Site-Directed Incorporation of Fluorescent Nonnatural Amino Acids

Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1. Tsushimanaka, Okayama 700-8530, Japan, and Department ...
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Biomacromolecules 2000, 1, 118-125

Site-Directed Incorporation of Fluorescent Nonnatural Amino Acids into Streptavidin for Highly Sensitive Detection of Biotin Hiroshi Murakami,† Takahiro Hohsaka,† Yuki Ashizuka,† Kimiko Hashimoto,‡ and Masahiko Sisido*,† Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan, and Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan Received December 13, 1999

Fluorescent nonnatural amino acids were incorporated into specific positions of streptavidin. The positions of the nonnatural amino acids were directed by a CGGG/CCCG four-base codon/anticodon pair. The nonnatural mutants with a single 2-anthrylalanine at the 22nd, 43rd, 54th, and 120th positions, respectively, were found to bind biotin, indicating that the mutants retained active conformation. The fluorescence intensities of the anthryl groups were relatively insensitive to the positions and the biotin binding when excited at 265 nm. When the anthryl group at the 120th position was excited through energy transfer from tryptophan units, the fluorescence intensity markedly decreased with biotin binding, because of a suppression of the energy transfer. Amino acids carrying 7-methoxycoumarine fluorophore were also incorporated at the 120th position. Their fluorescence quantum yields were very sensitive to the biotin binding. The high sensitivity of the coumarine-labeled streptavidin exemplifies potential applications of fluorescent nonnatural mutants for detecting specific molecules at very low concentrations. Site-directed incorporation of nonnatural amino acids into proteins through an in vitro biosynthesizing system is a versatile technique by which a variety of functional groups can be built into proteins with a designed spatial arrangement.1-10 The technique has been finding wide applications in the field of biochemistry and biomolecular engineering. We have extended the technique by incorporating a variety of nonnatural amino acids with large aromatic groups11 and using four-base codon/anticodon pairs that can assign multiple nonnatural amino acids onto a single protein.12-14 One of the potential applications of the nonnatural mutation is a fluorescence labeling of receptors, antibodies, and enzymes at specific positions. By using the properly labeled proteins, we can detect ligands, antigens, and inhibitors under extremely low concentrations. For this purpose, however, we have to find fluorescent nonnatural amino acids that can be incorporated into proteins in high yields. The fluorescent groups must be highly sensitive to the small change of the microenvironment caused by the binding of small molecules. Furthermore, the nonnatural amino acids must be introduced at specific positions where the binding ability of the protein is not suppressed. So far, however, only a few types of nonnatural amino acids bearing highly fluorescent groups have been successfully incorporated through the in vitro system. N-DansylL-lysine was incorporated into β-galactosidase,15 but its incorporation efficiency was not high. Higher incorporation * To whom correspondence should be addressed. Tel.: +81-86-2518218. Fax: +81-86-251-8219. E-mail: [email protected]. † Okayama University. ‡ Keio University.

efficiency was achieved for 5-hydroxytryptophan and 7-azatryptophan,15 but the fluorescence wavelengths of these amino acids are largely overlapped with the intrinsic tryptophan fluorescence. A fluorescein group was introduced into mutant T4 lysozyme through a keto-selective reaction of fluorescein hydrazide.16 Although the labeling was specific to the keto group, the reaction yield was not perfect (about 50%). Chollet and co-workers17 incorporated 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diamino-propionic acid (NBD-Dap) through in vivo synthesis in Xenopus oocytes. Similarly, NBD-lysine was introduced to an in vitro system by Johnson and co-workers.18 As related topics, NBD19 and 7-diethylaminocoumarine20 groups were covalently attached to thiol groups of proteins. We have shown that a simple fluorescent amino acid, L-2anthrylalanine (antAla), could be incorporated into streptavidin through the Escherichia coli in vitro system with reasonable efficiency (45% with respect to the wild-type protein).11 The anthryl group emits fluorescence at 390450 nm which may be detected without severe interference from the intrinsic fluorescence of tryptophan groups. The very strong absorption band at 257.5 nm ( ) 1.6 × 105 cm-1 L mol-1) is another advantage of the anthryl group that allows us to detect fluorescence in less than nM concentrations. In this study, antAla was incorporated into 24 different positions of streptavidin. As novel highly fluorescent nonnatural amino acids, three methoxycoumarine derivatives were prepared. The methoxycoumarine groups were linked by an ester linkage to glutamic acid or aspartic acid, or linked to the γ-position of homoalanine. The new amino acids were also successfully

10.1021/bm990012g CCC: $19.00 © 2000 American Chemical Society Published on Web 02/07/2000

Incorporation of Fluorescent Amino Acids into Proteins Chart 1. Structure of L-2-Anthrylalanine (antAla), γ-(7-Methoxycoumarin-4-yl)homoalanine (mchAla), β-(7-Methoxycoumarin-4-yl)methyl L-Aspartate [Asp(OMc)], and γ-(7-Methoxycoumarin-4-yl)methyl L-Glutamate [Glu(OMc)]

incorporated into streptavidin and, for some mutants, the biotin-binding property was retained. The methoxycoumarine group in protein was very sensitive to the change in the local environment and the biotin binding. Typically, the 120mchAla streptavidin was found to detect a biotin in nM concentrations. The molecular structures of antAla and the coumarine amino acids are shown in Chart 1. Experimental Section Materials. Preparation of antAla and its attachment to tRNA has been described before.11 γ-(7-Methoxycoumarin4-yl)-L-homoalanine (mchAla) was synthesized by one of the authors (K.H.) and the detailed procedure will be described elsewhere.21 β-(7-Methoxycoumarin-4-yl)methyl L-aspartate [Asp(OMc)] and γ-(7-methoxycoumarin-4-yl)methyl L-glutamate [Glu(OMc)] were newly synthesized in this study. β-(7-Methoxycoumarin-4-yl)methyl L-Aspartate Cyanomethyl Ester. Boc-L-aspartic acid R-benzyl ester (130 mg, 0.402 mmol, Kokusan Kagaku, Tokyo), 4-bromomethyl-7methoxycoumarine (100 mg, 0.372 mmol, Tokyo Kasei), and K2CO3 (1.0 g) were mixed in acetone (10 mL) and the mixture was stirred at 65 °C for 1 h. The mixture was evaporated to dryness and the residue was dissolved in ethyl acetate (10 mL). The latter mixture was washed with a 10mL portion of 4% aqueous NaHCO3 and then with brine. The organic phase was dried over MgSO4 and concentrated under reduced pressure. The residual oil was dissolved in THF (5 mL) and hydrogenated on palladium oxide hydrate for 5 min. The solution was filtered, evaporated to dryness, and redissolved in ethyl acetate (10 mL). The latter solution was washed with a 10-mL portion of 5% aqueous KHSO4 and then with brine. The solution was dried over MgSO4 and then concentrated under reduced pressure to give a Bocamino acid. The Boc-amino acid was dissolved in acetonitrile (300 µL) containing 102 µL (0.73 mmol) of triethylamine. Chloroacetonitrile (153 µL, 2.43 mmol) was added to the mixture at 0 °C and the mixture was stirred for 12 h at room temperature and then evaporated to dryness. The residue was dissolved in ethyl acetate (10 mL) and washed with a 10mL portion of 5% aqueous KHSO4, 4% aqueous NaHCO3, and then brine. The organic phase was dried over MgSO4 and concentrated under reduced pressure. The residue was

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purified by flash chromatography on a silica gel (ethyl acetate/hexane ) 1/1) to give 25.6 mg (0.056 mmol, 15%) of the Boc-amino acid cyanomethyl ester. 1H NMR (300 MHz, CDCl3) δ: 1.47 (s, 9H), 2.97-3.22 (m, 2H), 3.89 (s, 3H), 4.70-4.82 (m, 3H), 5.34 (m, 2H), 5.48 (d, 1H), 6.30 (m, 1H), 6.85-6.95 (m, 2H), 7.39 (d, 1H). N-Boc-β-(7-methoxy coumarin-4-yl)methyl L-Glutamate Cyanomethyl Ester. The title compound was synthesized in a manner similar to Boc-L-glutamic acid R-benzyl ester. 1 H NMR (300 MHz, CDCl3) δ: 1.45 (s, 9H), 1.98-2.08 (m, 1H), 2.25-2.35 (m, 1H), 2.62 (t, 2H), 3.89 (s, 3H), 4.45-4.50 (m, 1H), 4.82 (q, 2H), 5.12 (d, 1H), 5.29 (m, 1H), 6.85-6.90 (m, 2H), 7.41 (d, 1H). N-Boc-γ-(7-methoxycoumarin-4-yl)-L-homoalanine Cyanomethyl Ester. To a stirred solution of mchAla (10.0 mg, 36 µmol) and NaHCO3 (6.1 mg, 72 µmol) in a dioxane/H2O (2/1) mixed solvent (300 µL) cooled to 0 °C was added ditert-butyldicarbonate (4.3 mg, 40 µmol) in 50 µL of dioxane. The stirring was continued overnight at room temperature. The mixture was acidified to pH 2.0 with 5% aqueous KHSO4 and the organic component was extracted with chloroform (3 mL each) three times. The extract was washed with a 3-mL portion of brine and dried over MgSO4. The residue after evaporation of the solvent was redissolved in acetonitrile (1 mL) containing triethylamine (15 µL, 108 µmol). Chloroacetonitrile (45.2 µL, 720 µmol) was added at 0 °C and the mixture was stirred for 12 h at room temperature. The mixture was evaporated to dryness and the residue was dissolved in ethyl acetate (10 mL). The organic phase was washed with a 10-mL portion of 5% aqueous KHSO4, 4% aqueous NaHCO3, and then brine and dried over MgSO4. Evaporation of the solvent gave the title compound (10 mg, 24 µmol, 67%). 1H NMR (300 MHz, CDCl3) δ: 1.48 (s, 9H), 2.02-2.12 (m, 1H), 2.25-2.35 (m, 1H), 2.84 (t, 2H), 3.89 (s, 3H), 4.52 (m, 1H), 4.82 (q, 2H), 5.10 (d, 1H), 6.15 (s, 1H), 6.84-6.90 (m, 2H), 7.49 (d, 1H). pdCpA-Amino Acids and Aminoacyl-tRNACCCG. The Boc-amino acids were linked to a pdCpA and the pdCpAamino acid was coupled with the tRNACCCG(-CA) with T4 RNA ligase. The procedure was described before.11-14 The pdCpA-amino acids were identified by MALDI-TOF mass spectroscopy. pdCpA-mchAla: calcd for C33H40O17N9P2 (M + H) 896.202; found 896.193. pdCpA-Asp(OMc): calcd for C34H40O19N9P2 (M + H) 940.191; found 940.156. pdCpAGlu(OMc): calcd for C35H42O19N9P2 (M + H) 954.207; found 954.191. In Vitro Protein Synthesis. The tRNA with a CCCG fourbase anticodon charged with the nonnatural amino acid was mixed with the in vitro biosynthesizing system of E. coli S30 lysate together with the mRNA for mutant streptavidin.11-14 The mRNA was synthesized in vitro by T7 RNA polymerase from a plasmid (pGSH) that contained a sequence of T7 promoter, a T7 tag, a streptavidin including a single CGGG four-base codon, a His6 tag, and a T7 terminator.11-14 The synthesis of the full-length mutant streptavidins was confirmed on a SDS-PAGE followed by a Western blot analysis using an anti-T7-tag antibody (Novagen) and alkaline phosphatase-labeled antimouse IgG (Promega) as the marker. The binding activity of the mutant streptavidins

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against biotin was tested for the in vitro reaction mixture by a dot blot analysis by using biotin-linked alkaline phosphatase (Zymed). For spectroscopic studies, a larger amount of the mutant streptavidin was prepared by using 100 µL of the in vitro reaction mixture that contained 20 µL of S30 lysate. After the reaction mixture was centrifuged at 20 000 G for 20 min, the supernatant was loaded onto 10 µL of Co-NTA agarose column (Clontech) equilibrated with a buffer (A) containing 50 mM sodium phosphate and 300 mM NaCl at pH 7.0. The column was washed with seven 1-mL portions of a buffer (B) that contained 50 mM sodium phosphate, 1 M NaCl, and 5 mM imidazole (pH 7.0) and then with two 0.2-mL portions of buffer A. The protein was eluted with 40 µL of elution buffer that contained 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, and 0.05% PEG8000 at pH 7.0. The purity of the mutant was checked by SDS-PAGE with silver staining. The concentration of the purified mutant solution was determined by the densitometry of the Western blotting. The solution of wild-type streptavidin of known concentration (282 ) 4.3 × 104 cm-1 L mol-1) was used as the standard. Fluorescence Measurement. Fluorescence spectra were recorded on a Spex-Joan-Yvon Fluoromax2. The slit width was 1.5 nm for the excitation and 5 nm for the emission. The mutant solution of known concentration as described above was diluted by nine portions of buffer A and transferred into a microcuvette under argon. The fluorescence quantum yields were determined from a fluorescence spectrum and absorbance at the excitation wavelength. Because the absorbance was very low, it was calculated from the concentration of the mutant using the extinction coefficient of N-acetyl-DL-anthrylalanine (342 ) 5.2 × 103 cm-1 L mol-1) or that of 7-methoxycoumarin-4acetic acid (325 ) 1.4 × 104 cm-1 L mol-1). Results and Discussion Yields and Biotin-Binding Activities of antAla-Streptavidins. The yields of the antAla-streptavidins were checked by the Western blotting of the reaction mixture by using an alkaline phosphatase-linked anti-T7 antibody. The results for the antAla mutants at 24 different positions are shown in Figure 1 (top). The full-length streptavidins (19.0 kDa) are successfully produced for all mutants with similar yields, indicating that the CGGG four-base codon was correctly translated by the antAla-tRNACCCG. As described previously,11 if the CGGG four-base codon was translated as a CGG three-base codon by an endogenous Arg-tRNACCG, the translation would be stopped at a stop codon that appears after the CGG codon.11,12 The truncated proteins are actually detected at the low-molecular-weight side of the Western blotting. The yields of mutant streptavidins were about the same, irrespective of the mutation positions, indicating that the translation efficiency of the four-base codon/anticodon pair does not depend on the context of the base sequence. A qualitative test of the binding activity of each mutant was done by dot blotting by using alkaline phosphataselabeled biotin as the marker (Figure 1 (bottom)). Although the yields of the mutant streptavidins were about the same,

Murakami et al.

Figure 1. (Top) Western blot analysis on the wild-type and the antAlastreptavidins prepared in the S30 in vitro biosynthesizing system. The proteins were visualized with anti-T7-tag antibody combined with alkaline phosphatase-labeled antimouse IgG. The numbers on the top of each band indicate the positions of antAla. No mutant was formed in the absence of antAla-tRNACCCG. (Bottom) Results of dot blot analysis for the wild-type and the antAla streptavidins. The binding abilities are visualized with a biotin-labeled alkaline phosphatase. For a rough estimate of the incorporation efficiency of antAla, the dot blot analysis was made for the wild-type protein at different dilution factors.

the binding activities depended markedly on the positions of the mutation. For example, the mutations at the 21st, 79th, 80th, 85th, 87th, and 92nd positions, respectively, resulted in the loss of binding activity, indicating that these mutants are largely denatured. The 22antAla, 43antAla, 54antAla, and 120antAla mutants that retained strong binding activity were prepared in a large scale (100 µL of the reaction mixture containing 20 µL of the S30 lysate) and purified with the Co-NTA column that selectively binds the histidine hexamer at the C terminal. The yields of the purified mutants were evaluated from the densitometry of the Western blotting. For the purified mutants, the biotin binding activity was measured by using an FITC-labeled biotin. The fluorescence titration curves for the antAla mutants are shown in Figure 2. The abscissa of the figure is the concentration of each mutant that gives the best fit to the experimental points, assuming a 1:1 binding. The latter concentrations agreed with those determined from the densitometry of the Western blotting with an error range of about 10%. The binding constants were determined from the least-squares curve fitting and are listed in Table 1. The binding constants markedly depend on the mutation positions: that of the 120antAla mutant was very high (3.9 × 1011 L mol-1), whereas that of the 43antAla mutant (2.5 × 108 L mol-1) was lower by 3 orders of magnitude, indicating that the anthryl group disturbs the biotin binding. Positions and Orientations of Anthryl Groups in the Mutant Streptavidins. The positions and orientations of the

Incorporation of Fluorescent Amino Acids into Proteins

Figure 2. Fluorescence titration curves for the 22antAla (open circles), 43antAla (solid circles), 54antAla (open triangles), and 120antAla (solid triangles) streptavidins bound with FITC-labeled biotin. [FITC biotin] ) 1.0 nmol L-1. Solid lines are the calculated curves assuming a 1:1 binding with different binding constants as listed in Table 1. The abscissa is the concentration that gives the best fit to the experimental points. Table 1. Relative Yields with Respect to the Wild-type Streptavidin (y, %), Biotin Binding Constants (K, L mol-1), and Fluorescence Quantum Yields of the Anthryl Group in the Absence (q0) and Presence (qb) of Biotin, for antAla Streptavidins position 22 43 54 120

y

K

50 30 65 55

2.3 × 2.0 × 108 1.4 × 109 4.7 × 1010 1011

q0

qb

0.080 0.069 0.025 0.072

0.078 0.064 0.026 0.069

anthryl groups in the mutants that retain binding activity were predicted from molecular mechanics calculations,12,22,23 assuming that the backbone conformation is not altered by the mutation. The X-ray crystallographic structure of streptavidin tetramer with four biotins in the binding sites was used as the starting conformation for the bound protein.24 The crystallographic structure of apo-streptavidin at pH 7.5 was used for the free protein.25 In both cases, only the side-chain rotational angles (χ1, χ2, ...), including that of the anthrylalanine itself, were varied. The computer-predicted orientations of anthryl groups at the 22nd, 43rd, and 120′th positions in the biotin-bound streptavidin are illustrated in Figure 3. In the same figure, the orientations of tryptophan units are also shown. From the above spatial arrangements the centerto-center distances between the anthryl group and the indole groups were obtained and were used to calculate the energytransfer efficiency as will be discussed below. Some conclusions can be drawn from the conformational calculations. First, the calculation on the 54antAla mutant indicated that the antAla cannot be introduced unless the main chain conformation is significantly distorted. Because the prediction of the main-chain conformation is difficult, the conformation of the 54antAla mutant is not included in Figure 3. The high binding constant (6.2 × 109 L mol-1) of the 54antAla mutant, however, indicates that the structure of the biotin binding site is well-preserved. The anthryl group at the 43rd position (Figure 3, center) is predicted to be on the bottom of the biotin-binding site. The prediction is inconsistent with the low binding constant of the 43antAla mutant. The anthryl group at the 120th

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position is far separated from the main body of the subunit, but the anthryl group of the third subunit that is indicated as 120′antAla in Figure 3 (right) is predicted to be in close contact with biotin and the 108th tryptophan unit. The anthryl group at the 22nd position (Figure 3, left) is isolated from most of the tryptophan units and from biotin. As is obvious from the predicted conformations, the anthryl groups in different mutants are situated in very different microenvironments. Fluorescence Spectra of Anthryl Groups in Mutant Streptavidins. Fluorescence spectra were measured for the antAla-streptavidins in the presence and absence of biotin. The spectra of the 22antAla and 54antAla mutants are shown in Figure 4. The positions of the 0-0 peak show a small shift from 383.8 nm for the 22nd and 385.6 nm for the 120th anthryl group to 387.1 nm for the 43rd and 54th anthryl groups. The peak position of the 22antAla mutant agrees with that of N-acetyl-DL-anthrylalanine in water (383.5 nm), indicating that the anthryl group is exposed to the outer surface as predicted in Figure 3 (left). The red shift of the fluorescence of the 43antAla mutant (not included in Figure 4) suggests that the anthryl group is located inside the protein, as in Figure 3 (center). The effect of biotin binding on the fluorescence intensity was also shown in Figure 4. For all mutants, fluorescence intensity slightly decreased with the addition of biotin. In the case of the 54antAla mutant, however, the decrease of the fluorescence at 380-410 nm is accompanied with the increase around 450-500 nm. Although this spectral change was very small, it could be reproducible. The appearance of the new fluorescence band at a long wavelength region may indicate an exciplex-type interaction between the excited anthryl group and, presumably, an indole group located nearby. Because the location of the 54th antAla could not be predicted, we cannot specify the chromophoric group that forms an exciplex with the anthryl group. The exciplex-type fluorescence was observed only in the 54antAla mutant. The fluorescence quantum yield for each mutant was calculated from the fluorescence spectrum and the protein concentration determined from the Western blotting. The results are listed in Table 1. The quantum yields are not sensitive to the position and the biotin binding, except for the 54antAla mutant for which an exciplex-type interaction has been detected. The insensitiveness of the anthryl fluorescence to the environment may be attributed to the inherent property of the anthryl fluorophore. Figure 5 shows the quantum yields of N-acetyl-DL-anthrylalanine in various solvents of different dielectric constants. The fluorescent property is not sensitive to the change of the dielectric constant as compared with the coumarine derivative that will be described below. Energy Transfer from Tryptophans to Anthryl Groups in Mutant Streptavidins. Fluorescence spectrum of 120antAla-streptavidin excited at 290 nm is shown in Figure 6. At this excitation wavelength, the absorption of the anthryl group is much weaker than that of the tryptophan groups and the direct excitation of the anthryl group can be neglected. Indeed, the fluorescence spectrum of an equimolar mixture of wild-type streptavidin and N-acetyl-DL-anthryla-

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Figure 3. Computer-predicted orientations of 2-anthryl groups in the mutant streptavidins carrying a single antAla unit at the 22nd (left), 43rd (center), and 120th (right) positions. Orientation of each anthryl group has been optimized by varying the side chain angles (χ1, χ2) of the 2-anthrylalanine and the neighboring amino acids that are located within rcut ) 6 Å from the anthrylalanine unit. The orientations of tryptophan units are also shown. For the 120antAla mutant, a part of the third subunit that includes the 120th antAla (120′antAla) is added.

Figure 4. Fluorescence spectra of 22antAla (top) and 54antAla (bottom) streptavidin in aqueous solution in the absence (s) and presence (- - -) of biotin. [22antAla mutant] ) 15 nmol L-1, [54antAla mutant] ) 69 nmol L-1, [biotin] ) 1.0 µmol L-1, phosphate buffer, and pH 7.0. λex ) 263 nm for the 22antAla mutant and 265 nm for the 54antAla mutant.

Figure 5. Fluorescence quantum yields of N-acetyl-DL-antAla (open circles, triangle, and square) and 7-methoxycoumarine-4-acetic acid (solid circles, triangle, and square) in various solvents with different dielectric constants. Solvents: (solid and open circles) ethanol/water, 100/0, 80/20, 60/40, 40/60, 20/80, 0/100; (solid and open triangles) n-butanol; (solid and open squares) dioxane.

lanine shows negligible contribution of anthryl fluorescence (dashed lines), irrespective of the biotin concentration. Under the same conditions, the 120antAla mutant shows strong anthryl fluorescence that is accompanied with the marked decrease of the tryptophan fluorescence. The fluorescence

Figure 6. Fluorescence spectra of 120antAla streptavidin at λex ) 290 nm in the absence (s) and presence (- - -) of biotin. [protein] ) 22 nmol L-1, [biotin] ) 1.0 µmol L-1, phosphate buffer, and pH 7.0. The fluorescence spectrum of an equimolar mixture of wild-type streptavidin and N-acetyl-DL-anthrylalanine in the absence (s) and presence (- - -) of biotin are also shown.

behavior indicates very efficient energy transfer from the indole groups to the anthryl group in the mutant. The intramolecular energy transfers were also observed in other antAla mutants. The efficiencies of energy transfer were evaluated quantitatively from the excitation spectra that were monitored by the fluorescence of the anthryl group at the second vibronic peak (405-409 nm). The absorption spectrum of an equimolar mixture of the wild-type streptavidin and N-acetyl-DL-anthrylalanine was taken as a standard for the 100% energy transfer. The energy-transfer efficiencies thus evaluated are listed in Table 2. The energy-transfer efficiency showed complex change when biotin was bound to the mutants. The biotin-bound 120antAla mutant showed less efficient energy transfer than the free mutant as can be seen in Figure 6, whereas the energy transfer was slightly enhanced by the biotin binding to the 54antAla mutant. The observed efficiencies may be compared with the calculated efficiencies for the spatial arrangements of indole and anthryl groups in the tetramer conformations of mutant streptavidins, on the basis of the Fo¨rster theory.26,27 The calculations were carried out for the 22antAla, 43antAla, and

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Incorporation of Fluorescent Amino Acids into Proteins Table 2. Efficiencies of Energy Transfer from the Indole Groups to the Anthryl Group in Various antAla Streptavidins in the Absence (Eobsd,0) and Presence (Eobsd,b) of Biotin (Calculated Efficiencies in the Absence (Ecalc,0, r0 ) 19.0 Å) and Presence (Ecalc,b, r0 ) 17.8 Å) of Biotin Are Also Listed) position

Eobsd,0

Eobsd,b

Ecalc,0

Ecalc,b

22 43 54 120

0.68 0.77 0.95 0.65

0.68 0.76 1.00 0.32

0.68 0.99 a 0.81

0.60 0.98 a 0.77

a

Calculation predicted a significant conformational change in the main chain of this mutant.

120antAla mutants on which the orientations of the indole and anthryl groups in the tetramer were predicted as shown in Figure 3. One of the parameters required for the calculation is the inherent quantum yield of the tryptophan fluorescence of streptavidin in the absence and presence of biotin. The tryptophan fluorescence slightly decreased with a blue shift by the biotin binding.27-29 The quantum yield was evaluated for wild-type streptavidin in the absence and presence of biotin to be 0.055 and 0.041, respectively. In the case of the 120antAla mutant, the fluorescence quantum yield was multiplied by 5/6, since 120tryptophan is replaced by anthrylalanine. The second parameter required for the energytransfer calculation is the spectral overlap between the inherent tryptophan fluorescence and anthryl absorption. Because absorption of the anthryl group in each mutant could not be measured, the spectral overlap was calculated for the fluorescences of free and bound wild-type streptavidin and the absorption of N-acetylanthrylalanine. The orientational factor (κ2) was assumed to be 2/3. These parameters gave the critical distance of energy transfer, r0 ) 19.0 Å for free mutants and 17.8 Å for biotin-bound mutants. The theoretical transfer efficiencies were calculated with the above r0 values and the center-to-center distances between the indole and anthryl groups in the tetramer. Because there are four anthryl groups in a tetramer, energy-transfer efficiency of one of the six (or five) tryptophan units is calculated as27 4

E(i) )

(1/riI6) ∑ I)1 (1)

4

1/r0 + 6

(1/riI ) ∑ I)1 6

where riI is the center-to-center distance between the ith indole group in the first subunit and the anthryl group in the Ith subunit. The energy-transfer efficiencies for the six (or five) indole groups in the first subunit were averaged to give the average energy-transfer efficiency. The latter may be compared with the observed data because the four subunits are identical. The calculated energy-transfer efficiencies are listed in Table 2. For the 22antAla mutant in which the anthryl group is separated from most of the tryptophan units and from biotin, the agreement between the observed and calculated efficiencies is satisfactory. For the other two mutants, however, the observed efficiency is significantly lower than the calculated value. In the case of the 120antAla mutant, the experimental

Table 3. Relative Yields with Respect to the Wild-Type Streptavidin (y, %), Biotin Binding Constants against FITC-Labeled Biotin (KFITC, L mol-1), Biotin Binding Constants against Biotin (KBio, L mol-1), and Fluorescence Quantum Yields of the Methoxycoumarine Group in the Absence (q0) and Presence (qb) of Biotin, for Mutant Streptavidins Containing Methoxycoumarine Amino Acids at the 120th Position amino acid

y

KFITC

Kbio

q0

qb

mchAla Asp(OMc) Glu(OMc)

12 14 19

5.2 × 1010 1.9 × 1010 2.4 × 109

6.0 × 109

0.24 0.13 0.28

0.13 0.13 0.38

1.8 × 107

efficiency is markedly decreased by the biotin binding, although calculations suggested no significant conformational change. The disagreement may be partly due to an inappropriate assumption of the equivalence of all tryptophan units in the protein.28 If the six (or five) indole groups are not equally fluorescent, the anthryl group that is close to poor donors may receive less energy than average and vice versa. The suppression of the energy transfer in the 120antAla mutant by biotin binding may be interpreted in terms of the biotin-induced specific quenching of 108tryptophan that may be the major energy donor to the 120antAla. The marked decrease of the energy-transfer efficiency with the biotin binding in 120antAla mutant may be useful as a tool for the fluorescence detection of biotin. Although the anthryl group itself is not a sensitive fluorophore as indicated from Figure 5, its fluorescence intensity becomes sensitive to the ligand binding when it was photoexcited at 290 nm, because of the change of energy-transfer efficiencies from indole groups. Incorporation of Methoxycoumarine Derivatives and Fluorescence Spectra of the Mutants. Coumarine is another highly fluorescent group that can be linked to the side group of a nonnatural amino acid. In this study, three different types of amino acids carrying methoxycoumarine group (Chart 1) were synthesized and incorporated into streptavidin. A methoxycoumarine derivative of homoalanine (mchAla) was incorporated into various positions of streptavidin as in the case of antAla. Western blotting indicated that about twothirds of the mutants were successfully synthesized in full length with the yield of 10-25% with respect to the wildtype streptavidin. However, the yields of the mutant were very small when the nonnatural amino acid was introduced at the 44th, 49th, 65th, 79th, 114th, 117th, and 124th positions, respectively. Because the translation efficiency of the four-base codon was shown to be insensitive to the base context, the reason for the low yields is not clear at this moment. The dot blotting indicated that only the 25mchAla and 120mchAla mutants retained high biotin-binding activity. This indicates that the incorporation of mchAla influences the conformation of streptavidin more significantly than that of antAla. The 120mchAla mutant was synthesized at a large scale and purified by using the affinity of His6 tag to the Co-NTA column. Similarly, methoxycoumarine derivatives of aspartate [Asp(OMc)] and of glutamate [Glu(OMc)] were incorporated into the 120th position and the mutants were purified. The yields of the three coumarine mutants were, however, lower than the antAla mutants (Table 3).

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Figure 7. Fluorescence spectra of 120mchAla streptavidin in the presence of different amounts of biotin. [mutant] ) 9.0 nmol L-1, [biotin] ) 0-100 nmol L-1, phosphate buffer, and pH 7.0. λex ) 325 nm. The inset is the fluorescence titration curve against biotin with a least-squares fitting curve.

Figure 8. Fluorescence spectra of 120Asp(OMc) streptavidin in the presence of different amounts of biotin. [mutant] ) 12 nmol L-1, [biotin] ) 0-10 µmol L-1, phosphate buffer, and pH 7.0. λex ) 325 nm.

The binding constants of the mutants were evaluated from fluorescence titration curves against FITC-labeled biotin as in the case of antAla mutants (Table 3). The binding constants of the four mutants that contain a nonnatural amino acid at the 120th position depend on the type of amino acids in the following order: antAla > mchAla > Glu(OMc) > Asp(OMc). As will be discussed below, because the binding force to the FITC-labeled biotin includes the affinity of streptavidin to the FITC group, the binding constants cannot be simply related to the molecular structures of the mutants. Detection of Biotin Binding by the Change of Fluorescence Intensity. Fluorescence spectra of the 120mchAla, 120Asp(OMc), and 120Glu(OMc) mutants in the presence of various amounts of biotin are shown in Figures 7, 8, and 9, respectively. The quantum yields in the absence and presence of biotin are listed in Table 3. In the absence of biotin, high quantum yields were observed especially for the glutamate derivative. More interestingly, the quantum yields markedly decreased and increased with the binding of biotin for the mchAla and Glu(OMc) mutant, respectively. The fluorescence of the Asp(OMc) mutant was insensitive to the biotin binding. The change of fluorescence intensities of the methoxycoumarine mutants by the biotin binding is due to high

Murakami et al.

Figure 9. Fluorescence spectra of 120Glu(OMc) streptavidin in the presence of different amounts of biotin. [mutant] ) 16 nmol L-1, [biotin] ) 0-10 µmol L-1, phosphate buffer, and pH 7.0. λex ) 325 nm. The inset is the fluorescence titration curve against biotin with a leastsquares fitting curve.

Figure 10. Computer-predicted conformation of 120mchAla streptavidin in the absence (left) and presence (right) of biotin. A part of the third subunit that contains the 120th mchAla (120′mchAla) is included.

susceptibility of the fluorophore to the microenvironment. Figure 5 shows fluorescence quantum yields of 7-methoxycoumarine-4-acetic acid plotted against the dielectric constants of solvents. The quantum yield is very small in less polar media but it increases dramatically with the increase of water content because of the enhancement of the intramolecular electron polarization. Therefore, the decrease of fluorescence intensity of the 120mchAla mutant indicates that the effective polarity of the fluorophore decreased with the biotin binding. Indeed, the computer-predicted conformations in Figure 10 suggest that the methoxycoumarine group is oriented outside the tetramer protein in the absence of biotin, whereas it may become in close contact with the biotin added. Contrary to the mchAla case, the methoxycoumarine group of the glutamate esters may be expelled to the outer region by the biotin binding, resulting in the increase of the fluorescence intensity. In the case of the aspartate derivative, little change was detected by the biotin binding. The insets of Figures 7 and 9 show the titration curves plotted against the biotin concentration. The least-squares analysis gave the binding constants to biotin (Kbio), as are listed in Table 3. The binding constants are significantly different from those determined against the FITC-labeled biotin (KFITC). It seems somewhat curious that the binding

Incorporation of Fluorescent Amino Acids into Proteins

of FITC-biotin is easier than that of free biotin, especially in the case of Glu(OMc) mutant. A plausible explanation is that streptavidin has special affinity to FITC as reported by Gruber and co-workers.30 In the case of the 120mchAla streptavidin, the biotin-binding is not seriously suppressed by the incorporation of the fluorophore, but the latter fluorescence behavior is markedly influenced by the biotin binding. This situation is very appropriate for the fluorescence detection of biotin by the nonnatural mutant of streptavidin containing a fluorescent group. The 120mchAla streptavidin exemplifies the utility of the fluorescent mutants as diagnostic and environmental sensors. The fluorescent mutants of receptors, antibodies, and enzymes will be able to detect the ligands, antigens, and inhibitors under nM concentrations or less, provided that the binding constant is high enough. For these applications to be possible, the use of fluorophores that are highly sensitive to a small change in the microenvironment is essential. Furthermore, the fluorescent amino acids must be incorporated into a very specific position to accomplish sensitive detection of the ligands and to avoid the suppression of the ligand binding. The site-specific incorporation of fluorescent amino acids such as mchAla is a promising approach toward the fluorescence detection of specific ligands. Conclusions 2-Anthrylalanine was incorporated at several positions in streptavidin and some of the mutants were found to retain the biotin-binding activity. The quantum yield of the anthryl group was relatively insensitive to the biotin binding. However, when the anthryl group was excited through energy transfers from tryptophan groups, the fluorescence intensity became sensitive to the biotin binding. Methoxycoumarine groups were also introduced into streptavidins and they were found to be very sensitive to the biotin binding. These results suggest that nonnatural mutants incorporated with fluorescent amino acids will find applications as highly sensitive fluorescence sensors that can detect small molecules under nM concentrations or less. Acknowledgment. This work has been supported by a Grand-in-Aid for Specially Promoted Research from the Ministry of Education, Science, Sports and Culture, Japan (No. 11102003). References and Notes (1) Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182.

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