Dislocations in High-Quality Glucose Isomerase Crystals Grown from

Article pubs.acs.org/crystal

Dislocations in High-Quality Glucose Isomerase Crystals Grown from Seed Crystals H. Koizumi,*,† M. Tachibana,‡ I. Yoshizaki,§ S. Fukuyama,⊥ K. Tsukamoto,† Y. Suzuki,¶ S. Uda,† and K. Kojima∥ †

Institute for Materials Research, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai 980-8577, Japan Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan § Japan Aerospace Exploration Agency (JAXA), 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505, Japan ⊥ Advanced Engineering Services Co., Ltd., Tsukuba Mitsui Bldg., 1-6-1 Takezono, Tsukuba, Ibaraki 305-0032, Japan ¶ Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima, Tokushima 770-8506, Japan ∥ Department of Education, Yokohama Soei University, 1 Miho-tyou, Midori-ku, Yokohama 226-0015, Japan ‡

ABSTRACT: Glucose isomerase crystals with thicknesses of 0.5 mm were grown using seed crystals. The thickness required for better X-ray topographic images by kinematical theory is determined by the extinction distance, ξg. The seed crystals were cross-linked with glutaraldehyde to maintain stability for a long period before the growth experiments. Dislocations that were produced during the growth of such thick glucose isomerase crystals were observed as clearly as those in inorganic crystals via X-ray topography with monochromatic-beam synchrotron radiation, which is attributed to the high quality of the glucose isomerase crystals. This also indicates that high-quality protein crystals can be grown using seed crystals. The dislocations extended from the interface with the cross-linked seed crystal to the surface of the grown crystal; however, no dislocations were generated if the seed crystal was not cross-linked. We discuss the effect of the chemical cross-linking of the seed on the generation of dislocations from seed crystals.

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microgravity conditions.13 Microgravity experiments require the seed crystals to be kept stable for a long period before the start of growth experiments. Such a problem can be solved if the surfaces of the seed crystals are cross-linked with glutaraldehyde. These cross-linked seed crystals were confirmed not to dissolve in an undersaturated solution;14 however, the effects of the cross-linking procedure on the crystal quality, such as a change in the dislocation density, have not yet been clarified. Therefore, X-ray topography was employed to determine such effects in this study. X-ray topography is a powerful tool for the evaluation of crystal quality and observation of dislocations.15−18 Dislocations in protein crystals were investigated using synchrotron white- and later, monochromatic-beam X-ray topography.19−36 We have previously succeeded in the characterization of the Burgers vector of dislocations in tetragonal hen egg white (HEW) lysozyme crystals by using synchrotron white-beam Xray topography.28 Moreover, we have not only characterized the Burgers vectors of dislocations in orthorhombic HEW lysozyme crystals,33,36 but also observed loops and highly curved dislocations in monoclinic HEW lysozyme crystals.35 However, there have been very few X-ray topography results reported for proteins other than the HEW lysozyme26,27 because of the lack

INTRODUCTION It is important to determine the 3D structures of protein molecules in order to achieve structure-guided drug design and controlled drug delivery. However, high-quality protein crystals are required to achieve this; therefore, many researchers have investigated methods and conditions for obtaining suitable crystals. Crystallization in microgravity is one potential method, and thus research is being actively conducted on the growth of crystals in a microgravity environment.1−12 Two approaches may be possible for the growth of crystals under various growth conditions: (1) the use of varied salt and protein concentrations to determine the optimum conditions, or (2) to investigate the growth mechanism of a few crystals under well-controlled conditions and the measurement of the crystal growth rate versus the supersaturation of the solution using a seed crystal. In the former case, it is difficult to predict when the nucleation and crystal growth start during the concentration increase to supersaturation. However, a nucleation process is not necessary in the latter case, and thus supersaturation remains constantly low if the decrease in the concentration is negligible because of further growth. This case is suitable for kinetic measurements to elucidate the detailed growth mechanisms of protein crystals both on the ground and under “convection-free” microgravity conditions. Thus, a pioneering observation of the concentration-depletion zones around protein crystals was performed by using gelreinforced seed crystals both on the ground and under © 2014 American Chemical Society

Received: May 20, 2014 Revised: August 18, 2014 Published: September 11, 2014 5111

dx.doi.org/10.1021/cg500731v | Cryst. Growth Des. 2014, 14, 5111−5116

Crystal Growth & Design

Article

Figure 1. (a) Optical micrograph of a grown glucose isomerase crystal. The yellow contrast in the central part is the cross-linked seed crystal. (b) Morphology of the grown glucose isomerase crystal. (c) Glucose isomerase crystal sealed in an acrylic cell.

Table 1. Extinction Distance, ξg, for Glucose Isomerase Crystals and the Parameters Used To Calculate Its Value. Parameters for Tetragonal HEW Lysozyme Crystals Are Also Tabulated protein

refln

Bragg angle (deg)

|Fhkl|

Vc (Å3)

ξg (mm)

glucose isomerase HEW lysozyme

020 440

0.69 2.46

14 006 1669

962 547 237 133

0.64 1.32

from this crystallization solution at 20 °C via a hanging drop technique. The grown seed crystals were chemically fixed by a modification of the previously reported method.14 The solution used for chemical cross-linking was a mixture of 2.5 wt % glutaraldehyde and 0.91 M ammonium sulfate. The seed crystals were immersed in the cross-linking solution for 20 min at room temperature. After the cross-linking, the seed crystals were rinsed and used as reusable seeds because the seeds do not dissolve in undersaturated solution. The epitaxial growth layer can grow over the seed crystal. Therefore, the glucose isomerase crystals were grown again from the cross-linked seed crystals using the sitting drop system and the crystallization solution at 20 °C for 2 weeks. Figure 1, panel (a) shows an optical micrograph of a grown glucose isomerase crystal. The yellow contrast in the central part of the crystal (indicated by an arrow) is the cross-linked seed crystal. The crystals were orthorhombic with a space group of I222; lattice constants of a = 93.88 Å, b = 99.64 Å, and c = 102.90 Å; and two molecules per unit cell.40 The crystals were bounded by the crystallographic {110}, {101}, and {011} faces, as shown in Figure 1, panel b. Crystals with thicknesses greater than 0.5 mm were selected for the X-ray topographic experiments. X-ray Topography. Monochromatic-beam X-ray topography was conducted using synchrotron radiation at the BL20B beamline at the Photon Factory (PF) of the High Energy Accelerator Research Organization (KEK). A monochromatic beam with a wavelength of 1.2 Å was selected by an adjustment of the double-crystal monochromator. The grown glucose isomerase crystals were sealed in an acrylic cell, as shown in Figure 1, panel c. The sealed crystal was mounted on the goniometer and the crystallographic [101] direction was prealigned using a microscope to be almost parallel to the incident beam. An Xray flat panel sensor (C9732DK, Hamamatsu Photonics KK) was employed to find the target reflections for the X-ray topography. The exact Bragg angle for the target reflection was determined by the measurement of the peak intensity for the reflection spot using the flat panel sensor. The weak-beam condition was then determined. The camera length was 20 cm. Monochromatic-beam topographs were recorded on X-ray film (Agfa D2) or nuclear plates (Fujifilm Co. Ltd., Resolution: 15 μm) with exposure times of approximately 5 and 1.5 min, respectively.

of sufficient large and high-quality crystals for X-ray topography measurements that are necessary to obtain good contrast. Thus, the difficulties in the growth of large and high-quality protein crystals and the control over the dislocation densities are planned to be discussed by using seed crystals. The most common dislocation contrast in X-ray topographs is a kinematical contrast (direct image) produced by additional diffraction from a distorted lattice region close to a dislocation.17,37 Direct transmission topography images can be obtained when μt < 1, where μ is the linear absorption coefficient, and t is the thickness of the crystal sample. This condition determines the upper limit of the crystal thickness. A further condition is that t > αξg, where α varies from about 0.4 for low-order reflections to about 0.15 for high-order reflections, and ξg is the extinction distance.17,37 This indicates the lower limit of crystal thickness, that is, the dislocation contrast for low-order reflections typically disappears when the crystal thickness is less than 0.4ξg. For example, the extinction distance for 440 reflections in tetragonal HEW lysozyme crystals is calculated to be 1.32 mm when 1.2 Å of radiation is used. Therefore, for tetragonal HEW lysozyme crystals with thicknesses of less than 0.53 mm, the dislocation contrast would probably be either weak or absent in X-ray topographs. Since it is difficult to grow protein crystals greater than 1 mm in size, the dislocation contrast has not been observed for protein crystals other than the HEW lysozyme using X-ray topography. We previously demonstrated that it is possible to grow large tetragonal HEW lysozyme crystals using seed crystals.14 More recently, large lysozyme crystals were grown with seed crystals in microgravity experiments using the Foton-M3 satellite in 2007, in collaboration with Tohoku University, the University of Granada, the European Space Research and Technology Centre (ESTEC), and the Japan Aerospace Exploration Agency (JAXA).38 Such large crystals grown using seed crystals have also led to X-ray topographic observations of dislocations generated under microgravity conditions.39 In this article, we report details on the crystal quality and dislocation structure of glucose isomerase crystals using synchrotron monochromaticbeam X-ray topography.

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RESULTS AND DISCUSSION The extinction distance, ξg, for glucose isomerase crystals was calculated to determine whether the minimum crystal thickness criterion for X-ray topography was satisfied. The ξg for a symmetrical reflection in a perfect crystal is given as15,17

EXPERIMENTAL PROCEDURE

Crystal Growth. Glucose isomerase was purchased from Hampton Research and used without further purification. The solution included 33 mg/mL glucose isomerase, 6 mM tris(hydroxymethyl)aminomethane (TRIS) hydrochloride (pH 7.0), 0.91 M ammonium sulfate, and 1 mM magnesium sulfate. The seed crystals were grown

ξg = 5112

πVccos θ re|Fhkl|λ

(1)



Article

where θ is the Bragg angle, re is the classical electron radius (2.82 × 10−5 Å), λ is the wavelength of X-ray radiation, |Fhkl| is the structure factor, and Vc is the volume of the unit cell. Table 1 shows the parameters used to calculate ξg for glucose isomerase crystals. According to eq 1 and Table 1, ξg is calculated to be 0.64 mm for the 020 reflection, which is much smaller than that for tetragonal HEW lysozyme crystals, as shown in Table 1. This suggests that dislocation images can be observed even for relatively small glucose isomerase crystals. The minimum criterion, 0.4ξg, for the crystal thickness for the 020 reflection of glucose isomerase crystals can be estimated to be 0.256 mm, which is smaller than the thickness of the crystals used here for the X-ray topography of glucose isomerase crystals. Therefore, it was expected that glucose isomerase crystals with thicknesses greater than 0.5 mm would provide clear dislocation images. Figure 2, panels a and b show synchrotron monochromaticbeam X-ray topographs of glucose isomerase crystals taken using the 020 reflection with the incident beam almost normal

to the (101) face of the crystals grown from the cross-linked seed crystals. Clear, straight dislocations are seen to extend from the interface with the seed crystals to the outer surface of the larger grown crystals, which is similar to the case for organic crystals of small molecules.16 The contrast in the central part of Figure 2, panel (a) (indicated by an arrow) corresponds to the cross-linked seed crystal. The topographs are clearer than those of the tetragonal HEW lysozyme crystals,36 which suggests that the glucose isomerase crystals have high crystal quality. This may be attributable to the high symmetry of glucose isomerase, because it is a tetramer composed of four identical subunits.41 Concerning the image widths related to the dislocation contrasts, it is considered that the image widths of glucose isomerase crystals are narrower than those of tetragonal HEW lysozymes. The image width is inversely proportional to the Xray scattering power, |Fhkl|/Vc. Therefore, the |Fhkl|/Vc value of glucose isomerase crystals is larger than that of tetragonal HEW lysozymes (see Table 1). This tendency could also contribute to the clear dislocation contrasts of glucose isomerase crystals. Both dislocation images and clear fringes were observed, as shown in the magnified part of Figure 2, panel a. The fringes observed in this work were straight, although curved fringes were previously observed in protein crystals.24,26 The tapered crystals with wedge-like edges cause Pendellösung fringes,16 and moreover, the fringe spacing is twice the extinction distance. Therefore, the Pendellösung fringe spacing was calculated to be 1.28 mm, which is larger than the observed fringe spacing by approximately two orders of magnitude. This indicates that the observed fringes are not Pendellösung fringes; however, the observation of clear fringes is evidence that the glucose isomerase crystals have high crystal quality. In addition, etch pits were observed on the (011) surface, as indicated by the arrow in Figure 2, panel c. These are associated with dislocations that penetrate the (011) surface in Figure 2, panel b (indicated by an arrow). Therefore, the individual etch pits on the surface are derived from dislocations in the protein crystal. Such characteristics were previously observed in tetragonal HEW lysozyme crystals using synchrotron whitebeam X-ray topography;32 however, in that case, the individual etch pits could not be directly matched with dislocations, because the dislocation images were not sufficiently clear. In contrast, for the glucose isomerase crystals, the dislocation images could be individually resolved and directly assigned to etch pits on the crystal surface. To the best of our knowledge, this is the first report to clearly show etch pits and the corresponding dislocations in protein crystals. This is attributed to the high crystal quality of glucose isomerase. Two individual dislocations are seen to extend from the cross-linked seed crystal in the topograph in Figure 2, panel a, while dislocation bundles are found in the topograph in Figure 2, panel b. The difference in the generation of the dislocations could be attributed to differences in the roughness on the crystal surface between the cross-linked seed crystals, which would result in differences in the generation of dislocations from the cross-linked seed crystals. However, no dislocations were observed in the glucose isomerase crystals grown with non-cross-linked seed crystal, that is, native glucose isomerase crystals, as shown in Figure 2, panel d. This is thought to be because the surfaces of these seed crystals are smooth. Such a phenomenon is clearly observed in Ba(NO3)2 crystals, where inclusions trapped on the surface of the etched seed crystal play an important role in the generation of dislocations.42 Therefore, the density of the dislocations induced in the crystals may be

Figure 2. (a) and (b) Synchrotron monochromatic-beam X-ray topographs of glucose isomerase crystals taken using the 020 reflection with the incident beam almost normal to the (101) face. These two crystals were grown from cross-linked seed crystals. (c) Optical micrograph of a glucose isomerase crystal that corresponds to the Xray topograph of panel b. (d) Synchrotron monochromatic-beam Xray topograph of a glucose isomerase crystal taken using the 020 reflection with the incident beam almost normal to the (101) face. The crystal was grown from a non-cross-linked seed crystal. 5113



Article

isomerase crystal grown without a seed crystal, as shown in Figure 3, panel c. These curved dislocations may be induced to reduce the strain caused by crystal growth. We have determined the Burgers vector of dislocations in the glucose isomerase crystals. According to the dislocation theory,43 the dislocation energy, E, per unit length of a straight dislocation consists of two terms. One is the elastic energy, Ea, due to the long-range strain field that surrounds the dislocation, and the other is the dislocation core energy, Ec. However, Ec can be neglected without serious error, because it is usually estimated to be at least one order of magnitude smaller than Ea. Thus, E can be approximated by Ea as follows:

controllable by the regulation of the roughness of the crystal surface due to chemical cross-linking. Furthermore, a spot-like contrast was observed for glucose isomerase crystals grown with non-cross-linked seed crystals, as shown in Figure 3, panel a. Figure 3, panel b shows the (101)

E ≃ Ea = (Gb2 /4π )ln(R /r0)

(2)

where G is the shear modulus, b is the magnitude of the Burgers vector, and R and r0 are the outer and inner cutoff radii, respectively, of the dislocation concerned. Eq 2 shows that the dislocation energy is proportional to the square of the magnitude of the Burgers vector,44 which implies that shorter lattice translational vectors are favored as Burgers vectors. Glucose isomerase crystals are bound by the crystallographic {110}, {101}, and {011} faces; therefore, it is considered that there are dislocations on those crystallographic faces that are densely-packed planes, that is, the slip planes. Possible Burgers vectors on these slip planes are ⟨110⟩, ⟨101⟩, ⟨011⟩, and ⟨111⟩, where the shortest Burgers vector is ⟨110⟩. Here we focus on the character of the dislocation denoted as “A” in Figure 2, panel b. Dislocation A could be on the (101) slip plane, which is almost parallel to the [1̅21] direction; therefore, its possible that the Burgers vectors are [1̅01], [111̅], and [1̅11]. Figure 4, panel (a) shows a synchrotron monochromatic-beam X-ray topograph of a glucose isomerase crystal taken using the 011 reflection with the incident beam almost parallel to the (011) face. This topograph was taken of the same glucose isomerase crystal that is shown in Figure 2, panel b. The dislocation contrast is invisible for the 011 reflection. According to the invisibility criterion for dislocation images (g·b = 0; g and b are the diffraction vector and the Burgers vector, respectively), it can be determined that the dislocation has a mixed character with a Burgers vector of [111̅]. Figure 4, panel b shows the relationship between the dislocation geometry and the Burgers vector in a glucose isomerase crystal. The Burgers vector [111̅] observed in this work is not the shortest possible Burgers vector; however, such long Burgers vectors have also often been observed during the growth of tetragonal HEW lysozyme crystals.28,35,36 Moreover, dislocation A in Figure 2, panel b has a screw component because it has a Burgers vector of [111̅] on the (101) slip plane, which results in spiral growth on the (10̅ 1) face. This result is quite important for detailed observations of the growth kinetics of the hillocks on the surface of glucose isomerase crystals via a technique such as two-beam interferometry, which is similar to those in tetragonal HEW lysozyme crystals.45 A detailed determination of the Burgers vectors for dislocations in glucose isomerase crystals is now in progress.

Figure 3. (a) Synchrotron monochromatic-beam X-ray topograph of a glucose isomerase crystal taken using the 020 reflection with the incident beam almost normal to the (101) face. The crystal was grown from a non-cross-linked seed crystal. (b) The (101) surface on a glucose isomerase crystal after etching at 37 °C for 2 h, which corresponds to the X-ray topograph of panel (a). (c) Synchrotron monochromatic-beam X-ray topograph of a glucose isomerase crystal taken using the 020 reflection with the incident beam almost normal to the (101) face. The crystal was grown without a seed crystal.

surface of a glucose isomerase crystal after etching at 37 °C for 2 h. As shown in Figure 3, panel b, faceted inclusions were observed, which indicate that microcrystals were incorporated into the glucose isomerase crystal. The microcrystals that are formed by three-dimensional nucleation in solution could be transported to the crystal surface by convection and then be incorporated into the crystal. Such an incorporation process was also observed for tetragonal HEW lysozyme crystals, although no spot-like contrast was detected in the X-ray topographic images. Rather than straight dislocations that extend from the seed crystal, curved dislocations were also observed for the glucose

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CONCLUSION Synchrotron monochromatic-beam X-ray topography was conducted using glucose isomerase crystals with thicknesses greater than the minimum criterion of 0.4ξg. Straight, grown-in dislocations were observed as clearly in the glucose isomerase crystals as in the inorganic crystals. The dislocations extended 5114



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a Grant-in-Aid for Scientific Research (C) (No. 25420694) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Figure 4. (a) Synchrotron monochromatic-beam X-ray topograph of a glucose isomerase crystal taken using the 011 reflection with the incident beam almost parallel to the (011) face. This topograph was taken using the same glucose isomerase crystal as that for Figure 2, panel b. (b) Illustration of the dislocation geometry and Burgers vector in a glucose isomerase crystal.

from the interface with the cross-linked seed crystals to the surface of the grown crystals. Clear fringes were also observed using a seed crystal, which suggests that the grown glucose isomerase crystals have high crystal quality. Furthermore, differences in the dislocation density were observed when different cross-linked seed crystals were used. This could be attributed to differences in the roughness of the crystal surface grown from different cross-linked seed crystals and could be used to control the dislocation density by regulating the roughness on the crystal surface due to chemical cross-linking. The characterization of the dislocations observed in the glucose isomerase crystals led to an estimation of the Burgers vector to be ⟨111⟩ with a screw component. These results are a useful contribution to future detailed observations of the growth kinetics of the hillocks on the surface of glucose isomerase crystals.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Monochromatic-beam X-ray topography was performed at the Photon Factory (PF) under the auspices of the Photon Factory Program Advisory Committee of KEK (Proposal No. 2012G504, 2013G116). This work was supported in part by 5115



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(32) Yoshizaki, I.; Fukuyama, S.; Koizumi, H.; Tachibana, M.; Kojima, K.; Matsuura, Y.; Tanaka, M.; Igarashi, N.; Kadowaki, A.; Rong, L.; Adachi, S.; Yoda, S.; Komatsu, H. J. Cryst. Growth 2006, 290, 185−191. (33) Koizumi, H.; Shimizu, M.; Tachibana, M.; Kojima, K. Phys. Status Solidi A 2007, 204, 2688−2693. (34) Koizumi, H.; Tachibana, M.; Kojima, K.; Yonenaga, I. Physica B 2007, 401−402, 691−694. (35) Koishi, M.; Ohya, N.; Mukobayashi, Y.; Koizumi, H.; Kojima, K.; Tachibana, M. Cryst. Growth Des. 2007, 7, 2182−2186. (36) Mukobayashi, Y.; Kitajima, N.; Yamamoto, Y.; Kajiwara, K.; Sugiyama, H.; Hirano, K.; Kojima, K.; Tachibana, M. Phys. Status Solidi A 2009, 206, 1825−1828. (37) Tanner, B. Phys. Status Solidi A 1972, 10, 381−386. (38) Tsukamoto, K.; Sazaki, G.; Kojima, K.; Tachibana, M.; Yoshizaki, I. Space Util. Res. 2008, 24, 6−7. (39) Yoshizaki, I.; Tsukamoto, K.; Tachibana, M.; Kojima, K. Abstracts. In Third International Symposium on Physical Sciences in Space, 2007, Nara, Japan; p 129. (40) Carrell, H.; Glusker, J. P.; Burger, V.; Manfre, F.; Tritsch, D.; Biellmann, J. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 4440−4444. (41) Suzuki, Y.; Sazaki, G.; Visuri, K.; Tamura, K.; Nakajima, K.; Yanagiya, S.-I. Cryst. Growth Des. 2002, 2, 321−324. (42) Maiwa, K.; Tsukamoto, K.; Sunagawa, I. J. Cryst. Growth 1987, 82, 611−620. (43) Hirth, J. P.; Lothe, J. Theory of Dislocations; John Wiley & Sons: NewYork, 1982; p 59. (44) Kojima, K. Progress in Crystal Growth and Characterization of Materials; Niizeki, N., Ed.; Pergamon Press: Oxford, UK, 1991; Vol. 23, pp 369−420. (45) Yoshizaki, I.; Tsukamoto, K.; Yamazaki, T.; Murayama, K.; Oshi, K.; Fukuyama, S.; Shimaoka, T.; Suzuki, Y.; Tachibana, M. Rev. Sci. Instrum. 2013, 84, 103707.

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Dislocations in High-Quality Glucose Isomerase Crystals Grown from

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