A Novel Approach for Protein Crystallization by a Synthetic Hydrogel

Apr 2, 2013 - This approach will provide a wide variety of possibilities for protein crystallization in hydrogels. ... Global Challenges 2018 2 (1), 1...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/crystal

A Novel Approach for Protein Crystallization by a Synthetic Hydrogel with Thermoreversible Gelation Polymer Published as part of the Crystal Growth & Design virtual special issue on the 14th International Conference on the Crystallization of Biological Macromolecules (ICCBM14) Shigeru Sugiyama,*,†,‡,# Noriko Shimizu,† Gen Sazaki,†,‡,⊥ Mika Hirose,†,# Yoshinori Takahashi,†,‡ Mihoko Maruyama,†,‡ Hiroyoshi Matsumura,†,‡,§ Hiroaki Adachi,†,‡,§ Kazufumi Takano,†,‡,§ Satoshi Murakami,‡,§,∥ Tsuyoshi Inoue,†,‡,§ and Yusuke Mori†,‡,§ †

Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan Japan Science and Technology Agency, CREST, Suita, Osaka 565-0871, Japan § SOSHO Inc., 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan ∥ Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan ‡

S Supporting Information *

ABSTRACT: Agarose has been utilized in protein crystallization to control nucleation of protein crystals. It reduces convection, prevents crystal sedimentation, and increases tolerance to environmental perturbations, resulting in high-quality protein crystals. However, crystallographers have seldom used agarose hydrogel because it requires preincubating the crystallization solution at high temperatures where a high-temperature-sensitive protein may be inactivated or aggregated. To overcome this disadvantage, we used a thermoreversible gel polymer (TGP) made from synthetic polymer. TGP turns into hydrogel upon warming and liquefies upon cooling. This novel approach enabled us to prepare the crystallization solutions at low temperature (277−283 K) and to crystallize elastase, glucose isomerase, and lysozyme with TGP. We also found that TGP clearly increased the number of elastase, glucose isomerase, and lysozyme crystals. This approach will provide a wide variety of possibilities for protein crystallization in hydrogels.

1. INTRODUCTION

Agarose has been utilized in protein crystallization to control nucleation of the protein crystals.13−15 It reduces convection and prevents crystal sedimentation,16 resulting in high-quality protein crystals.17 In addition, the crystals grown in agarose solution have increased tolerance to environmental perturbations such as evaporation and temperature change as well as any vibration generated during transportation.18,19 We recently developed a new method for growing protein crystals in a high-concentration, high-strength agarose hydrogel.20−27 Furthermore, our recent study demonstrated that this method enabled us to increase the mechanical stability of the crystals while considerably reducing the osmotic shock.28 For instance, the micro-Vickers hardness was estimated to be an average of 21.6 MPa for the crystals grown in agarose hydrogel, but the hardness of solution-grown crystals could not be measured because they broke immediately even under the

High-throughput protein X-ray crystallography offers an unprecedented opportunity to facilitate drug discovery. Already, X-ray crystallography has facilitated the development of drugs for immunosuppression and for treating of HIV, influenza, glaucoma, and cancer.1,2 Structural information of the protein− ligand complex also has the potential to find ways to improve lead compounds. The ability to actually see how lead compounds bind to target proteins makes drug development faster and more cost efficient.1 The best way is to determine the threedimensional (3D) structure of the protein−ligand complex by soaking the ligand in apoprotein crystals.3−5 However, the soaking step poses a problem in many cases because many lead compounds are not readily water-soluble. Such lead compounds must be dissolved in concentrated organic solvents such as alcohol and dimethyl sulfoxide (DMSO). These solutions fatally damage protein crystals by osmotic shock during soaking.6−12 The problem arises from the influence of osmotic shock on crystal packing during soaking. © XXXX American Chemical Society

Received: October 31, 2012 Revised: March 30, 2013

A

dx.doi.org/10.1021/cg301588b | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

minimum load. Also, the hydrogel-grown crystals are more tolerant of temperature changes than those grown in solution in part because hydrogel fibers were incorporated into the crystal during growth, significantly strengthening the crystal. Thus, the series of recent studies of crystal growth in hydrogel raises new possibilities for X-ray protein crystallography.29 However, a high-temperature-sensitive protein may be inactivated or aggregated by the crystallization technique because we need to keep the temperature above 308 K when mixing the protein solution and agarose sol solution.26 To overcome this shortcoming, we used a thermoreversible gelation polymer (TGP)30 made from synthetic polymer instead of agarose hydrogel from natural polymer. TGP is a water-soluble polymer originally developed as a cell culture medium for tissue engineering.31−34 TGP can also be used as a drug delivery vehicle for biologically active substances or chemotherapy.35

40

Agarose turns into hydrogel upon cooling and liquefies upon heating. In contrast, TGP is in the fluid state at low temperature (277−283 K) and is gelated at room temperature or above. Therefore, this novel approach enabled us to prepare crystallization solutions by mixing the protein solution and the TGP sol solution at low temperature (277−283 K), significantly reducing high-temperature damage to the protein. Here, we report the first application of the TGP gel for protein crystallization. To assess the feasibility and efficacy of this novel approach, we crystallized elastase (ELS), glucose isomerase (GI), and lysozyme (LZM) in TGP. These crystals were used for X-ray diffraction (XRD) measurements, followed by a structural analysis. The crystals yielded data statistics comparable to controls obtained by conventional convective solution growth. The resulting electron densities were clear for the entire polypeptide consisting of these model proteins. Additionally, according to our previous study,24 TGP increased the number of ELS, GI, and LZM crystals. We believe that this novel approach for protein crystallization by TGP opens the possibility of accelerating structural studies of protein crystallography and subsequent structure-based drug discoveries.

0.5 M NaCl

2. MATERIALS AND METHODS

0.1 M Na acetate 4.5

2.1. Sample and Gel Preparations. Porcine pancreas ELS (Sigma-Aldrich), Streptomyces rubiginosus GI (Hampton Research), and chicken egg-white LZM (Seikagaku Kogyo) were purchased and

Table 1. Crystallization Conditions in the Presence or Absence of TGP protein

ELS

GI

protein conc 20 10 (mg/mL) precipitant agent 0.1 M (NH4)2SO4 5%(w/v) PEG6000 0.1 M CaCl2 buffer 0.1 M Na acetate 0.1 M Tris-HCl pH 5.0 7.0

LZM

Table 2. Comparison of Crystal Data between TGP-Grown and Solution-Grown Crystals protein crystals grown in cryoprotectant source Beamline space group unit-cell parameters (Å) a b c resolution (Å) (high-resolution shell) no. of reflections oscillation angle (°) total rotation angle (°) no. of unique reflections redundancy ⟨I/σ(I)⟩ completeness (%) Rmerge (%)a mosaicity (°) refinement resolution range (Å) no. of reflections Rcrystb Rfreec no. of water molecules RMSD bond length (Å) RMSD bond angle (°)

ELS TGP

TGP

GI solution

TGP

TGP

LZM solution

TGP

TGP

solution

5 M Li acetate PF BL17A P212121

30% glycerol 30% glycerol 5 M Li acetate PF SPring-8 PF BL17A BL44XU BL17A P212121 P212121 I222

30% glycerol 30% glycerol 5 M Li acetate PF SPring-8 SPring-8 BL17A BL44XU BL44XU I222 I222 P43212

30% glycerol 30% glycerol SPring-8 BL44XU P43212

PF BL5A P43212

49.8 58.0 74.2 50−0.97 (0.99−0.97) 898,138 1.0 200 126,105 7.1 (6.2) 10.8 99.0 (99.6) 5.8 (37.6) 0.15

50.0 57.9 74.4 50−1.03 (1.05−1.03) 716,150 1.0 250 107,164 6.7 (4.5) 8.9 99.7 (99.3) 8.5 (40.5) 0.15

50.0 58.2 74.3 50−1.03 (1.05−1.03) 742,614 1.0 180 105,754 7.0 (7.0) 12.2 97.9 (100) 5.8 (32.0) 0.10

93.0 97.5 102.1 50−1.13 (1.15−1.13) 584,889 1.0 90 164,540 3.6 (2.4) 8.2 95.5 (71.4) 6.9 (36.2) 0.11

92.7 97.6 102.2 50−1.15 (1.17−1.15) 1,067,292 1.0 200 163,696 6.5 (5.3) 8.3 99.8 (99.7) 8.5 (37.2) 0.19

93.2 98.2 102.4 50−1.13 (1.15−1.13) 818,182 0.4 170 161,858 5.1 (3.9) 9.3 93.6 (93.5) 8.3 (35.8) 0.34

78.1 78.1 37.2 50−1.10 (1.12−1.10) 384,646 1.0 120 46,839 8.2 (7.7) 8.2 99.1 (99.9) 10.2 (39.3) 0.17

78.1 78.1 37.0 50−1.18 (1.22−1.18) 403,870 1.5 135 38,059 10.6 (7.9) 12.5 99.0 (81.9) 5.4 (36.7) 0.17

77.1 77.1 38.2 50−1.21 (1.23−1.21) 371,013 1.0 180 35,780 10.4 (6.2) 10.0 99.9 (99.6) 5.4 (37.5) 0.26

26.48−0.97 119,481 0.18 0.20 167 0.028 2.2

45.74−1.03 101,516 0.18 0.19 190 0.025 2.1

25.04−1.03 100,183 0.18 0.20 230 0.025 2.1

40.68−1.13 156,028 0.16 0.18 360 0.032 2.6

40.69−1.15 155,167 0.16 0.18 317 0.029 2.3

35.46−1.13 151,822 0.21 0.22 305 0.029 2.2

27.61−1.10 44,271 0.18 0.19 125 0.026 2.2

27.64−1.18 35,998 0.18 0.20 110 0.026 2.1

38.56−1.21 33,865 0.19 0.22 126 0.026 2.2

Rmerge = ∑hkl∑i |Ii(hkl) − |/ ∑hkl∑i Ii(hkl), where Ii(hkl) is the ith observed intensity of reflection hkl and is the average intensity over symmetry-equivalent measurements. Values in parentheses are for the highest resolution shell. bRcryst =∑∥Fo| − |Fc∥/∑|Fo| calculated from 95% of the data, which were used during refinement. cRfree =∑∥Fo| − |Fc∥/∑|Fo| calculated from 5% of the data, which were used during refinement. a

B

dx.doi.org/10.1021/cg301588b | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

used without further purification. TGP called Mebiol gel30−33 was used in this study. Mebiol gel was purchased from Mebiol Inc. (Kanagawa, Japan, through Ikeda Scientific Co. Ltd., Tokyo, Japan). TGP is a copolymer composed of thermoresponsive polymer blocks [poly (N-isopropylacrylamide-co-n-butyl methacrylate) (poly NIPAAm-coBMA)] and hydrophilic polymer blocks [polyethylene glycol (PEG)].34 This polymer block is hydrophilic at temperatures below 293 K and hydrophobic at temperatures above 293 K forming crosslinking points and a homogeneous 3D network of Mebiol. The gelling temperature of TGP is 293 K. TGP was dissolved in distilled water at 277 K for 3 days. The 24% (w/v) TGP sol solutions were prepared and then stored at 277 K. 2.2. Crystallization. Crystals of the proteins were grown by the batch method at room temperature (295 K) using 96-well microbatch plates (Hampton Research). First, the ELS (LZM) protein powder was dissolved in 0.1 M sodium acetate buffer at pH 5.0 (pH 4.5) to protein concentrations of 60 mg/mL (120 mg/mL). The GI solution was adjusted to 30 mg/mL by 0.1 M Tris buffer (pH 7.0). These protein solutions were passed through a 0.22 μm pore filter (Advantec DISMIC-25). Second, the crystallization solutions were prepared by mixing equal volumes of protein solution, precipitating agent, and TGP sol solution [24% (w/v)] at 277 K. The final TGP concentration was 8.0% (w/v). TGP concentration for the crystallization experiments is almost the same as the average concentration used in tissue and cell cultures.35,36 Third, the mixed drop solutions (6.0 μL) were immediately loaded onto the microbatch plates at 277 K, and the plates were covered with 0.055 mm-thick sealing tape (NITTO DENKO CORPORATION). Table 1 summarizes the crystallization conditions of the proteins used in this study. The mixed-drop solutions were completely gelled at room temperature (295 K). 2.3. Data Collection. First, we processed the TGP surrounding ELS, GI, and LZM crystals in closed vessels using a femtosecond laserprocessing system.21,22 Next, the crystals were carefully removed with Microknife and CrystalRemover tools (SOSHO Inc., Osaka, Japan). The processed TGP containing crystals could then be captured using a cryoloop with little damage to the crystals. The crystals were soaked in cryoprotectant solution and then directly flash-cooled in a stream of cold nitrogen gas at 100 K on the goniometer head of the XRD equipment. XRD data were collected by beamlines from the SPring-8 and Photon Factory (PF) synchrotron radiation sources (Harima and Tsukuba, Japan) and a Rigaku R-AXIS IV++ imaging plate using CuKα radiation produced with a Rigaku ultra X18 rotating anode generator. These data were processed and scaled using HKL-2000.37 Tables 2 and S1 summarize these crystal data.

Figure 1. Photographs of protein crystals grown in TGP with concentrations of 8.0% (w/v) and grown in solution at 295 K. (a) TGP-grown ELS crystals. (b) Solution-grown ELS crystals. (c) TGPgrown GI crystals. (d) Solution-grown GI crystals. (e) TGP-grown LZM crystals. (f) Solution-grown LZM crystals.

the TGP concentration (Figure 2). The TGP increased nucleation linearly up to a concentration of 2.0 or 2.5% (w/v).

3. RESULTS AND DISCUSSION 3.1. Protein Crystal Growth in TGP. Porcine pancreas ELS, GI from Streptomyces rubiginosus, and chicken egg-white LZM crystals were successfully grown in TGP using the batch method at 295 K (Figure 1). The crystallization conditions were almost the same as in the solutions, except for the presence of TGP. Furthermore, these crystals were obtained over a wide range of pH values (4.5−9.0) using a variety of crystallization methods, such as hanging-drop and sitting-drop vapor-diffusion methods. These results indicate that TGP is fully compatible with conventional crystallization techniques. 3.2. Crystal Nucleation by TGP. We examined the correlation of the ELS, GI, and LZM crystal-nucleation rate with the concentration of TGP. These proteins were crystallized in the mixtures of precipitant agents and TGP at 0.5% increments up to 4.0% (w/v). The experiments for each condition were repeated three times and averaged. The number, appearance, and size of the nucleated ELS, GI, and LZM crystals were observed after the crystallization setup. Although the appearance and size of the nucleated ELS, GI, and LZM crystals did not differ significantly, the number of crystals was considerably affected by

Figure 2. (a−c) Graph of the mean number of ELS, GI, and LZM crystals as a function of TGP concentration. The number of nucleated ELS, GI, and LZM crystals was observed three days after the crystallization setup.

However, the number of crystals seems to be inversely proportional to the TGP concentration between 2.5 and 3.5% (w/v). These results indicate that the number of ELS, GI, and LZM crystals increases in the presence of TGP, suggesting a similar C

dx.doi.org/10.1021/cg301588b | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

solutions, which are virtually different from those crystallization conditions.37 LZM crystals grown in TGP with concentrations of 8.0% (w/v) were transferred to a buffer-free solution containing nearly saturated 5 M lithium acetate to examine the influence of osmotic shock (Figure 3). We also soaked the crystals in 100% glycerol (Figure S1). TGP-grown LZM crystals did not crack or dissolve for more than 10 min. In addition, we confirmed that ELS and GI crystals grown in TGP do not crack or dissolve under the same conditions. In contrast, solutiongrown crystals dissolved immediately in these solutions. These results indicate that the TGP-grown crystals can sufficiently withstand osmotic pressure in the presence of 5 M lithium acetate or 100% glycerol. However, the TGP-grown crystals cracked a few minutes after soaking in 60% (v/v) DMSO or 100% ethanol. In general, protein crystals are unable to tolerate osmotic pressure under these extreme conditions. At that time, we observed that the gelated TGP surrounding the crystals gradually solates or dissolves in 60% (v/v) DMSO or 100% ethanol at room temperature. To reconfirm this phenomenon, we transferred only the gelated TGP into 100% ethanol under a stereoscopic microscope. After a few minutes, the gelated TGP gradually became soluble or solated at room temperature. The result indicates that the crystals grown in TGP are not resistant to osmotic shock caused by extremely concentrated organic

tendency with the previous results that agarose hydrogel increased the number of protein crystals.24 3.3. Soaking in High-Concentration Solutions. Protein crystals are fatally damaged by osmotic shock when soaked in extremely concentrated organic solvents and high-ionic-strength

Figure 3. Soaking experiments with LZM crystal grown in TGP. (a) TGP-grown crystal before it was transferred to high-ionic-strength solution. (b−d) Time course of LZM crystal in 5 M lithium acetate. The crystal was transferred to this solution at room temperature. The white dotted lines indicate the border between the TGP and the solution.

Figure 4. XRD patterns of TGP-grown and solution-grown crystal at 100 K mounted by cryoloop. (a) TGP-grown ELS crystal after soaking in 5 M lithium acetate. (b) TGP-grown ELS crystal after soaking in 30% glycerol. (c) Solution-grown ELS crystal after soaking in 30% glycerol. (d) TGPgrown GI crystal after soaking in 5 M lithium acetate. (e) TGP-grown GI crystal after soaking in 30% glycerol. (f) Solution-grown GI crystal after soaking in 30% glycerol. (g) TGP-grown LZM crystal after soaking in 5 M lithium acetate. (h) TGP-grown LZM crystal after soaking in 30% glycerol. (i) Solution-grown LZM crystal after soaking in 30% glycerol. D

dx.doi.org/10.1021/cg301588b | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

determined by the molecular replacement method using the program MOLREP.41 Structural refinements were performed with a stereochemically restrained least-squares refinement method using Refmac software42 as implemented within the CCP4 package. Representative portions of the (2|Fo| − |Fc|) electron-density map after refinement and refinement statistics are depicted in Figure 5 and Table 2, respectively. These maps reflect clear, well-separated electron densities without ambiguity, corresponding to individual atoms. They demonstrate the high quality of the structural analysis. The 3D structures determined from the TGP-grown crystals were superimposed very well on those of the solution-grown crystals. The root-mean-square deviation (rmsd) between the ELS structures was an average of 0.23 Å for 230 Cα atoms. The rmsd between the GI structures was an average of 0.19 Å for 382 Cα atoms, and that between the LZM structures was 0.30 Å for 129 Cα atoms. These results demonstrate that the TGP-grown crystal structures are essentially the same as the solution-grown crystal structures.

solvents. It also suggests that the stability of hydrogel-grown crystals against osmotic shock is improved by incorporating of the polymeric network formed by the gelatification. 3.4. Data Collection Using TGP-Grown Crystals. To confirm the crystallinity of the TGP-grown crystals, XRD experiments were performed under cryogenic conditions using the TGP-grown crystals soaked in each crystallization solution containing 30%(w/v) glycerol. The XRD pattern featured sharp, clear diffraction spots without streaks or breaks (Figure 4), enabling us to collect full data sets with the crystals. The crystal quality was also satisfactory for diffraction experiments. These results demonstrate that the XRD quality of crystals is not significantly affected by TGP or laser irradiation. Next, XRD experiments were carried out to examine the diffraction quality of the TGP-grown crystals soaked in a bufferfree solution containing nearly saturated 5 M lithium acetate. This solution severely damages protein crystals by osmotic shock during soaking. Typically, high lattice stresses are introduced into crystals soaked in high-ionic-strength solutions, resulting in shrinking of the unit cell.7−9 The unit-cell dimensions of the TGP-grown crystals were almost the same as those of the solution-grown crystals (Tables 2 and S1). These results indicate that the TGP-grown crystals can be safely soaked in high-ionicstrength solutions without suffering damage. 3.5. Structures of the TGP-Grown Crystals. Structural analyses were carried out to clarify the difference in structures between TGP-grown and solution-grown crystals, and to investigate the effect of osmotic shock on crystal packing during soaking. The deposited protein coordinates for ELS [Protein Data Bank (PDB) ID code 1GVK; space group P212121],38 GI (PDB ID code 2GLK; space group I222),39 and LZM (PDB ID code 193L; space group P43212)40 were used as starting models for structural analysis after removing any ligands and all water molecules. The structures of TGP-grown crystals were

4. CONCLUSION In this study, we successfully crystallized three proteins (ELS, GI, and LZM) in TGP, which turns into hydrogel upon warming and liquefies upon cooling. Our analysis indicates that TGP provides new possibilities for overcoming the inherent problem of high-temperature damage to the proteins when using agarose. We also investigated the nucleation rate of protein crystals in the presence of TGP and found that TGP increased the number of protein crystals. Furthermore, the TGP surrounding the crystals reduces the influence of osmotic shock in high-ionic-strength solutions, although the TGPgrown crystals are unable to withstand osmotic pressure due to the solation or dissolution of TGP during soaking in the extremely concentrated organic solvents. This technique will open up a new dimension for the crystallization of biological macromolecules in hydrogels.

■

ASSOCIATED CONTENT

S Supporting Information *

Table S1: Crystal data of TGP-grown ELS crystals. Figure S1: Soaking experiments with TGP-grown LZM crystal. This information is available free of charge via the Internet at http:// pubs.acs.org/.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Addresses

# (S.S.) JST, ERATO, Murata Lipid Active Structure Project, Graduate School of Science, Osaka University, Japan ⊥ (G.S.) Laboratory for Phase Transition Dynamics of Ice, The Institute of Low Temperature Science, Hokkaido University, JAPAN

Figure 5. Electron density maps contoured at 2.0σ with (2|Fo| − |Fc|) amplitudes. These maps are calculated using the diffraction data from TGP-grown and solution-grown crystals. (a) TGP-grown ELS crystal after soaking in 5 M lithium acetate. The map resolution is 0.97 Å. (b) TGP-grown ELS crystal after soaking in 30% glycerol. The map resolution is 1.03 Å. (c) Solution-grown ELS crystal after soaking in 30% glycerol. The map resolution is 1.03 Å. (d) TGP-grown GI after soaking in 5 M lithium acetate. The map resolution is 1.13 Å. (e) TGP-grown GI after soaking in 30% glycerol. The map resolution is 1.15 Å. (f) Solutiongrown GI after soaking in 30% glycerol. The map resolution is 1.13 Å. (g) TGP-grown LZM after soaking in 5 M lithium acetate. The map resolution is 1.10 Å. (h) TGP-grown LZM after soaking in 30% glycerol. The map resolution is 1.18 Å. (i) Solution-grown LZM after soaking in 30% glycerol. The map resolution is 1.21 Å.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Satoshi Sano and Masaru Sato in JAXA for helpful advice. We also thank Eiki Yamashita and Atsushi Nakagawa on BL44XU for the data collection at SPring-8 (Hyogo, Japan) and the staff members on BL17A and BL5A for data collection at PF-AR (Tsukuba, Japan). The synchrotron radiation experiments E

dx.doi.org/10.1021/cg301588b | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

(27) Sugiyama, S.; Hirose, M.; Shimizu, N.; Niiyama, M.; Maruyama, M.; Sazaki, G.; Murai, R.; Adachi, H.; Takano, K.; Murakami, S.; Inoue, T.; Mori, Y.; Matsumura, H. Jpn. J. Appl. Phys. 2011, 50, No. 025502. (28) Sugiyama, S.; Maruyama, M.; Sazaki, G.; Hirose, M.; Adachi, H.; Takano, K.; Murakami, S.; Inoue, T.; Mori, Y.; Matsumura, H. J. Am. Chem. Soc. 2012, 134, 5786−5789. (29) Lorber, B.; Sauter, C.; Theobald-Dietrich, A.; Moreno, A.; Schellenberger, P.; Robert, M. C.; Capelle, B.; Sanglier, S.; Potier, N.; Giege, R. Prog. Biophys. Mol. Biol. 2009, 101, 13−25. (30) Madhavan, H. N.; Malathi, J.; Joseph, R. P.; Mori, Y.; Abraham, S. J. K.; Yoshioka, H. Curr. Sci. 2004, 87, 1275−1277. (31) Yoshioka, H.; Mikami, M.; Mori, Y.; Ttsuchida, E. J. Macromol. Sci., Part A 1994, 31, 113−120. (32) Yoshioka, H.; Mikami, M.; Mori, Y.; Tsuchida, E. J. Macromol. Sci., Part A 1994, 31, 121−125. (33) Yoshioka, H.; Mori, Y.; Cushman, J. A. Polym. Adv. Technol. 1994, 5, 122−127. (34) Parveen, N.; Khan, A. A.; Baskar, S.; Habeeb, M. A.; Babu, R.; Abraham, S.; Yoshioka, H.; Mohammed, H. C. Hepatitis Mon. 2008, 8, 275−280. (35) Yoshioka, H.; Mori, Y.; Shimizu, M. Anal. Biochem. 2003, 323, 218−223. (36) Arai, T.; Joki, T.; Akiyama, M.; Agawa, M.; Mori, Y.; Yoshioka, H.; Abe, T. J. Neurooncol. 2006, 77, 9−15. (37) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307− 326. (38) Katona, G.; Wilmouth, R. C.; Wright, P. A.; Berglund, G. I.; Hajdu, J.; Neutze, L.; Schofield, C. J. J. Biol. Chem. 2002, 277, 21962− 21970. (39) Katz, A. K.; Li, X.; Carrell, H. L.; Hanson, B. L.; Langan, P.; Coates, L.; Schoenborn, B. P.; Glusker, J. P.; Bunick, G. J. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 8342−8347. (40) Pugliese, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J. Mol. Biol. 1993, 231, 698−710. (41) Vagin, A.; Teplyakov, A. J. Appl. Crystallogr. 1997, 30, 1022− 1025. (42) Murshudov, G. N.; Vagin, A. A.; Lebedev, A.; Wilson, K. S.; Dodson, E. J. Acta Crystallogr., Sect. D 1999, 55, 247−255.

were performed at SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal 2012A6724 and 2012B6724). This work was partially supported by a Core Research for Evolutional Science and Technology (CREST) Grant to Y.M., Grants-in-Aid for Research Activity Start-up (Grant 23860028 to S.S.) from the Japan Society for the Promotion of Science, and the research grant of Astellas Foundation for Research on Metabolic Disorders.

■

REFERENCES

(1) Bailey, D.; Brown, D. Drug Discovery Today 2001, 6, 57−59. (2) Sintchak, M. D.; Nimmesgern, E. Immunopharmacology 2000, 47, 163−184. (3) Carr, R.; Jhoti, H. Drug Discovery Today 2002, 7, 522−527. (4) Stewart, L.; Clark, R.; Behnke, C. Drug Discovery Today 2002, 7, 187−196. (5) Goodwill, K. E.; Tennant, M. G.; Stevens, R. C. Drug Discovery Today 2001, 6, S113−S118. (6) Mitchell, E. P.; Garman, E. F. J. Appl. Crystallogr. 1994, 27, 1070− 1074. (7) Lopez-Jaramillo, F. J.; Moraleda, A. B.; Gonzalez-Ramirez, L. A.; Carazo, A.; Garcia-Ruiz, J. M. Acta Crystallogr., Sect. D 2002, 58, 209− 214. (8) Frauenfelder, H.; Hartmann, H.; Karplus, M.; Kuntz, I. D.; Kuriyan, J.; Parak, F.; Petsko, G. A.; Ringe, D.; Tilton, R. F.; Connolly, M.; Max, N. Biochemistry 1987, 26, 254−261. (9) Kurinov, I. V.; Harrison, R. W. Acta Crystallogr., Sect. D 1995, 51, 98−109. (10) Kriminski, S.; Caylor, C. L.; Nonato, M. C.; Finkelstein, K. D.; Thorne, R. E. Acta Crystallogr., Sect. D 2002, 58, 459−471. (11) Yao, M.; Yasutake, Y.; Tanaka, I. Acta Crystallogr., Sect. D 2004, 60, 39−45. (12) Berejnov, V.; Husseini, N.; Alsaied, O. A.; Thorne, R. E. J. Appl. Crystallogr. 2006, 39, 244−251. (13) Vidal, O.; Robert, M. C.; Boué, F. J. Cryst. Growth 1998, 192, 257−270. (14) Biertumpfel, C.; Basquin, J.; Suck, D.; Sauter, C. Acta Crystallogr. 2002, D58, 1657−1659. (15) García-Ruiz, J. M. Methods Enzymol. 2003, 368, 130−154. (16) Robert, M. C.; Lefaucheux, F.; Jannot, B.; Godefroy, G.; Garnier, E. J. Cryst. Growth 1988, 88, 499−510. (17) Zhu, D. W.; Lorber, B.; Sauter, C.; Ng, J. D.; Benas, P.; Grimellec, C. L.; Giege, R. Acta Crystallogr. 2001, D57, 552−558. (18) Sauter, C.; Lorber, B.; Giege, R. Proteins 2002, D48, 146−150. (19) Gavira, J. A.; Garcia-Ruiz, J. M. Acta Crystallogr. 2002, D58, 1653−1656. (20) Sugiyama, S.; Tanabe, K.; Hirose, M.; Kitatani, T.; Hasenaka, H.; Takahashi, Y.; Adachi, H.; Takano, K.; Murakami, S.; Mori, Y.; Inoue, T.; Matsumura, H. Jpn. J. Appl. Phys. 2009, 48, No. 075502. (21) Sugiyama, S.; Hasenaka, H.; Hirose, M.; Shimizu, N.; Kitatani, T.; Takahashi, Y.; Adachi, H.; Takano, K.; Murakami, S.; Inoue, T.; Mori, Y.; Matsumura, H. Jpn. J. Appl. Phys. 2009, 48, No. 105502. (22) Hasenaka, H.; Sugiyama, S.; Hirose, M.; Shimizu, N.; Kitatani, T.; Takahashi, Y.; Adachi, H.; Takano, K.; Murakami, S.; Mori, Y.; Inoue, T.; Matsumura, H. J. Cryst. Growth 2010, 312, 73−78. (23) Yoshikawa, H. Y.; Murai, R.; Sugiyama, S.; Sazaki, G.; Kitatani, T.; Takahashi, Y.; Adachi, H.; Matsumura, H.; Murakami, S.; Inoue, T.; Takano, K.; Mori, Y. J. Cryst. Growth 2009, 311, 956−959. (24) Tanabe, K.; Hirose, M.; Murai, R.; Sugiyama, S.; Shimizu, N.; Maruyama, M.; Takahashi, Y.; Adachi, H.; Takano, K.; Murakami, S.; Mori, Y.; Mizohata, E.; Inoue, T.; Matsumura, H. Appl. Phys. Express 2009, 2, No. 125501. (25) Murai, R.; Yoshikawa, H. Y.; Takahashi, Y.; Maruyama, M.; Sugiyama, S.; Sazaki, G.; Adachi, H.; Matsumura, H.; Murakami, S.; Inoue, T.; Takano, K.; Mori, Y. Appl. Phys. Lett. 2010, 96, No. 043702. (26) Matsumura, H.; Sugiyama, S.; Hirose, M.; Kakinouchi, K.; Maruyama, M.; Adachi, H.; Murai, R.; Takano, K.; Murakami, S.; Mori, Y.; Inoue, T. J. Synchrotron Radiat. 2011, 18, 16−19. F

dx.doi.org/10.1021/cg301588b | Cryst. Growth Des. XXXX, XXX, XXX−XXX