene and its mutant was performed according to a previous method (22). The preparation of mRNA was carried out in a solution (50 µL) containing the template DNA (5 µg), T7 RNA polymease (147 units), RNase inhibitor (40 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, and then the mRNA was purified using an RNeasy Protect Mini Kit (Qiagen). The E. coli S30 Extract System (Promega) was employed for the in vitro protein synthesis following the reported method (28, 29). The translation reaction was carried out in a 10 µL of reaction mixture

Nakayama et al.

containing mRNA (2 µg), aminoacyl-tRNACCCG (1 µg), 0.10 mM amino acid mixture (lacking methionine and arginine), a 0.01 mM arginine, premix (4 µL), E. coli S30 extract (3 µL), and L-[35S]-methionine (3 µCi) at 30 °C for 2 h. The wild-type BamHI was prepared by the same method without aminoacyl-tRNACCCG. The reaction was quenched by the addition of a mixture containing 50 mM Tris-HCl (pH 6.8), 0.1 M DTT, 2% SDS, and 10% glycerol and loaded onto an 15% SDS-polyacrylamide gel for electrophoresis. The SDS-PAGE gels were visualized and quantified using an imaging analyzer. For the cold samples, the same procedure described above was employed by just replacing the [35S]-methionine with 0.1 mM methionine, and the concentration was quantified by Western blotting using a hexahistidine antibody as the primary antibody. The generation of the full-length proteins was about 10 ng from a 10 µL scale synthesis of the in vitro translation system. Photoirradiation to Phenylazophenylalanine Derivatives. The phenylazophenylalanine derivatives (azoAla) 1 and 2 were prepared by treatment of the NRBoc-phenylazophenylalanine and compound 5 with TFA, respectively. After removal of TFA under reduced pressure, the deprotected azoAla 1 and 2 were dissolved in DMSO. Photoirradiation was carried out using a 500 W ultrahigh-pressure mercury lamp equipped with a monochromator. Samples (1 mL) containing azoAla 1 or 2 (0.01 mM), 10 mM Tris-HCl (pH 8.0), and 10% DMSO were placed in a quartz cell with a 1 cm path length. Photoirradiation and Measurements of the BamHI Activities. Photoirradiation was carried out using the same instrument as described above. The trans to cis photoisomerization was carried out using the translation mixture prepared above by irradiation with 366 nm light on ice for 10 min. The translation mixtures (0.25 µL) before or after the photoirradiation were diluted by a reaction buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 100 mM NaCl, 0.01% BSA, and 1 mM DTT, and then linearized pBR322 (6 nM) digested by NdeI was added. The reaction mixtures were incubated at 30 °C for 1 h. The reaction mixtures were loaded onto a 1.2% agarose gel in TBE buffer. The gels were visualized by ethidium bromide staining and quantified by the ImageJ program (NIH). RESULTS AND DISCUSSION

Synthesis of 4′-Carboxyphenylazophenylalanine. The synthesis of the active ester of 4′-carboxyphenylazophenylalanine (azoAla 1) was carried out according to Scheme 1. The transesterification of the 4-nitrobenzoic acid methyl ester to the tert-butyl ester was carried out by reaction with n-butyllithium and tert-butyl alcohol to give 3 (24). The reduction of the nitro group of 3 was carried out using Zn in ammonium hydroxide to produce 4 (25). For production of the azo group, compound 4 was oxidized to tert-butyl nitrosobenzoate using tert-butyl hypochlorite in THF at -20 °C (26), and the crude product was then coupled with NR-Boc-4-aminophenylalanine in 10% AcOH/MeOH solution to yield 5. The esterification of compound 5 with chloroacetonitrile gave the 4′-tert-butoxycarbonyl-NR-Boc-phenylazophenylalanine cyanomethyl ester 6. The active ester 6 was coupled with a nucleoside dimer 5′-phospho-2′-deoxycytidylyl-(3′5′)-adenosine (pdCpA) tetrabutylammonium salt at room temperature for 3 h to give aminoacyl-pdCpA 7. Photochemical Properties of Phenylazophenylalanine Derivatives. To characterize the photochemical behaviors of the azoAla 1 and azoAla 2 monomers, the

Photochemical Control of a Restriction Enzyme BamHI Scheme 1 a

a Reagents and conditions: (a) tert-BuOH, tert-BuLi, THF, rt; (b) Zn, NH4OH, MeOH, H2O, rt; (c) i. tert-BuOCl, THF, -20 °C, ii. NR-Boc-4-aminophenylalanine, AcOH, MeOH, rt; (d) chloroacetonitrile, CH3CN, Et3N, rt; (e) pdCpA tetra-n-butylammonium salt, DMF, rt.

absorption changes by photoirradiation were examined. 4′-tert-Butoxycarbonyl-NR-Boc-azoAla 1 (compound 5) and NR-Boc-azoAla 2 were treated with TFA for deprotection. Due to the introduction of carboxylate to the 4′-position, azoAla 1 was solubilized in water at neutral pH, while azoAla 2 needed at least 10% DMSO for solubilization in water. The azoAla derivatives (0.01 mM) were dissolved in 10% DMSO and 10 mM Tris-HCl buffer (pH 8.0), and photoirradiation was carried out at 25 °C using a high-pressure mercury lamp equipped with a monochromator. Photoisomerization was monitored by a UV/ vis spectrometer. The spectrum of the untreated azoAla 1 showed two major peaks at 242 and 334 nm, which are identified as the characteristic π-π*, and at 432 nm as the characteristic n-π* absorption band of the trans isomer of the azobenzene derivatives, respectively (Figure 2A) (16, 17). By photoirradiation to azoAla 1 at 366 nm, the peak at 334 nm rapidly decreased and largely shifted to 310 nm, while the peak at 432 nm increased and shifted to 429 nm (Figure 2A). The reaction were completed within 20 min. When irradiation to this product (cis-azoAla 1) was carried out using 436 nm light, the increasing peak at 310 nm shifted to 334 nm and the decreasing one at 429 nm shifted to 432 nm (Figure 2B). This behavior is also characteristic of the cis to trans isomerization of the azobenzene derivatives, indicating that the reversible trans-cis photoisomerization of azoAla 1 occurred under this condition. In the case of azoAla 2, a similar photoisomerization was observed by the photoirradiation. The ratios of the cis isomers of azoAla 1 and 2 after the 20 min irradiation at 366 nm were 81% and 79% under this condition (quantified by HPLC), respectively. The half-lives for the trans to cis and cis to trans isomerizations of the azoAla 1 and azoAla 2 monomers are summarized in Table 1. They show similar values, indicating that the substitution of the 4′-position by a

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carboxylate does not change the basic behavior of the photoisomerization as compared to the normally used azoAla 2. cis-azoAla 1 showed a good thermal stability at 30 °C (τ1/2 ) 27 h) as similar to the cis-azoAla 2 (τ1/2 ) 35 h), meaning that most of the cis isomer is retained under the reaction conditions of the BamHI with a DNA substrate (30 °C, 1 h) when these azoAla derivatives were incorporated into the enzyme. The photochemical properties of these azoAla derivatives indicate that these have substantial potentials for photochemical switches in the protein. Preparation of Phenylazophenylalanyl-BamHI. The incorporation of the modified amino acids was performed using a four-base codon-anticodon method according to a previously reported method (28, 29). The protected azoAla 1-pdCpA and azoAla 2-pdCpA were treated with TFA followed by ligation onto a tRNACCCG(-CA) using T4 RNA ligase to give azoAla 1-tRNACCCG and azoAla 2-tRNACCCG. The azoAla derivatives 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 cell-free translation system with the mutant mRNA containing a four base codon and the synthesized aminoacyl-tRNACCCG. In vitro translation was carried out in a mixture containing the mutant mRNA, unnatural aminoacyl-tRNACCCG, E. coli S30 extract, and amino acid with [35S]-methionine at 30 °C for 2 h. After the reaction, the mixtures were loaded onto a 18% SDS-PAGE, and the gel was visualized using an imaging analyzer (Figure 3). Wild-type BamHI was expressed as a full-length form at 27 kDa (lane 1). For the translation with the mutant mRNA containing a fourbase codon mutation, an incomplete length of BamHI was observed in the absence of aminoacyl-tRNACCCG (lane 2). In contrast, the full-length BamHI was obtained in the presence of the specific aminoacyl-tRNACCCG (lanes 3 and 4). The efficiencies of incorporation of azo-Ala 1 and azoAla 2 at position 132 were 40% and 36% quantified by the radioactivity of [35S]-methionine, respectively. The incorporation efficiencies for both azoAla derivatives were similar, indicating that the steric hindrance and negative charge of the carboxyl group in the 4′-position did not affect the protein synthesis using this cell-free translation system. We employed these azoAla-BamHI mutants for the investigation of the photochemical control of the enzymatic activities without further purification. Photochemical Control of the Activity of Phenylazophenylalanine-Bearing BamHI Mutants. To examine the properties of the azoAla functionalized BamHI mutants, photoirradiation was performed at 0 °C for 10 min using a high-pressure mercury lamp (500 W) equipped with a monochromator for generating the 366 nm light. The photoirradiated enzymes were incubated with a substrate DNA to examine the activity. As shown in Figure 4, both azoAla 1 and azoAla 2 mutants suppressed the activities after the preparation of the protein without irradiation (lanes 3 and 5, respectively). In addition, the intrinsic activities were recovered (both 44% substrate cleaved) in the azoAla 1 and 2 mutants after photoirradiation (lanes 4 and 6, respectively). This sequence selectivity was similar to that of the wild-type BamHI (lane 2). The results show that the activities of the azoAla 1 and azoAla 2 mutants can be regulated through the photoisomerization of the azobenzene moiety. In the previous study, we identified that the key amino acid regulating the BamHI activity is the K132 residue using the protection and deprotection of the -amino group with a photoremovable group (22). The result

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Nakayama et al.

Figure 2. Absorption spectral changes of azoAla 1 with photoirradiation at 25 °C in pH 8.0 solution (water/DMSO ) 9/1 v/v). (A) Trans to cis photoisomerization was carried out using 366 nm light for 0, 2, 5, 10, and 20 min. (B) Cis to trans isomerization was carried out. After the 20 min irradiation at 366 nm, the cis to trans photoisomerization was performed using 436 nm light for 0, 2, 5, 10, and 20 min. Table 1. Photochemical Properties of Azobenzene Derivatives. Half-lives (min) of the Azobenzene Derivatives 1 and 2 at pH 8.0a photoisomerzation

thermal isomerization

azobenzene derivatives

trans to cis (366 nm)

cis to trans (436 nm)

cis to trans (30 °C)

1 2

5.5 5.8

11.3 7.3

1.6 × 103 2.1 × 103

a Detailed experimental conditions are described in the Experimental Section.

Figure 4. Cleavage of a substrate DNA (pBR322 digested by NdeI) with wild-type and photofunctionalized BamHI mutants before and after photoirradiation at 366 nm. Lane 1, substrate DNA (4361 bp); lane 2, DNA fragments (1920 and 2441 bp) cleaved by wild-type BamHI (New England BioLabs); lanes 3 and 4, DNA cleavage with azoAla 1-BamHI before (-) and after (+) photoirradiation, respectively; lanes 5 and 6, DNA cleavage with azoAla 2-BamHI before (-) and after (+) photoirradiation, respectively. Table 2. Kinetic Parameters of BamHI Mutants Containing Azobenzene Derivativesa

Figure 3. In vitro translation of azoAla-BamHI mutants labeled with [35S]-methionine. Lane 1, wild-type BamHI; lane 2, translation with K132 mutant mRNA in the absence of aminoacyl-tRNACCCG; lane 3, translation with K132 mutant mRNA in the presence of azoAla 1-tRNACCCG; lane 4, translation with K132 mutant mRNA in the presence of azoAla 2-tRNACCCG.

obtained in this experiment indicates that both trans isomers of azoAlas 1 and 2 would induce a local structural change of the dimer interface to the inactive form. The BamHI mutant having an o-nitroveratry group at the K132 position employed in the previous study showed activity, suggesting that the flexible n-butyl side chain of lysine would be excluded to outside of the dimer interface (22). The activities of azobenzene-bearing mutants after photoirradiation (366 nm, 10 min) were evaluated by kinetic parameters according to the previously reported method (30). Using the pBR322 restriction fragment as a substrate, kinetic parameters were obtained and summarized in Table 2. Although the cisazoAla-BamHI mutants showed lower activity than the wild-type, these mutants preserved the same level of the affinity and activity as compared to the H133A BamHI mutant (22). We tried the cis to trans photoisomerization using the cis forms of both azoAla-BamHI mutants which were generated by irradiation with 366 nm light for 20 min. When these cis-azoAla-BamHI mutants were irradiated

BamHI mutants

KM (nM)

Vmax (nM min-1)

Vmax/KM (min-1)

1-BamHI + hv 2-BamHI + hv Wild-typeb H133Ab

2.2 2.9 1.7 2.3

5.1 × 10-2 7.2 × 10-2 8.8 × 10-1 6.5 × 10-2

2.3 × 10-2 2.5 × 10-2 5.2 × 10-1 2.8 × 10-2

a Detailed experimental conditions are described in the Experimental Section. b Data from ref 22.

with 436 nm, suppression of the DNA cleavage was not observed. In the previous study, the enzyme activity of Horseradish peroxidase was successfully controlled in switchable way using the site-selectively incorporated azophenylalanine (12). In this case, the phenylazophenylalanine introduced close to the substrate binding site effectively controls the binding and activity for the small substrate. In our case, once the cis forms of the azoAla derivatives are removed from the dimer interface for formation of the active dimer complex, the regenerated trans-azobenzene moieties would not be forced to change the conformation of the dimer to an inactive conformation. Effect of the Carboxylate Group in the Azobenzene on the Recovery of the Activity. We examined the relationship between the recovery of the activities and irradiation time for the azoAla-BamHI mutants during the trans to cis photoisomerization using 366 nm light. The irradiation time-dependent activitation of the enzyme was observed for both the azoAla 1 and azoAla 2 BamHI mutants (Figure 5). During photoirradiation for 5 min, the activity of the enzymes gradually increased, and the trans to cis photoisomerization in both azoAla

Photochemical Control of a Restriction Enzyme BamHI

Figure 5. Recovery of the activity of the azoAla 1-BamHI and azoAla 2-BamHI mutants by photoirradiation (366 nm). Photoirradiation was carried out at 0 °C for 0, 1, 2, 5, 10, and 20 min, and then the samples were incubated with a substrate DNA at 30 °C for 1 h.

monomers was completed in 20 min. For an irradiation of more than 10 min, the activities reached the saturated level. Thus, the activity level of these azobenzenefunctionalized enzymes can be regulated depending on the irradiation time within 10 min. The cis-trans thermal isomerization at 30 °C is slow enough to retain the cis form azoAla during the enzymatic reactions with substrates at 30 °C for 1 h (see Table 1). Therefore, the activity of the enzymes depends on the population of the active (cis isomer) and the inactive (trans isomer) BamHI, namely the concentration of active BamHI, can be purposely controlled by photoirradiation time. Effect of the negatively charged carboxylate group on the azobenzene moiety for the activity was examined. As shown in Figure 5, we observed a faster recovery of the activity of azoAla 1-BamHI in shorter irradiation times (1-5 min) as compared to that of azoAla 2-BamHI. This means that the negatively charged azoAla 1 can rapidly produce the active BamHI with correct dimer formation after photoirradiation. Since the properties of the photoisomerization between azoAla 1 and 2 were basically the same as the monomer level in solution (Table 1), the expression of the enzyme activity using the azoAla derivatives would be affected by the environment of the protein surface when the azoAla derivatives possessing different electrostatic properties were incorporated into the 132 position of the dimer interface. In the present case, there are two possibilities for the regulation of the enzyme activity using the trans-cis photoisomerization of the azobenzene. One possibility is that the azobenzene moiety directly interferes the interaction between the two BamHI monomers for the correct formation of the dimer. The short methylene chains of the azoAla residues constrain the orientations of the bulky trans-azobenzene moieties, which would cause the steric hindrance between the azobenzene moieties and the counterpart R-helix for consequent suppression of the activity. Recovery of the activities after the photoirradiation means the correct formation of the BamHI dimer with the cis isomers. The compact cis-azobenzene moieties may reduce the steric interference in the dimer interface and exhibit the activity after the correct BamHI dimer formation. The negatively charged carboxylate in the cis-isomer of the azoAla 1-BamHI would cause rapid dimer formation in the dimer interface as compared to the neutral azoAla 2-BamHI. The other possibility is that the azobenzene moiety would change the local structure of the monomer around

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the azobenzene residue because of the interaction of the aromatic ring with the protein surface in order to avoid the water-accessible environment. The short methylene chains of the azoAla residues limit the movement of the trans-azobenzene moieties, and aromatic azobenzene may interact with the surface of BamHI, which induces local structural changes for misalignment of the dimer structure and consequent suppression of the activity. Since the possible contact area of the cis-azobenzene moiety with the protein surface is smaller than that of the trans form, the cis isomer may weaken the interaction with the protein surface, and the BamHI dimer forms an active one. Negative charge of the cis-isomer would work on the correct dimer formation in the environment on the surface of the BamHI monomer. In these possibilities, the carboxylate group in the azobenzene affects the rapid expression of the activity in the protein scaffold as compared to the neutral azobenzene moiety without changing the potential of the activity of both cis-azoAlaBamHI after a long time irradiation. CONCLUSIONS

We designed and synthesized a novel hydrophilic and negatively charged azobenzene-bearing amino acid, 4′carboxyphenylazophenylalanine, and investigated the photochemical regulation of BamHI where the azobenzene derivatives were selectively incorporated. We found that the negative charge of the azobenzene moiety affects the initial activation of the enzyme in the trans-cis photoisomerization. The result suggests that these azobenzene derivatives may be a new tool for investigation of the environment such as solvent-accessibility, hydrophobicity, and electrostatic state in the protein. ACKNOWLEDGMENT

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