Thermoresponsive Controlled Association of Protein with a Dynamic

Nov 3, 2004 - Kyoto University, Yoshida-Hommachi, Sakyo-ku, Kyoto 606-8501, Japan, Depertment of ... Tokyo Medical and Dental University, Japan...
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Biomacromolecules 2005, 6, 447-452

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Thermoresponsive Controlled Association of Protein with a Dynamic Nanogel of Hydrophobized Polysaccharide and Cyclodextrin: Heat Shock Protein-Like Activity of Artificial Molecular Chaperone Yuta Nomura,† Yoshihiro Sasaki,† Masaki Takagi,‡ Tadashi Narita,‡ Yasuhiro Aoyama,† and Kazunari Akiyoshi*,§,| Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Hommachi, Sakyo-ku, Kyoto 606-8501, Japan, Depertment of Materials Science and Engineering, Graduate School of Engineering, Saitama Institute of Technology, 1690 Fusaiji, Okabe, Saitama 369-0293, Japan, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, Japan, and Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Japan Received August 22, 2004

Dynamic CHP-CD nanogels, which consisted of a self-assembly of cholesteryl-group-bearing pullulan (CHP) and β-cyclodextrin (CD), were characterized by SEC and SEC-MALS methods. The nanogels prevented the thermal aggregation of carbonic anhydrase B (CAB) by selective trapping of the heat-denatured protein. After the complex between the CHP-CD nanogels and CAB was cooled, the enzyme activity of CAB spontaneously recovered upon release from the complex. The dynamic nanogels self-regulated an association of heat denatured protein and dissociation of native protein depending on the concentration of CD. The thermal stability of CAB was improved by thermoresponsive controlled association between the proteins and the artificial molecular chaperone. Introduction The dynamic self-assembly of biopolymers can be controlled by binding to suitable effector molecules or by coupling with enzymatic reactions. Chaperonin GroEL, for example, which is a well-investigated family of molecular chaperones, is a dynamic self-assembly that controls protein folding.1,2 GroEL acts as a host that can selectively bind heatdenatured proteins or a folding intermediate in the central cavity of its cage-like structure to prevent protein aggregations. The host GroEL then changes its conformation upon binding ATP and the co-chaperone. Finally, the denatured protein folds within the cavity of the GroEL and is released as a native protein. This host-guest interaction is controlled by cyclic conformational changes in GroEL upon the binding of ATP and the hydrolysis of ATP to ADP.3-6 This process is an excellent example of a molecular nanomachine. Molecular chaperone systems have inspired us to explore new concepts in designing artificial molecular chaperones to assist protein folding.7-15 To simulate the function of molecular chaperones, the design of an artificial host with a * To whom correspondence should be addressed. Fax number: +81-35280-8020. E-mail: [email protected]. † Kyoto University. ‡ Saitama Institute of Technology. § Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University. | Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University.

nano-cage to bind denatured proteins and the control of the dynamics of catching and releasing proteins are indispensable. Interest in stimuli-sensitive hydrogels as functionally smart materials for biotechnological and biomedical applications has been growing.16,17 We designed self-assembled nanogels as artificial molecular chaperones.18-20 Hydrophobized polysaccharides such as the cholesteryl-group-bearing pullulan (CHP) spontaneously form nanogels in water by intermolecular self-aggregation.21-25 In addition, the amphiphilic nanogels form complexes with various soluble proteins or enzymes in water and behave as hosts for guest proteins.26-29 In a previous communication, we reported that the irreversible aggregation of carbonic anhydrase B (CAB) upon heating was prevented by complexation between the heat-denatured enzyme and the CHP nanogels. 18,19 The complexed CAB was released as a native form by dissociation of the CHP nanogels upon the addition of β-cyclodextrin. In the artificial system, the capture and release of proteins are sequential and separate processes “two-step”. In a molecular chaperone system, however, the two processes periodically and dynamically proceed in “one-pot” in the presence of ATP and the co-chaperone. Here, we described property of dynamic nanogels, which is a host-guest complex of CHP and cyclodextrins (CD). The function of one-pot system using dynamic nanogels as artificial molecular chaperon was compared with two-step system. The dynamic nanogels of CHP-CD self-regulated an association of heat denatured protein and dissociation of native protein depending on the concentration of CD, and they showed high

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heat shock protein-like activity as one-pot system of artificial molecular chaperone. Experimental Section Materials. The CHP, which was substituted with 1.4 cholesteryl groups per 100 glucose units of the parent pullulan (Mw 108 000), was obtained as previously reported. Dimethyl sulfoxide (DMSO, analytical grade), R-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were purchased from Wako Pure Chemical Industry, Co. Ltd. Hydroxy-propyl-βcyclodextrin (HP-β-CD) was purchased from Mitsubishi Chemicals. Methyl-β-cyclodextrin (Methyl-β-CD) was purchased from Tokyo Kasei Kogyo. Bovine carbonic anhydrase (CAB) and p-nitrophenyl acetate (p-NPA) were purchased from SIGMA. Methods. Preparation of CHP Nanogels. Cotton-like flakes of dried CHP were weighed and dissolved in 50 mM Tris-sulfate buffer (pH 7.5) for 6-12 h at 50-60 °C. The solution was then sonicated using a probe-type sonifier at 40 W in water for 10 min (TOMY UR200P, tip diameter 2 mm.). The resulting suspension was filtered through three different types of membrane filters (Super Acrodisc 25, Gelman Science, pore size 1.2 µm, 0.45 µm, and 0.2 µm). The final result was a clear aqueous suspension of CHP nanogels. Thermal Stability of Carbonic Anhydrase B (CAB). CAB (Mw 30 000) is a monomeric enzyme. This protein has no disulfide bridge. Therefore, the principal interactions during denaturation would be the breaking and making of hydrogenbonding and hydrophobic interactions. The protein concentration of native CAB in 50 mM Tris-sulfate buffer (pH 7.5) was determined by its absorbance at 280 nm using an extinction coefficient of 1.83 (mg/mL protein)-1 cm-1. The CAB activity was determined using a pNPA esterase assay in which 20 µL of 100 mM pNPA in dry acetonitrile was added to 0.8 mL of assay solution to make a solution of 59-9.8 mg/mL CAB, 50 mM Tris-sulfate (pH 7.5), and 2.4 mM pNPA. After mixing for 10 s, the increase in the hydrolysis of product p-nitrophenolate was monitored by measuring the increase in absorbance at 400 nm as a function of time. The absorbance of the solution was measured every 0.5 s for 2 min by U-2010 (HITACHI, Tokyo, Japan). The yield of reactivated CAB was determined by comparison to the initial rate of pNPA hydrolysis by the native enzyme at the same concentration. For typical thermal denaturation, sample aliquots containing CAB (0.06, 0.03, or 0.01 mg/ mL) in 50 mM Tris-sulfate (pH 7.5) were heated at 70 °C for 10 min and then allowed to cool for 10 min. After the mixture was allowed to stand at 25 °C for 24 h, the enzyme activity was checked. High Performance Size Exclusion Chromatography (HPSEC). The HPSEC system (Tosoh, Tokyo, Japan) consisted of a CCPS dual pump (Tosoh, Tokyo, Japan), a SD-8022 degasser (Tosoh, Tokyo, Japan), a CO-8020 column oven (Tosoh, Tokyo, Japan), a RI-8020 R.I. detector (Tosoh, Tokyo, Japan), a UV-8020 UV detector (Tosoh, Tokyo, Japan), and a MALS detector (DAWN DSP, Wyatt Technology, Santa Barbara, CA). All of the sample that was applied

Figure 1. Chemical structure of cholesteryl-group-bearing pullulan (CHP).

to the column (TSK-Gel-4000SWXL column, Tosoh, Tokyo, Japan) was passed through a 0.22 µm filter (Ekicrodisk 3, Gelman Science Japan, Ltd.) before injection. The sample mixture was eluted with 50 mM Tris-sulfate buffer (pH 7.5) in the absence and presence of cyclodextrins. The flow rate was 0.75 mL/min. Elution of the sample aliquot was monitored by UV (at 280 nm), R.I., and MALLS detector. The molecular weight and the z-average root-mean-square radius of gyration were determined using ASTRA software based on Zimm’s equation. We used 0.142 mL/g as the value of refractive index increment (dn/dc) of CHP. Results and Discussion Interaction between CHP Nanogels and CAB: TwoStep System. The preparation and characterization of CHP nanogels have been reported elsewhere.21,22 Cholesterylgroup-bearing pullulan (CHP, Figure 1) form nanogels by self-assembly in water. The domains of the associated cholesteryl groups of CHP provide noncovalent hydrophobic cross-linking points in the gel structures. The aggregation number of cholesteryl moieties in one hydrophobic domain is 4-5. The average hydrodynamic radius (Rh) of the nanogels used in this experiment is approximately 18 nm by dynamic light scattering. Carbonic anhydrase B (CAB) was selected as a model enzyme to evaluate heat shock protein-like activity. The heatinduced aggregation of CAB (molecular weight 30 000, 0.06 mg/mL) was investigated by the light scattering method at 400 nm in the absence and presence of CHP nanogels. When CAB was heated at 63 °C, which is above its denaturation temperature (55 °C), aggregation began within 5 min. Upon partial unfolding of the protein under heating, the hydrophobic portions of globular proteins are exposed. The aggregation of CAB was significantly prevented in the presence of CHP nanogels. When the molar ratio of CHP nanogels to CAB was above 0.2, the solution was optically clear even after heating at 63 °C for 40 min. In contrast, the parent pullulan did not show the same behavior even in the presence of higher concentrations of pullulan. Quantitative complexation between CAB and CHP nanogels was confirmed by high-performance size-exclusion chromatography (Figure 2). Figure 2a shows a chromatograph of the mixture of CHP nanogels (1.6 mg/mL) and native CAB (0.06 mg/ mL) at 25 °C. The CHP nanogels did not complex with the native CAB at 25 °C even after incubation for 32 h. The

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Figure 2. HPSEC analysis of the interaction between CHP nanogels (2 µM) and CAB (2 µM) in a two-step system. (a) Mixture of CHP and CAB at 25 °C after incubation in 50 mM Tris-sulfate buffer (pH 7.5) for 32 h. (b) After incubation of CHP and CAB at 70 °C for 10 min, then cooled to 25 °C in 50 mM Tris-sulfate buffer (pH 7.5). (c) After addition of β-CD (5 mM) to CHP-CAB complex samples and incubation at 25 °C for 24 h in 50 mM Tris-sulfate buffer (pH 7.5). All measurements were performed on an HPSEC system (Tosoh, Tokyo, Japan) and a Superdex 200 HR column (Pharmacia) using 50 mM Tris-Sulfate (pH 7.5) as an elution buffer at 25 °C. The flow rate was 1.0 mL/min.

Figure 3. Recovery of enzyme activity of CAB after addition of various CDs to CHP nanogel-CAB complexes as a function of the concentration of various types of cyclodextrins (two-step system) CAB was heated to 70 °C for 10 min in the presence or absence of CHP nanogels (4.83 mg/mL). After denaturation, the sample solution was cooled to 25 °C and allowed to stand for 2 h at 25 °C. Two hours latter, various cyclodextrins were added to the solution and then allowed to stand for 24 h at 25 °C. The yield of the reactivated CAB was determined by comparing the initial rate of enzyme activity with that of the native CAB. The enzyme activity of CAB was determined by a p-nitrophenyl acetate hydrolysis assay. A total of 20 µL of 100 mM pNPA solution was added to 0.8 mL of the final concentration of the CAB solution (0.01 mg/mL). The increase in absorbance at 400 nm was measured as a function of time.

mixture was incubated at 70 °C for 10 min and then cooled to 25 °C. As shown in Figure 2b, the CHP nanogels complexed with the CAB. No spontaneous dissociation of CAB was observed even after the mixture was maintained for a week at room temperature. Thus, the CHP nanogel prevented protein aggregation by trapping the denatured CAB. This effect is similar to that of a molecular chaperone in nature. The dynamics of molecular chaperones are controlled by the binding of suitable effectors such as ATP and cochaperones.3 In our artificial system, cyclodextrins were used as modulators of the structure of nanogels. The main driving force of the self-aggregation of CHP is the association of the hydrophobic cholesteryl groups of CHP in water. Cyclodextrins can solubilize various hydrophobic compounds in water by incorporation into hydrophobic cavities.23 Since the cholesteryl group is a suitable guest for β-CD,30 the CHP nanogels dissociated upon complexation with β-cyclodextrin (β-CD) to yield a dissociated CHP-CD complex. Dissociation of the CHP nanogel-protein complex by cyclodextrin subsequently induced the release of CAB (Figure 2c). Figure 3 shows the recovery of the enzymatic activity of CAB after the addition of various types and concentrations of cyclodextrins. Various concentrations of cyclodextrins were added to the CHP-CAB complex, which was prepared by mixing CHP nanogels and CAB at 70 °C for 10 min. The mixture was kept at 25 °C for 24 h, and then the residual enzyme activity was measured. The enzyme activity recovered in high yields only in the presence of β-CD and its derivatives. The dissociation of the nanogels depended on the concentration and types of cyclodextrins. The affinity of β-CD for cholesteryl is higher than that of both R-CD

and γ-CD. 31 Therefore, β-CD was the most effective CD for achieving the dissociation of the CHP nanogels. We also investigated β-CD derivatives such as 2-hydroxy-propyl-βcyclodextrin (HP-β-CD) and methyl-β-cyclodextrin (methylβ-CD). Methyl-β-CD was the most effective refolding reagent probably due to the more effective dissociation of the nanogel-protein complex. Interaction between CHP-CD Nanogels with CAB: One-Pot System. The thermal stability of CAB was investigated in the presence of dynamic equilibrium mixtures of CHP nanogels and β-CD: a one-pot system. A mixture of CAB (0.06, 0.03, or 0.01 mg/mL) and CHP nanogels (4.8 mg/mL) was incubated at 70 °C for 10 min in the presence of various concentrations of β-CD, methyl-β-CD, and HPβ-CD, and then cooled to 25 °C. Recovery of the enzyme activity of CAB after incubation for 24 h at 25 °C was investigated (Figure 4). The thermal stability of CAB depended on the concentration of CD. Higher concentrations of β-CD resulted in decreased enzyme activity. It is interesting that the enzyme activity spontaneously recovered up to about 80% after cooling with an appropriate concentration of CD. β-CD systems show especially higher chaperon-like activity in a one-pot system. Figure 5 shows chromatograms of CHP nanogels in the presence of various types and concentrations of cyclodextrins as eluents. In the presence of higher concentrations of CDs in every system, there was a peak in the low molecular weight region which corresponds to a nonaggregated complex of CHP-CD (Mw 1.5 × 105 by SEC-MALLS). This was almost the same molecular weight as the CHP monomer (Mw 1.2 × 105 by SEC-MALLS). As shown Figure 4, the dissociated CHP-CD complex did not show chaperon-like

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Figure 4. Recovery of enzyme activity of CAB (0.06-0.01 mg/mL) after heat denaturation in a one-pot system. In the one-pot system, CAB (0.06-0.01 mg/mL) was heated at 70 °C for 10 min in the presence or absence of CHP-CD nanogels that contained CHP nanogels (4.83 mg/mL) and various types and concentrations of cyclodextrins. After denaturation, the sample solution was cooled to 25 °C and allowed to stand for 24 h at 25 °C. The yield of the reactivated CAB was determined by comparing the initial rate of enzyme activity with that of the native CAB. The enzyme activity of CAB was determined by a p-nitrophenyl acetate hydrolysis assay. A total of 20 µL of 100 mM pNPA solution was added to 0.8 mL of the final concentration of the CAB solution (0.01 mg/mL). The increase in absorbance at 400 nm was measured as a function of time.

Figure 5. HPSEC analysis of CHP nanogels in the presence or absence of various types of β-cyclodextrins. CHP nanogels (4.83 mg/ mL) were mixed with various types and concentrations of β-cyclodextrins. ((a) β-CD, (b) HP-β-CD, and (c) Methyl-β-CD). After dissolution, the CHP nanogel and cyclodextrin solution was applied to the column (TSK-Gel-4000SWXL column, Tosoh, Tokyo, Japan) at 30 °C. The flow rate was 0.75 mL/min. The eluent was Tris-Sulfate (pH 7.5) containing the same types and concentrations of cyclodextrins as those of the sample solutions.

activity. In contrast, especially in the presence of relatively lower concentrations of β-CD, the new peak was observed in the high molecular weight region at a slightly shorter elution time compared to the CHP-nanogels. For example, the molecular weight of the species in the presence of 1 mM of β-CD (Mw 6.8 × 105 [CHP] ) 4.8 mg/mL) did not change much compared to that of the CHP nanogels (Mw

6.0 × 105: [CHP] ) 4.8 mg/mL) by SEC-MALLS. However, Rg of the species significantly increased from 20.8 to 38.0 nm. This indicates that still associated CHP-CD complex may exist in an equilibrium mixture depending on the concentration of cyclodextrins. The associated CHPCD complex may still have a hydrogel structure (CHP-CD nanogels) so that CD partly binds to the CHP-nanogels

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Figure 6. Schematic representation of interaction between CHP nanogels and β-CD (CHP-CD nanogels).

Figure 8. Mean change in residual ellipticity (at 222 nm) as a function of temperature in 50 mM Tris-sulfate buffer (pH 7.5) in the presence of CHP nanogels or CHP-CD nanogels. Native CAB (0.01 mg/mL) was heated to 70 °C from 30 °C at increments of 10 °C/hour in the presence of CHP nanogels ([CHP] ) 4.8 mg/mL) or CHP-CD nanogels ([CHP] ) 4.8 mg/mL, [β-CD] ) 4 mM).

Figure 7. Circular dichroism (c.d.) spectra of CAB in the presence or absence of CHP-β-CD nanogels. (a) Native CAB at 30 °C. (b) Native CAB in the presence of CHP-β-CD (4 mM) nanogels at 30 °C. (c) Denaturation of CAB in the presence of CHP-β-CD (4 mM) nanogels at 70 °C for 10 min and then measured at 70 °C. (d) Denaturation of CAB in the presence of CHP nanogels at 70 °C for 10 min and then measured at 70 °C. (e) Denaturation of CAB in the presence of CHP-β-CD (4 mM) nanogels at 70 °C for 10 min and then cooled to 25 °C. After incubation for 24 h at 25 °C, measured at 30 °C. In these measurements, the CHP concentration was 4.83 mg/ mL.

(Figure 6). That species was not observed in the CHP methylβ-CD system that showed only low chaperon-like activity. The associated CHP-CD complex (CHP-β-CD nanogels) probably plays an important role in increasing higher heatshock activity in the one-pot system. Selective Trapping of Denatured CAB in CHP-CD Nanogels. To obtain further information about the higher activity of a CHP-β-CD one-pot system, a circular dichroism (c.d.) spectroscopic study was performed. The c.d. spectrum of CAB (0.01 mg/mL) in the presence of CHP nanogels ([CHP] ) 4.8 mg/mL) or CHP-β-CD nanogels ([CHP] ) 4.8 mg/mL β-CD 4 mM) upon heating at 70 °C for 10 min are shown in Figure 7. The secondary structure of CAB changed drastically upon the complexation with CHP nanogels (Figure 7c) or CHP-β-CD nanogels (Figure 7d). The c.d. spectra in both systems at 70 °C were nearly the same. CHP-β-CD nanogels probably form a complex with denatured CAB similar to that with CHP nanogels. The secondary structure of native CAB consists of 5.0% R-helical and 35% β-forms. Upon complexation, the R-helical form drastically increased, whereas the β-form decreased. Both hydrogen bonding with the polysaccharide skeleton and hydrophobic interaction with the cholesteryl groups of CHP might be responsible for this increase in the R-helical content. Another possibility might involve the intermediate structure during the refolding of the thermally denatured native protein. Goto et al. previously pointed out that the R-helical intermediate might be a nonhierarchical intermediate of β-sheet proteins

such as CAB.32 Thus, CHP nanogels and CHP-β-CD nanogels might capture the intermediate of CAB in thermal denaturation. In CHP-β-CD nanogels (one-pot system), which show high chaperone-like activity, the change in the mean residual ellipticity was investigated as a function of temperature at 222 nm. CAB mixed with CHP nanogels or CHP-β-CD nanogels was gradually heated from 30 to 70 °C at a rate of 0.17 °C/min (Figure 8). In both cases, no significant spectral changes were observed up to 40 °C. At a higher temperature, however, the θ222 value changed sigmoidally. This temperature coincides with the denaturation temperature of CAB (transition temperature ) 50-60 °C). In the presence of 4 mM β-CD, however, the transition temperatures significantly increased. CHP-β-CD nanogels thermally stabilized the structure of CAB. Both nanogels selectively and effectively complex only with heat-denatured CAB. In this process, the aggregation of protein was effectively prevented. In living systems, molecular chaperones selectively trap denatured protein. This is an important function as a molecular chaperon machine. It is noteworthy that simple amphiphilic nanogels can simulate this function. The hydrophobicity of the surface in denatured protein is higher than that in native protein. Hydrophobic unfolded denatured protein might prefer complexation with amphiphilic nanogels than with hydrophilic compact native protein. By cooling the sample of CHP nanogel-CAB complex, no spectral change was observed in the absence of β-CD. The CHP-CAB complex was stable, as previously mentioned. Upon cooling of the CHP-β-CD nanogel system, however, the value of θ222 spontaneously recovered accompanied by a slight change in the CD spectra (Figure 7e). CHP-β-CD nanogels react more weakly with denatured protein than do CHP nanogels because of the dynamic property of the CHP-β-CD nanogels. Therefore, below the transition temperature, the relatively loose binding of the denatured CAB to the CHP-β-CD nanogels enables it to refold to native CAB. The driving force of the dissociation of the CAB from the CHP-β-CD nanogels might be the different binding constants of the CHP-β-CD nanogels for native and denatured CAB. β-CD regulates the ability of CHP nanogels to bind the proteins.

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Figure 9. Schematic representation of thermostability of CAB in CHP nanogels (two-step system) and CHP-CD nanogels (one-pot system).

In conclusion, we showed that a dynamic equilibrium system of CHP-CD nanogels could control the selective capture of denatured protein and release of protein (Figure 9). The dynamic self-assembled nanogels act as a host for proteins in heat denaturation. Cyclodextrin is an effector molecule that controls the binding ability of the host for the protein. This is a novel example of a thermoresponsive controlled association between a protein and an artificial chaperone. CHP-CD nanogel systems are useful as proteinstabilized reagents. Acknowledgment. This work was supported by a Grantin-Aid for Scientific Research from the Japanese government (No. 15300158). K.A. acknowledges the financial support from Sekisui Chemical Co., Ltd. References and Notes (1) Hartl, F. U. Nature 1996, 381, 571. (2) Ranson, N. A.; White, H. E.; Saibil, H. R. Biochem. J. 1998, 333, 33. (3) Roseman, A. M.; Chen, S.;White, H.; Braig, K.; Saibil, H. R. Cell 1996, 87, 241. (4) Martin, J.; Langer, T.; Boteva, R., Schramel, A.; Horwich, A.; Hartl, F. U. Nature 1991, 352, 36.

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