Photochemical Construction of Coumarin Fluorophore on Affinity

Feb 24, 2011 - r 2011 American Chemical Society. 315 ... January 6, 2011. Revised: .... tion, Culture, Sports, Science and Technology of Japan and by...
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Photochemical Construction of Coumarin Fluorophore on Affinity-Anchored Protein Takenori Tomohiro,* Kenichi Kato, Souta Masuda, Hiroyuki Kishi, and Yasumaru Hatanaka* Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

bS Supporting Information ABSTRACT: A simple and unique construction of a coumarin fluorophore on a prey protein has been achieved by sequential photoreactions using a nonfluorescent bait protein. The bait protein was first modified with a novel diazirine-based photo-cross-linker containing an o-hydroxycinnamoyl unit. The strategy involves two wavelength-dependent photoreactions: photolysis of the diazirine group and E to Z photoisomerization of the cinnamoyl group. The cross-linked interacting partners were cleaved by the consecutive steps of photoisomerization and thermal lactonization accompanied with the creation of a coumarin derivative on the prey protein as a fluorescent tag. Finally, the methodology was successfully applied for coumarin labeling of antilysozymes expressed on living B cells by using photoactivatable lysozymes.

fluorescent beacon which is concomitantly constructed in the releasing step. The reagent for this purpose includes two types of reactive groups: N-hydroxysuccinimide ester and diazirine for cross-linking of the interacting proteins. The structure of the small-sized cross-linker 3-trifluoromethyl-3H-diazirin-3-yl cinnamoyl derivative 1 is illustrated in Scheme 1. The N-hydroxysuccinimidyl group chemically reacts with amines, and the diazirinyl group photochemically cross-links to spatially closed molecules. Among the photophores such as arylazide and benzophenone, 3-phenyl-3-trifluoromethyldiazirine is employed in the system because it has been recognized as a photophore that satisfies the chemical and biological criteria for most photoreagents owing to its stability in general synthetic and biological conditions, relatively longer excitation wavelength, rapid photolysis to generate an electron-deficient carbene capable of reacting with a full range of functional groups, and formation of a stable covalent bond between interacting molecules.20,21 Briefly, the short-lived carbene intermediate reacts with molecules, including water, with minimal diffusion,22 which minimizes nonspecific cross-linking. This should be a crucial feature for specific labeling of interacting partners, although the yield is usually low. The strategy involves cross-linking of interacting proteins in two reaction steps: chemical modification of the bait protein with the reagent, and then photochemical trapping of the prey protein under irradiation. These steps are the same as those in the general label transfer methodology. An interesting feature of this compound is the E to Z photoisomerization of the cinnamoyl group existing between two reactive groups. Once the isomerization proceeds, intramolecular nucleophilic substitution (lactonization) by the hydroxyl group at the ortho position will easily occur at the carbonyl group. In our case, the cross-linked

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dentification of interacting partners has been invaluable in the field of life science for understanding complex biological processes. Biological or chemical protein tagging has often been used for detection or isolation of interacting proteins. In particular, selective incorporation with small fluorophores has remarkably progressed to reducing structural perturbations to the target protein1-3 by utilizing enzymatic systems,4 biological machineries such as metabolism5 or translation,6-9 and affinity-based chemical methodologies.10-12 In contrast to the advances in fluorophore-tagging of an individual protein, only a few methods have been reported for direct tagging of “unknown” interacting proteins. Photoaffinity cross-linking is a tool for making a covalent bond between interacting partners without disturbing the interaction by chemical treatment, and it has been applied to identification of interacting proteins, especially for proteins that interact weakly or transiently. Recently, site-directed unnatural amino acid mutagenesis in vitro has been successfully applied for incorporating an amino acid bearing a photo-cross-linker into a protein of interest.13 Alternatively, the label-transfer technique is known as a simple and valuable chemical tagging method of an unknown “prey” protein that utilizes a “bait” protein modified with a cleavable photo-cross-linker.14-16 The specific formation of photo-cross-link followed by the transfer of a tag from the bait protein to the prey protein by cleaving the cross-link accomplishes the identification of the protein-protein interaction. However, the method usually requires preparation of the crosslinker accompanied by a radioisotope, fluorescent, or biotin label in advance.17,18 It sometimes causes high background noise, loss of affinity, and problems in probe synthesis.19 Moreover, it also needs chemical treatment in the cleavage step of the cross-link to accomplish the label transfer. Here, we report a unique photochemical strategy for creating a fluorophore as a chemical tag on the prey protein from a nonfluorescent bait protein. The concept of our method is to enable the mild photochemical release of the cross-linked prey protein installing a r 2011 American Chemical Society

Received: January 6, 2011 Revised: February 7, 2011 Published: February 24, 2011 315

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afford a free active hydroxyl of the serine residue, producing a coumarin derivative.24 While they utilized the cinnamoyl group in a protein for the photoreaction, we combined this reaction with the photo-cross-linking technique for fluorescent labeling of target protein. The cross-linker 1 can be incorporated into lysine residues of the protein of interest via an amide bond. Since a cinnamoyl group has been installed to serine protease via an ester linkage, the photolysis of cinnamide derivative 3 was investigated by comparison to the ester derivative 2, prior to its application to the protein. Figure 1A shows the time course of coumarin formation from cinnamoyl derivatives (2 and 3) by photoreaction with light at around 315 nm. Emission was detected at 437 nm upon excitation at 320 nm, and the intensity at 437 nm reached the maximum in 10 min in the case of derivative 2. The coumarin derivatives were formed with approximately 40% yield, with the value estimated from the relative intensity of the corresponding coumarin compound synthesized separately. These compounds were identified by 1H, 13C, and 19F NMR and high-resolution MS (see Supporting Information). As shown in Figure 1A, the initial reaction rate of derivative 2 was approximately 19 times faster than that of derivative 3. However, the emission intensity of the solution of derivative 3 steadily increased with irradiation time to yield a comparable amount of the coumarin derivative in 30 min. On the other hand, photolysis of the diazirine group was then carried out with 360 nm light, which was monitored by measuring the absorption decay at 370 nm due to the n-π* transition of the diazirine group.25,26 Figure 1B showed that the photolysis was almost accomplished in 10 min. We applied this strategy to fluorescent labeling of antilysozyme (anti-LYZ) expressed on the cell membrane by using a photoactivatable lysozyme (DA-LYZ). DA-LYZ was prepared by a reaction of compound 1 and LYZ in a manner similar to a previously described method.27 Approximately 1.1 equiv mol of diazirine units was incorporated into an LYZ containing 6 lysine residues. The value was spectrometrically determined from the electronic absorption at 367 nm, relative to that of compound 3 (Supporting Information Figure S2). The photochemical reactivity of the DA-LYZ was first examined. Figure 2 shows the fluorescence spectral change of phosphate-buffered solution (PBS; 5 mM, pH 6.8, containing 2.5 mM NaCl) of DA-LYZ obtained by photoirradiation. An emission was observed at around 440 nm after irradiation of a DA-LYZ solution with 315 nm light at 0 °C. The intensity was increased by irradiation and reached a maximum in 2 h. In contrast, very little emission was observed after 1 h irradiation with 360 nm light, in which photolysis of the diazirine group occurred. The results showed the wavelength dependence of the two photoreactions of DALYZ, indicating that the coumarin fluorophore should be formed after cross-linking the DA-LYZ and anti-LYZ by changing the irradiation wavelength. The first photoreaction “cross-linking” of DA-LYZ with antiLYZ was evaluated in vitro by Western blot analysis of the products. A phosphate buffered solution of DA-LYZ and antiLYZ was irradiated with 360 nm light for 10 min at 0 °C. Figure 3 shows the Western blot analysis of the photoproducts after SDSPAGE by using anti-LYZ as the first antibody and IgG-horseradish peroxidase conjugate as the second antibody. Hence, the observed bands should be containing LYZ (14 kDa) or the heavy chain of anti-LYZ (50 kDa). The band around 40 kDa, indicated by an arrowhead, appeared in the irradiated sample (lane 2), and the emission intensity of the 40 kDa protein decreased in

Scheme 1. Strategy for Photochemical Fluorescent Labeling of the Interacting Protein with Multifunctional Cross-Linker 1

interacting partners should be cleaved by the consecutive steps of photoisomerization and thermal lactonization accompanied with the creation of a coumarin derivative on the prey protein as a fluorescent tag (Scheme 1). Because of the different excitation wavelengths as well as the thermal property of coumarin formation, the two photochemical steps can be controlled with good independence by selection of irradiation frequency. Moreover, the coumarin is formed from the simple linker unit by adding a double bond to the general phenyldiazirine photophore. There is no need for prior attachment of a bulky fluorophore to the diazirine unit. Since the cinnamoyl group is not fluorescent, the fluorescent proteins must be derived from the cross-linked prey proteins. Martinek et al. previously applied cinnamoyl derivatives to regulate protein functions by utilizing photoisomerization.23 While the approach relies on steric effects to differentiate cis/ trans photoisomers for the hydrolysis, Porter et al. have developed a more active chemical system by using an o-hydroxycinnamoyl derivative for the photoactivation of irreversibly inhibited serine proteinases. In this system, internal deacylation in the protein is achieved by lactonization via photoisomerization to 316

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Figure 1. (A) Time course of coumarin formation from cinnamoyl derivatives. Photolysis of cinnamoyl derivatives was carried out with 315 nm light at 0 °C in ethanol solution (1.00  10-4 M; filled circle for 2 and open circle for 3) in a sealed quartz cell and monitored by measuring the emission intensity at 437 nm (λex 320 nm) at 20 °C. (B) UV-vis spectral change of ethanol solution containing compound 2 (1.00  10-4 M) under irradiation of 360 nm light at 0 °C in a sealed quartz cell.

Figure 4. Fluorescent imaging of live B cells expressing monoclonal anti-LYZ after labeling with a coumarin derivative by sequential irradiations: optical (panel A) and fluorescence (λex 334 nm) microscopic images after the second irradiation (panel B) and a magnified view of the image (panel C). Scale bar, 30 μm.

Figure 2. Emission spectra of the photoproducts of DA-LYZ (λex 320 nm) were measured in a mixed solvent of PBS-ethanol (2:1, v/v) after irradiation with light at around 315 nm (spectra 1-3) or 360 nm (spectrum 4) at 0 °C in a sealed quartz cell.

was carried out continuously with 315 nm light for 60 min at 0 °C. In this reaction step, the small coumarin derivative was also produced from the non-cross-linked DA-LYZ in addition to the formation of coumarin-labeled anti-LYZ. Gel filtration was then performed to exclude the small compounds, and blue emission was observed in the protein fractions. Finally, the photoproducts were subjected to SDS-PAGE and the light chain was isolated by extraction from the gel. The emission was clearly observed for the products with DA-LYZ, but not detected from the sample after the same treatment without DA-LYZ (Supporting Information Figures S4 and S5). These data indicated that the light chain of anti-LYZ was coumarinlabeled by the sequential photoreactions. Finally, we applied this methodology to fluorescent labeling of B cells expressing hen-egg lysozyme-specific antibody on the cell surface. The cells were prepared from the spleens of transgenic C57BL/6 mice according to the previous report.28 DA-LYZ (2.5 μM) was incubated with B cells (6.0  107 cells/mL) in a PBS solution (pH 7.4) and was irradiated with 360 nm light at 0 °C for 3-10 min. After being washed with PBS, the cells were irradiated with 315 nm light at 0 °C for 15-60 min. Blue emission was clearly detected from the membrane of B cells after the second photoreaction (Figure 4, panels B and C) and the intensity was increased with the irradiation time. When the same treatment was applied to a mixture of cells, the emissions were only observed for the B cells, but not for the other cells (Supporting Information Figure S6). These data suggested that DA-LYZ specifically cross-linked to the anti-LYZ expressed on the cell membrane surface in response to the photo-cross-linking step and formed coumarin on the cell after the photoisomerization/ cyclization step. In conclusion, we have newly designed and synthesized a fluorescent label-transfer reagent containing a cinnamoyl unit.

Figure 3. Western blot analysis of the photo-cross-linked products of DA-LYZ and anti-LYZ. Sample solutions (10 μL) in lanes 1-7 contain anti-LYZ (40 pmol) and DA-LYZ as 0, 1, 0.5, 0.1, 0.05, 0.01, and 0.001 equiv mol of anti-LYZ (40 pmol), respectively. Lane 8 includes LYZ (20 pmol) instead of DA-LYZ. The photoproducts were denatured in sodium dodecyl sulfate (SDS) sample buffer at 95 °C for 5 min and then subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE), followed by blotting onto a polyvinylidene fluoride membrane. The products were detected by a chemiluminescence method.

response to the amount of DA-LYZ (lanes 2-7). The 40 kDa protein band was not detected in the photoproducts in the absence of DA-LYZ (lanes 1 and 8). It indicates that it is not derived from the photoreaction of anti-LYZ or LYZ itself. These results suggest that it is a cross-linked product of DA-LYZ and the light chain of anti-LYZ (∼25 kDa). The cross-linking efficiency in lane 2 was calculated as approximately 16% from the emission intensity of the 40 kDa protein, determined by comparing it with that of the 14 kDa non-cross-linked DA-LYZ, which is presumably a photoproduct with a water molecule. Following the cross-linking, the E to Z photoisomerization/lactonization 317

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The simple and facile creation of coumarin fluorophore onto a prey protein (anti-LYZ) using a photoactivatable bait protein (DA-LYZ) has been achieved at the interacting site (light chain) by regulating two reactions: cross-linking and lactonization. This method was applied for the labeling of membrane proteins in living cells. Although improvement of the labeling efficiency and fluorescent properties of the coumarin unit is required for in situ imaging analysis of proteins, this novel photochemical fluorophore-tagging technique will simplify the identification procedure of interacting proteins.

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’ ASSOCIATED CONTENT

bS

Supporting Information. Details on experimental protocols for synthesis of materials, preparation of DA-LYZ, photoreactions, and fluorescent imaging of cells. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Corresponding authors. Takenori Tomohiro: telephone þ81 764347516; fax þ81 764345063; E-mail address: ttomo@ pha.u-toyama.ac.jp. Yasumaru Hatanaka: telephone þ81 764347515; fax þ81 764345063; E-mail address: [email protected].

’ ACKNOWLEDGMENT This research was supported by Grants-in-Aid for Scientific Research (20390032, 21310138) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization). ’ REFERENCES (1) Andresen, M., Schmitz-Salue, R., and Jakobs, S. (2004) Short tetracysteine tags to β-tubulin demonstrate the significance of small labels for live cell imaging. Mol. Biol. Cell 15, 5616–5622. (2) Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002) Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918. (3) Prescher, J. A., and Bertozzi, C. R. (2005) Chemistry in living systems. Nat. Chem. Biol. 1, 13–21. (4) Chen, I., and Ting, A. Y. (2005) Site-specific labeling of proteins with small molecules in live cells. Curr. Opin. Biotechnol. 16, 35–40. (5) Prescher, J. A., Dube, D. H., and Bertozzi, C. R. (2004) Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877. (6) Cornish, V. W., Hahn, K. M., and Schultz, P. G. (1996) Sitespecific protein modification using a ketone handle. J. Am. Chem. Soc. 118, 8150–8151. (7) Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., and Schulte, P. G. (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188. (8) Suchanek, M., Radzikowska, A., and Thiele, C. (2005) Photoleucine and photo-methionine allow identification of protein-protein interactions in living cells. Nat. Methods 2, 261–268. (9) Summerer, D., Chen, S., Wu, N., Deiters, A., Chin, J. W., and Schultz, P. G. (2006) A genetically encoded fluorescent amino acid. Proc. Natl. Acad. Sci. U.S.A. 103, 9785–9789. (10) Soh, N. (2008) Selective chemical labeling of proteins with small fluorescent molecules based on metal-chelation methodology. Sensors 8, 1004–1024. (11) Nagase, T., Nakata, E., Shinkai, S., and Hamachi, I. (2003) Construction of artificial signal transducers on a lectin surface by 318

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