Photoswitching of Ligand Association with a Photoresponsive Polymer

SEPTEMBER/OCTOBER 2002 Volume 13, Number 5 © Copyright 2002 by the American Chemical Society

ARTICLES Photoswitching of Ligand Association with a Photoresponsive Polymer-Protein Conjugate Tsuyoshi Shimoboji, Zhongli L. Ding, Patrick S. Stayton,* and Allan S. Hoffman* Department of Bioengineering, University of Washington, Seattle, Washington 98195. Received May 10, 2001; Revised Manuscript Received December 18, 2001

Light-regulated molecular switches that reversibly control biomolecular function could provide new opportunities for controlling activity in diagnostics, affinity separations, bioprocessing, therapeutics, and bioelectronics applications. Here we show that site-specific conjugation of light-responsive polymers near the biotin-binding pocket of streptavidin provides control of ligand binding affinity in response to UV and visible light irradiation. Two different light-responsive polymers were utilized that display opposite photoresponsive solubility changes under UV or visible (vis) light irradiation in aqueous solutions. At 40 °C, the N,N-dimethylacrylamide (DMA)-co-4-phenylazophenyl acrylate (AZAA) copolymer (DMAA) was soluble under UV irradiation and precipitated under visible light, while the DMA-co-N-4-phenylazophenyl acrylamide (AZAAm) copolymer (DMAAm) was soluble under visible irradiation and precipitated under UV light. Both polymers were synthesized with a vinyl sulfone terminus and conjugated to the Glu116Cys (E116C) streptavidin mutant via thiol coupling. The DMAA-streptavidin conjugate bound biotin efficiently when the polymer was in the soluble state under UV irradiation, but under visible irradiation, the polymer collapsed and blocked free biotin association. Furthermore, if biotin was allowed to bind when the polymer was in the soluble state under UV irradiation, then when the polymer was collapsed by visible light, the streptavidin released the bound biotin. The DMAAm-streptavidin conjugate showed the opposite response, with association of biotin allowed under visible light irradiation and blocked under UV irradiation. The photoresponses of the streptavidin conjugates thus correspond to the original photoresponsive phase transition properties of the polymer switches triggered by the cis-trans isomerization of the diazo chromophores.

INTRODUCTION

An important aspect of many technologies that utilize biomolecular components is the control of their recogni* Correspondence should be addressed to Allan S. Hoffman, Department of Bioengineering, Box 352255, University of Washington, Seattle, WA 98195. Telephone: 206-543-9423, fax: 206-543-6124, e-mail: [email protected], and Patrick S. Stayton, Department of Bioengineering, Box 352125, University of Washington, Seattle, WA 98195, Telephone: 206685-8148, Fax: 206-685-8256, E-mail: [email protected].

tion properties. For example, changes in solution conditions such as temperature or pH are commonly used to release target antigens from antibodies. These solution changes are often damaging to the biomolecules, and there is thus considerable interest in developing molecular switches that control recognition processes with mild signals. We have been developing molecular switches composed of stimuli-responsive polymers that control protein recognition processes in response to small changes in pH and temperature. Light represents another attrac-

10.1021/bc010057q CCC: $22.00 © 2002 American Chemical Society Published on Web 07/19/2002

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Figure 1. Schematic illustration showing the use of different wavelengths of light to switch the bioactivity of a protein “on” or “off”, as provided by switching the conformation of a photoresponsive polymer that has been conjugated at a specific site near the active site of the protein.

tive signal because it would trigger these switches without requiring changes in solution conditions, and it is externally reversible; thus, it is amenable to device design and automation. The control of enzymatic reactions and ligand affinity with light has been actively investigated by many researchers (1-5). In previous studies, chemical modification of biomolecules by direct conjugation of photochromomes such as azobenzene (1-3) or spiropyran (3-5) have been reported and have provided some photoregulation of catalytic activities and affinity binding processes. Although these bioconjugates of small molecule photochromes have demonstrated the potential of light regulation, their photoresponsiveness has been relatively weak. Intrinsic problems include (a) the thermally activated reversible isomerization of the photochromes that leads to the dyes existing as a mixture of two distinct physical states/conformations, (b) the limited size of the photochromes, and (c) their conjugation to the protein at random, nonideal positions for switching activity. These results suggested that photoresponsive polymers might be conjugated to proteins at defined sites to control protein function more efficiently than single photochromes (Figure 1). We have described the site-specific conjugation of pH and temperature-sensitive polymers to control ligand association and dissociation in the model streptavidin-biotin system (6-9). These smart polymers change their size and physical properties reversibly between an extended hydrophilic random coil and a collapsed hydrophobic state over narrow changes of the defined stimulus. Here we report that photoresponsive polymers can serve as light-activated switches to reversibly control biotin-binding by site-specific conjugation to a critical site on streptavidin. The photoresponsive polymers rely on the cis-trans isomerization of diazobenzene chromophores to trigger the change in physical properties. The change in properties is reversible with distinct visible or far UV wavelengths of light, and these bioconjugates can thus be activated and deactivated by external light. EXPERIMENTAL PROCEDURES

Materials. 4-Aminoazobenzene and 4-hydroxyazobenzene (Aldrich, Milwaukee, WI) were recrystallized from ethanol and water and dried in vacuo. N,N-Dimethylacrylamide (DMA) (Fluka, Milwaukee, WI), diethyl ether, acryloyl chloride, triethylamine, and 2-mercaptoethanol (MEO) (Aldrich, Milwaukee, WI) were purified by distillation under reduced pressure. 2,2′-Azobisbutyronitrile (AIBN) (J. T. Baker, Phillipsburg, NJ) was recrystallized from methanol. Ethanol, dimethylformamide (DMF), tetrahydrofuran (THF), methylene chloride, potassium ter-butoxide, divinyl sulfone (DVS), ethylenediaminetet-

Shimoboji et al.

raacetic acid (EDTA), 4-phenylazomaleinanil (PAM) (Aldrich, Milwaukee, WI), tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (Pierce, Rockford, IL), Microcon-30 (Millipore, Bedford, MA), and [3H]Biotin (d-[8,93 H(N)]-Biotin, Dupont, Wilmington, DE) were used as received. All other reagents were of analytical grade. Azobenzene Monomer Synthesis. Two azobenzenecontaining monomers, N-4-phenylazophenylacrylamide (AZAAm) and 4-phenylazophenyl acrylate (AZAA), were synthesized. 4-Aminoazobenzene or 4-hydroxyazobenzene (0.2 mol) and triethylamine (0.26 mol) were dissolved in diethyl ether (200 mL). Acryloyl chloride (0.24 mol) dissolved in 80 mL of diethyl ether was added dropwise at 0 °C under nitrogen atmosphere with stirring. The reaction mixture was allowed to come to room temperature and stirred for 4 h. The reaction solution was filtered to remove triethylammonium chloride, washed with water, and evaporated. The product was recrystallized from ethanol-water mixture and dried in vacuo. The structures were confirmed by 1H NMR (d-DMSO). Photoresponsive Polymer Synthesis. Two photoresponsive polymers with hydroxyl termini, DMAAm (DMA-co-AZAAm copolymer) and DMAA (DMA-co-AZAA copolymer), were synthesized by chain transfer initiated free-radical polymerization in DMF at 60 °C for 20 h, using MEO as a chain transfer regent and AIBN as an initiator (monomer concentration ) 2 mol/L, monomer/ MEO/AIBN ) 100/1/0.2 molar ratio). The feed ratios of AZAAm and AZAA were 7.0 mol % and 4.0 mol %, respectively. The products were purified by precipitation into diethyl ether three times and dried in vacuo. The contents of AZAAm or AZAA incorporated in the copolymers were determined by 1H NMR (Spectrospin & Bruker, dpx200), comparing the ratio of aromatic and aliphatic hydrogens. The molecular weights (MWs) of the polymers were determined by gel permeation chromatography (GPC, Water, Styragel HR3 and 4) in THF, using polystyrene as standards. The hydroxyl terminus of DMAA or DMAAm was converted to a vinyl sulfone group for conjugation to the protein thiol. DMAA and DMAAm were dissolved in 20 mL of methylene chloride with 0.03 g of potassium tert-butoxide and 100 µL of divinyl sulfone (DVS) (DVS/OH ) 10/1 molar ratio). The solution was stirred for 12 h at room temperature under nitrogen atmosphere. The polymers were precipitated, washed in diethyl ether, and dried in vacuo. Conjugation of Polymers to Streptavidin. The streptavidin mutant E116C was constructed by the same method as reported previously (7). Conjugation of streptavidin (E116C) and polymers (DMAA-VS, DMAAm-VS) were carried out in 100 mM sodium tetraborate buffer, pH 8.0, containing 50 mM sodium chloride and 5 mM EDTA. Cysteine residues in E116C form disulfide bonds during storage. To reduce disulfide bonds to sulfhydryl groups, TCEP at 50-fold molar excess to the mutants was added into the solution, which was then rotated at room temperature for 20 min. The DMAA-VS or DMAAm-VS was added in 50-fold molar excess to the mutants and reacted for 1 h at room temperature, followed by the reaction for 24 h at 4 °C. The conjugates were separated from the unconjugated mutants by thermally induced precipitation at 60 °C. The unconjugated protein was retained in the supernatant. The thermal-induced precipitation was repeated three times. Conjugation of Azobenzene to Protein. PAM was reacted with cysteine in E116C in solution at pH 7.0 in phosphate-buffered saline, with TCEP as a disulfide reducing agent, mixing a 50:1 molar excess of 4-phenylazomaleinanil dissolved in DMF (10 vol % of protein

Ligand Photoswitching in Polymer−Protein Conjugate

Figure 2. Photoresponsive polymers, DMAAm and DMAA, with vinyl sulfone end groups.

solution). The conjugate was purified from the unreacted PAM by ultrafiltration. The MW of the conjugate was determined as 13523 by electrospray ionization mass spectrometry (ESI-MS), which corresponds to one azobenzene conjugated to one monomer of E116C SA. Photoirradiation. A UV Cure Lamp (Thorlabs, Newton, NJ) was used with a UG-1 band-pass filter (Edmund Scientific, Barrington, NJ) to select UV light between 300 and 400 nm, and a Fiber-Lite Illuminator (Edmund Scientific, Barrington, NJ) was used with a VG-1 bandpass filter to select for wavelengths greater than 420 nm. Biotin-Binding Assay. [3H]Biotin (d-[8,9-3H(N)]-Biotin, Dupont, Wilmington, DE) was employed to quantitate biotin binding. A 10 µg amount of the conjugate was suspended in 1200 µL of 100 mM of sodium phosphate buffer, pH 7.4. A mixture of 3H-biotin and unlabeled biotin (100 µL) was added into the conjugate solution (total volume was 1 mL) after incubation at 52 °C for 1 h. The ratio of total biotin/biotin binding sites was 1.5 (molar ratio). The solution temperature was reduced to 4 °C stepwise, incubating for 15 min at each temperature. A 50 µL aliquot was taken at the end of each incubation. The unbound biotin was separated from the bound biotin by ultrafiltration through a Microcon30 (MW cutoff: 30 kDa) at the incubation temperature of the solution. A 30 µL aliquot of the filtrate, which contained unbound biotin, was measured in a β-counter (LS7000 Liquid scintillation system, Beckman Instruments, Inc. Fullerton, CA), to determine the unbound biotin. The biotin binding could be quantitated by the decrease of free biotin in the solution. RESULTS AND DISCUSSION

Temperature- and Light-Dependent Polymer Properties. We have prepared two different copolymers

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that display both temperature- and light-sensitive properties. These copolymers differ mainly in the type of bond (ester vs amide) by which the azobenzene groups are attached to the main backbone chain. (Figure 2) These copolymers are similar to previously described photoresponsive polymers (10-15). The temperature- and lightresponsive properties of copolymers of N-isopropylacrylamide (NIPAAm) and N-4-phenylazophenylacrylamide (AZAAm) have been characterized by Irie and co-workers (10, 11). Kro¨ger et al. (12, 13) reported the largest differences in lower critical solution temperature (LCST) detected in aqueous solution using N,N-dimethylacrylamide (DMA)-co-4-phenylazophenyl acrylate (AZAA) copolymer. The photoresponsive properties of the copolymers described here depend on temperature, and conversely, the temperature-responsive properties depend on whether the diazo chromophore is in the cis or trans state. Previous work of Morishima and co-workers (14, 15) suggests that hydrophobic interactions of pendant groups such as pyrene and cholesterol will predominantly be intramolecular if the group is attached to the backbone via amide bonds, and intermolecular if it is attached via ester bonds. The trans configuration of azobenzene also permits stacking of the azobenzene moieties, which will enhance such hydrophobic interactions in the trans state. Thus, these very different hydrophobic interactions of the pendant azobenzene groups will depend on whether the groups are attached via ester or amide bonds, and this may explain the opposite visible vs UV photoresponses seen here for the DMAA and DMAAm copolymers. This is demonstrated for the free copolymers in Figure 3, which shows the temperature dependence under visible or UV irradiation of the phase separation. The UV light had a peak at 350 nm and a range from 300 to 400 nm, and the visible light had a peak at 520 nm while light less than 420 nm was filtered. When the LCST of the polymer was determined as the temperature of 10% of maximum biotin-binding, the DMAA and DMAAm exhibited LCST’s under visible irradiation of 37 °C and 39 °C, respectively. Upon UV irradiation, the LCST of the DMAA increased to 43 °C, and that of the DMAAm decreased to 34 °C. The LCST transition of these polymers is relatively broad, and the polymers exhibited a maximal difference in the degree of phase separation under UV vs visible irradiation at around 40 °C. A significant feature of these diazo-chromophore-based polymers is that the thermal reconversion of the cis-state to the trans-state is not simultaneously accompanied by a conversion of the polymer from the collapsed state to extended state, or vice versa. This conformational con-

Figure 3. Effect of photoirradiation on the phase transition temperature of DMAA (AZAA: 5.9 mol %) and DMAAm (AZAAm: 9.6 mol %). The absorbance was measured at 600 nm. The polymer concentration was 2 mg/mL in 100 mM PB pH 7.2. The rate of heating was 0.5 °C/min. The UV and visible (vis) light irradiations were for 10 min and 3 h, respectively, before the measurement.

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Figure 4. Three-dimensional model of streptavidin with a cysteine at amino acid position 116, showing the spacial relationship between the biotin-binding site and the cysteineSH polymer conjugation site on E116C SA (16).

version of the polymer happens very slowly, on the time scale of days, unlike many diazo compounds where the cis-trans reconversion rate can often be fast, depending on the temperature. Conjugation of Photoresponsive Polymers to Streptavidin. The general design of the streptavidin-

Shimoboji et al.

polymer conjugate for light-responsive switching is schematically shown in Figure 1. We have found previously that the E116C SA mutant locates the unique thiol residue in an appropriate position to control both biotin binding and release (7, 16). This conjugation site is on the loop close to Trp120, which is critical for biotinbinding (17) (Figure 4). The polymers were specifically conjugated to E116C by the reaction of the vinyl sulfone group of the polymer with the thiol group of the protein under conditions that favored the vinyl group addition to the -SH over the -NH26. The conjugates were separated from the unconjugated E116C by thermally induced precipitation. The conjugation efficiency was estimated by the depletion of the mutant after thermally induced precipitation as 12-20%. For the E116C conjugates reported here, we have utilized two types of photoresponsive polymers (Figure 2) that exhibit phase transitions in aqueous solution in response to UV or visible light irradiation: (1) N,N-dimethylacrylamide (DMA)-co-4-phenylazophenyl acrylate (“AZAA”) copolymer (“DMAA”) (MW: 18 kDa, AZAA: 5.9 mol %) and (2) DMA-co-N-4-phenylazophenyl acrylamide (“AZAAm”) copolymer (“DMAAm”) (Mw: 10 kDa, AZAAm: 9.6 mol %) (Shimoboji et al., manuscript submitted). These polymers also exhibit thermoresponsive phase transitions in aqueous solutions (16). The DMAA polymer becomes soluble upon UV irradiation and insoluble upon visible irradiation, while the DMAAm polymer becomes soluble upon visible light and insoluble upon UV light. Thus, they

Figure 5. Photoresponsive switching of streptavidin biotin-binding activity. (A) The biotin binding activities for the SA-DMAA and SA-DMAAm conjugates were compared to controls of their physical mixtures, an SA-AZ conjugate, and SA (E116C), in response to UV/VIS cyclic irradiation under isothermal conditions at 40 °C. The biotin-binding activity represents the percentage change in the biotin-binding capacity of the conjugates from 4 °C to 52 °C, assuming 100% binding at 4 °C and 0% binding at 52 °C. (B) The normalized biotin-binding activities for SA-DMAA and SA-DMAAm as a function of temperature, under constant visible (>420 nm) or far-UV (360 nm) light irradiation. The biotin-binding activity represents the percentage change in the biotin-binding capacity of the conjugates with 100% normalized to maximal binding and 0% normalized to the minimal binding activity for each of the conjugates.

Ligand Photoswitching in Polymer−Protein Conjugate

exhibited opposite photoresponses, as shown in Figure 3 and discussed above. Photoresponsive Switching of Biotin Binding Activity. In order to characterize the time dependence of biotin association and release as a function of the photo-dependent polymer transition, biotin binding and release studies were carried out under isothermal conditions at 40 °C where the polymers displayed their maximum photoresponsiveness. As a control, azobenzene (AZ) was also conjugated site-specifically to E116C (SAAZ conjugate). Figure 5a shows how UV/VIS photocycling alters the biotin binding and release properties of the SA-DMAA and SA-DMAAm conjugates. The unconjugated SA (E116C), a physical mixture of SA and the DMAA or DMAAm, and the SA-AZ conjugate did not exhibit any significant alteration in biotin binding affinity as a function of light irradiation. However, the SADMAA conjugate exhibited a 47% greater increase in biotin-blocking or release with VIS light than with UV light. Conversely, the SA-DMAAm conjugate displayed a 38% greater increase in the blocking of biotin association, and the release of bound biotin, with UV irradiation compared to VIS irradiation. These opposite photoresponsive phenomena agree with the photoresponsive phase transitions of the original polymerssi.e., DMAA collapses and becomes insoluble under VIS irradiation, while DMAAm collapses and becomes insoluble under UV irradiation. The temperature dependence of the photoswitch was also characterized and is shown in Figure 5b. The general trends follow the LCST behavior of the free polymers (Figure 3), but are shifted to slightly higher temperatures for the streptavidin conjugates. Mechanism of Photoresponsive Switches. The time-dependence for the release of the bound biotin was measured, and the dissociation rate was determined to be 1.3 × 10-4 (s-1) at 52 °C. This is within experimental error of the native streptavidin dissociation rate at this temperature. This result implies that biotin dissociates at the normal off-rate when the photoresponsive polymers are in the collapsed state near the binding pocket, but reassociation of biotin with streptavidin is blocked. The collapsed polymer does not interfere with dissociation or induce a faster off-rate through steric interactions at the binding pocket, but it does sterically retard access of free biotin to the binding pocket. The diazo monomers are not capable of modulating the access of biotin to the pocket when conjugated to E116C SA, consistent with the need for the larger polymer to achieve this steric blocking mechanism. CONCLUSIONS

We have described the use of thermal and photoresponsive diazo-containing polymers that provide photoswitching of ligand-binding properties when conjugated near a protein’s recognition binding site. This provides a general approach to the design and development of photoswitchable proteins, where function is controlled by external light signals of distinct wavelengths. In addition to the control of ligand capture and release, this approach should be applicable to a wide-range of technologies, such as photoswitching of enzyme-substrate catalytic reactions, and the optical blocking and de-blocking of recognition sites for light-based patterning of molecules and cells.

Bioconjugate Chem., Vol. 13, No. 5, 2002 919 ACKNOWLEDGMENT

This work was supported by NIH Grant No. GM53771. LITERATURE CITED (1) Willner, I., Rubin, S., and Zor, T. (1991) Photoregulation of R-chymotrypsin by its immobilization in a photochromic azobenzene copolymer. J. Am. Chem. Soc. 113, 4013-4014. (2) Liu, D., Karanicolas, J., Yu, C., Zhang, Z., and Woolley, G. A. (1997) Site-specific incorporation of photoisomerizable azobenzene groups into ribonucleases. Bioorg. Med. Chem. Lett. 7, 2677-2680. (3) Willner, I., and Rubin, S.(1996) Control of the structure and functions of biomaterials by light. Angew. Chem., Int. Ed. Engl. 35, 367-385. (4) Willner, I., Rubin, S., Shatzmiller, R., and Zor, T. (1993) Reversible light-stimulated activation and deactivation of R-chymotrypsin by its immobilization in photoisomerizable copolymers. J. Am. Chem. Soc. 115, 8690-8694. (5) Rathi, R. C., Kopeckova, P., and Kopecek, J. (1997) Biorecognition of sugar containing N-(2-hydroxypropyl)methacrylamide copolymers by immobilized lectin. Macromol. Chem. Phys. 198, 1165-1180. (6) Stayton, P. S., Shimoboji, T., Long, C., Chilkoti, A., Chen, G., Harris, J. M., and Hoffman, A. S. (1995) Control of protein-ligand recognition using a stimuli-responsive polymer. Nature 378, 472-474. (7) Ding, Z. L., Long, C., Hayashi, Y., Bulmus, E. V., Hoffman, A. S., and Stayton, P. S. (1999) Temperature control of biotin binding and release with a streptavidin-poly(N-isopropylacrylamide) site-specific conjugate. Bioconjugate Chem. 10, 395400. (8) Bulmus, E. V., Ding, Z., Long, C. J., Stayton, P. S., and Hoffman, A. S. (2000) A pH- and temperature-sensitive copolymer-streptavidin site-specific conjugate for pH-controlled binding and triggered release of biotin. Bioconjugate Chem. 11, 78-83. (9) Ding, Z., Fong, R. B., Long, C. J., Hoffman, A. S., and Stayton, P. S. (2001) “Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield” Nature 411, 59-62. (10) Kungwatchakun, D., and Irie, M. (1988) Macromol. Chem. Rapid Commun. 9, 243. (11) Irie, M., and Kungwatchakun, D. (1992) Proc. Jpn. Acad. 68, 127. (12) Kro¨ger, R., Menzel, H., and Hallensleben, M. L. (1994) Macromol. Chem. Phys. 195, 2291. (13) Menzel, H., Kro¨ger, R., and Hallensleben, M. L. (1995) Macromol. Rep. A32, 779. (14) Morishima, Y., Seki, M., Nomura, S., and Kamachi, M. (1994) Amphiphilic Polyelectrolytes and their Coulombic Complexes with Surfactants as Novel Photochemical Systems, pp 243-256, ACS Symposium Series 548, American Chemical Society: Washington D.C. (15) Yusa, S., Kamachi, M., and Morishima, Y. (1998) Hydrophobic self-association of cholesterol moieties covalently linked to polyelectrolytes: Effect of spacer bond. Langmuir 14, 6059-6067. (16) Shimoboji, T., Ding, Z., Stayton, P. S., and Hoffman, A. S. (2001) Mechanistic investigation of smart polymer-protein conjugates. Bioconjugate Chem. 12, 314-319. (17) Chilkoti, A., Tan, P. H., and Stayton, P. S. (1995) Sitedirected mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120. Proc. Natl. Acad. Sci. U. S. A. 92, 1754-1758.

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