Development of an Embedding Method for Analyzing the Impurity Distribution in Protein Crystals Yoshikazu Iimura,† Izumi Yoshizaki,*,‡ Shinichi Yoda,‡ and Hiroshi Komatsu‡,§ Advanced Engineering Services Co., Ltd., Tsukuba Mitsui Building, 1-6-1 Takezono, Tsukuba, Ibaraki 305-0032, Japan, Japan Aerospace Exploration Agency (JAXA), 2-1-1 Sengen, Tsukuba, Ibaraki, 305-8505, Japan, and Iwate Prefectural University, Takizawa-mura, Iwate 020-0193, Japan
CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 1 295-300
Received March 1, 2004
ABSTRACT: An embedding method was developed for analyzing the impurity distribution inside protein crystals. Tetragonal lysozyme crystals contaminated with a fluorescence-labeled impurity were chemically fixed with glutaraldehyde and embedded with resins. The crystals were cut into slices and observed by confocal laser scanning microscopy. The internal crystal structure remained as grown in the slices, and the impurity distribution inside the protein crystals was visualized. This new technique enabled us to qualitatively measure the impurity distribution inside the protein crystals. Tetragonal lysozyme crystals contaminated with a fluorescence-labeled impurity were chemically fixed with glutaraldehyde and embedded with resins. The crystals were cut into slices, and the impurity distribution inside the crystals was visualized by confocal laser scanning microscopy. 1. Introduction Crystals must often be cut and polished for analyzing the inner structure, such as defects and growth striation, and for studying the distribution of chemical elements. However, the application of these conventional processing techniques to protein crystals is limited for two reasons: (i) the fragility of protein crystals and (ii) the restrictions for preserving solutions. Protein crystals must be kept in a specific solution under controlled conditions (i.e., precipitant and protein concentrations, temperature, and buffer agent) to maintain the crystal structure. The development of a new processing technique for protein crystals is needed. Quiocho and Richards demonstrated that cross-linking of protein crystals by glutaraldehyde was effective for coping with the fragility.1 They showed that the cross-linked protein crystals were prevented from dissolution in various conditions of solution irrespective of temperature, pH, and precipitant concentration. It was also shown that both the enzyme activity1,2 and the molecule conformation3 were well-maintained. Although their original purpose of cross-linking was to obtain stable protein crystals for X-ray diffraction experiments, this cross-linking technique was extended to various fields.4 Cross-linking is already utilized for measuring physical properties of protein crystals. Morozov and Morozova processed cross-linked triclinic lysozyme crystals into needlelike plates and succeeded in measuring the elastic coefficient.5 On the basis of these studies, we intend to apply this cross-linking technique to crystal growth research, focusing on the observation of impurity distribution inside protein crystals. We previously reported a method for studying the distribution coefficients of fluorescence* To whom correspondence should be addressed. Tel: +81-29-8683654. Fax: +81-29-868-3956. E-mail:
[email protected]. † Advanced Engineering Services Co., Ltd.. ‡ Japan Aerospace Exploration Agency (JAXA). § Iwate Prefectural University.
labeled impurities by observing the impurity distribution in a crystal under confocal laser scanning microscopy (CLSM).6 However, our observation system has the following restrictions. (i) Every crystal should be observed at the same focal point along the Z-axis (just above the slide glass). Otherwise, optical aberrations induce obscure images. (ii) Every crystal should be observed in the same crystallographic direction to equalize the polarization effect. (iii) The crystal center plane should be observed to analyze the total impurity incorporation through the crystallization. Protein crystals tend to nucleate at the wall of a crystallization chamber. We took advantage of this and prepared a crystallization chamber made of two slide glasses. Crystals nucleated primarily on the slide glass, so we could quantitatively observe the crystal center plane by setting the focus just above the slide glass and turning the slide glass around to set the same crystallographic direction.7 However, this observation method cannot be applied to crystals obtained by the hanging drop method. In the hanging drop method, which is the most popular protein crystallization method, crystals often nucleate at the vapor-liquid interface and grow in the solution equally in all directions. If we try to observe the crystal center plane, the optical path length to the focus plane will be different in each crystal (the first restriction cannot be met). This causes optical aberration differences,6-8 which make even qualitative analysis impossible. To extend the application of CLSM, we developed a processing procedure from chemical fixing (cross-linking) and embedding to slicing protein crystals. Embedding was necessary to successively slice crystals of hundreds of micrometers in size. The slice containing the crystal center was set on a slide glass and used for CLSM. We named this processing technique the “embedding method”. In this paper, we introduce the details of this embedding method, focusing on the fixation step, and demonstrate its validity.
10.1021/cg049918i CCC: $30.25 © 2005 American Chemical Society Published on Web 07/17/2004
296
Crystal Growth & Design, Vol. 5, No. 1, 2005
Iimura et al.
2. Development of the Embedding Method 2.1. Materials. Tetragonal lysozyme crystals were used for this technical development as a model protein crystal. Crystallization was performed by the batch method using 6× recrystallized lysozyme purchased from Seikagaku Kogyo. The crystallization condition of lysozyme crystals used for sections 2.3 and 2.4 was 15 mg/mL lysozyme and 6% NaCl in 50 mM sodium acetate buffer (pH 4.5). Crystals of 500 µm size were used. Chemical reagent grade (CRG) and electron microscopy grade (EMG) glutaraldehyde were used for the fixing reagent. To easily handle the fixed crystal, the crystal was embedded in resin. Among various embedding materials, a popular resin historesin (LKB),9 a methacrylate resin, was selected for use. The embedded sample was sliced and prepared as a permanent preparation. The slices were enclosed with an enclosure reagent. Entellan new, a generally used enclosure reagent, was purchased from Merck. 2.2. Characterization of Glutaraldehyde. Glutaraldehyde had been used for chemical fixation of biological samples before Quiocho and Richards’ research. After the introduction by Sabatini,10 glutaraldehyde was used primarily to prepare biological samples for electron microscopy observation.11 Complicated denaturation mechanisms exist in glutaraldehyde itself. Impurities, such as glutaric acid and glutaraldehyde oligomer, are generated in the glutaraldehyde solution.12,13 The purity of glutaraldehyde is judged by the absorbance ratio A235/A280. This is because glutaraldehyde exhibits an absorption maximum at 280 nm (A280), but as oligomers increase, an absorption maximum appears at 235 nm (A235). To obtain a constant result, it is important to use a pure reagent since impurities are known to influence the enzyme activity.12 Oligomers are also reported to have a positive effect on fixation. Because the glutaraldehyde chain is longer, oligomers are thought to be able to cross-link a wider range.14-16 The wide range cross-linking may be effective for protein crystal fixation, irrespective of the impurity influence, because the distance between two lysine residues, which mainly react with glutaraldehyde,3 varies in protein crystals. The important role of oligomers in protein crystal fixation is supported by the report of Haas.17 He reported that the pH of glutaraldehyde solution must be 8 or more for complete crosslinking of monoclinic lysozyme crystals. At a pH of 8 or more, the oligomerization of glutaraldehyde is promoted.13,18 We decided to try both monomers and oligomers. The purity of EMG and CRG was checked by the absorption ratio. The UV-vis absorption spectra of 0.5% glutaraldehyde solutions are illustrated in Figure 1. The absorption peak at 235 nm is seen only in CRG. The absorbance ratios are 0.27 for EMG and 3.73 for CRG, confirming that the purity of EMG is very high and that CRG contains much impurity. Thus, EMG was used as a representative of glutaraldehyde monomers, and CRG was used as a reagent containing oligomers. In fact, EMG contains another oligomer that has no reactivity so that only the glutaraldehyde monomer reacts.19 2.3. Optimization of the Fixation Condition. The purpose of fixation is to fix the three-dimensional position of the protein molecules inside the crystals.
Figure 1. Absorption spectra of 0.5% glutaraldehyde solutions. A solid line indicates EMG, and a dashed line indicates CRG.
Figure 2. Observation of the cut surface of the fixed crystals. The grade of glutaraldehyde and the fixation time were (a) EMG, 3 h; (b) EMG, 12 h; (c) EMG, 2 days; (d) EMG, 8 days; (e) CRG, 3 h; (f) CRG, 12 h; (g) CRG, 2 days; and (h) CRG, 8 days.
Among the three procedures employed in the embedding method (fixation, embedding, and slicing), fixation is the key step in preventing the crystals from cracking or dissolving. Embedding and slicing procedures are described in section 2.5. Tetragonal lysozyme crystals were cross-linked in fixation solution containing 2.5% EMG or CRG, 6% NaCl, and 50 mM buffer agent for 3 h to 8 days. We then embedded and cut the crystals according to the method described in section 2.5. Figure 2 displays the cut surfaces of the embedded crystals. When the crystals were fixed with EMG, cracks occurred in the cut surface at all reaction times. In contrast, when the crystals were fixed with CRG, the cut surface was smooth with a reaction time exceeding 2 days. Cracks occurred with a reaction time of 3 and 12 h. It is difficult to visualize the impurity distribution in cracked crystals. Therefore, we decided to fix crystals with CRG for 2 days, because the cut surface was smooth without cracks. This difference between EMG and CRG is assumed to correlate with the solidity of fixed crystals. The crystals fixed under conditions in which cracks occurred in cut surfaces were quite rigid. When we crushed the crystals, they broke into pieces. In contrast, crystals fixed with CRG for more than 2 days did not break up, even if we crushed them; they were rather elastic. This means that the oligomers in CRG loosely cross-linked the crystals over a spacially wide range.14-16 2.4. Osmotic Pressure and Solubility. The effect of the osmotic pressure should be carefully examined
Protein Crystal Fixation for Growth Studies
because cracks occur easily when protein crystals are moved into a solution with a different osmotic pressure. The osmotic pressure of the glutaraldehyde solution can be estimated from the measurement result of Maser et al.20 The pressure increase due to 2.5% glutaraldehyde corresponds to about 0.9% NaCl. When fixing a crystal grown in 6% NaCl, it is best to fix it in 2.5% glutaraldehyde with 5.1% NaCl to equalize the osmotic pressure. There was also a possibility that the crystal may dissolve in this fixation solution because the equilibrium concentration tends to rise with the decrease of NaCl concentration. Therefore, we checked if the crystal dissolved in such solution or not. As a result, we found that crystals did not crack or dissolve in 2.5% glutarldehyde with 4-8% NaCl. This can be explained by the fact that the lysozyme solubility does not change largely over 4% NaCl.21 Thus, the same precipitant concentration with the crystallization solution was used. 2.5. Optimized Procedures for Tetragonal Lysozyme Crystals. The crystals were fixed for 2 days with 2.5% CRG of glutaraldehyde. To prevent osmotic shock, the concentration of the fixation solution was kept the same as that of the crystallization precipitant and buffer. The solution volume was 20× or more of the sample volume. The fixed crystals were rinsed three times with the crystallization precipitant solution. Before they are embedded, the crystals should be dehydrated. This is to enhance the permeability of historesin into the crystal. Ethanol solutions of 50, 70, 80, 90, and 95% were prepared. The crystals were soaked in the solutions sequentially from 50 to 95% ethanol for 30 min each. The embedding procedures described here follow the general instructions.9 The fixed crystals are soaked for 1 h in a 1:1 mixture of ethanol and historesin, followed by overnight soaking in 100% historesin. After they were soaked, the crystals were set in a container with a desired direction. Resin polimerization was performed by mixing 20× the amount of historesin with a hardener at room temperature for 2 h. The embedded crystals were cut into slices passing through the crystal center. Slices were cut by a microtome (RM2145 Leica) with a tungsten carbide blade. The slices were extended in water, dried on a slide glass, and then enclosed by a generally used enclosure, Entellan new, and covered by a cover glass. Note that the thickness of the slices is not accurate enough to guarantee quantitative measurement of the fluorescence. Therefore, further fluorescence observations are qualitative. 3. Application of the Embedding Method To Impurity Distribution Analysis In this section, we demonstrate the validity of the embedding method in the observation of impurity distributions inside protein crystals. Lysozyme was crystallized with a fluorescence-labeled impurity, processed by the embedding method, and observed by CLSM. 3.1. Impurity Distribution Inside Tetragonal Lysozyme Crystals. Figure 3a illustrates the morphology of a tetragonal lysozyme crystal.22,23 The crystal consists of four {110} sectors and eight {101} sectors. Every sector is shown in Figure 3b. The impurity
Crystal Growth & Design, Vol. 5, No. 1, 2005 297
Figure 3. Morphology of a tetragonal lysozyme crystal. (a) Morphology with crystal axes. (b) Morphology with sectors.
incorporation rate differs between the two sector types. For this reason, the sector structure can be visualized as a different fluorescence intensity by one or two photon fluorescence microscopy or CLSM when a fluorescence-labeled impurity is added to the crystallization solution.6,7,23-25 A different sector structure is observed in each crystal slice since sectors are formed three-dimensionally, as illustrated in Figure 3b. The sector structures expected when the crystal is cut perpendicular to the c-axis or parallel to the c-axis and {110} face are illustrated in Figure 4. There are only {110} sectors in Figure 4a1 that are the slice containing the crystal center. {101} sectors appear in slices far from the center and become larger with the distance from the center. Also, in the direction of Figure 4b, the sector structure changes with the slicing position. Figure 4a1,b1 shows slices including the crystal center. Thus, these slices contain information of the impurity distribution according to the distribution coefficient. For example, when the distribution coefficient exceeds 1, the incorporated impurity concentration decreases from the center to the surface.6 3.2. Crystallization. Lysozyme was crystallized in 1% agarose gel (low melting point) with crystallization conditions of 15 mg/mL lysozyme, 0.075 mg/mL fluo-
298
Crystal Growth & Design, Vol. 5, No. 1, 2005
Iimura et al.
Figure 4. Expected sector structures in slices at different positions.
Figure 5. Images of slices observed by CLSM. The crystal was cut perpendicular to the c-axis. The photograph is 300 µm2.
rescence-labeled lysozyme dimer (FL dimer), and 6% NaCl in 50 mM sodium acetate buffer (pH 4.5). Highly purified dimer was labeled with a labeling kit (Alexa Fluor 488, Molecular Probes)6,7 according to the instruction of the kit. The labeling efficiency (the number of Alexa molecules attached to a dimer) was 0.68. FL dimer is incorporated into tetragonal lysozyme crystals, and the effective distribution coefficient (K) at 6% NaCl is about 1.2.6 To demonstrate the validity of the embedding method, we tried to obtain crystals in agarose gel where crystals grow equally in all directions. Because the gel net structure captures small crystals nucleated homogeneously in the solution, crystals do not sediment and tend to grow equally in all directions. The hanging drop
method was not used here because we wanted to keep the precipitant concentration constant since the distribution coefficient changed with the precipitant concentration.6 3.3. Embedding Crystals in Agarose Gel. The crystals were caught in agarose gel, so the fixation was performed in gel by adding the fixation solution, 20× volume of the gel. The whole gel became cloudy because of the aggregation of lysozyme that existed in the solution. After 2 days of fixation, the gel was dissolved by 10 min of incubation at 70 °C and removed by pipetting. The fixed crystals were collected and treated according to the procedure in section 2.5. The embedded crystals were sectioned into slices along the directions to obtain slices as shown in Figure 4. The thickness of slices is variable from 0.5 to 60 µm in our system. Here,
Protein Crystal Fixation for Growth Studies
Crystal Growth & Design, Vol. 5, No. 1, 2005 299
Figure 6. Images of slices observed by CLSM. The crystal was cut parallel to the c-axis. The photograph is 350 µm2.
we sectioned the slices in 10 µm thicknesses to limit the number of slices to 20-30. 3.4. CLSM Observation. The impurity distributions in the slices were visualized by CLSM (TCS-4D Leica) with Ar 488 nm. Images corresponding to Figure 4a,b are depicted in Figures 5 and 6. Crossing of the sector boundary indicates that the slice including the center is in Figure 5n. It is apparent that this corresponds to Figure 4a1. {101} sectors appeared, and the sector shape changed with distance from the center. Such sector structures are shown in Figure 4a2 and are visualized from panels k to m and from panels p to r in Figure 5. The change of the morphology expected as Figure 4a3 was observed in Figure 5f,g,v,w. Images corresponding to Figure 4a4 containing the end of the crystal were visualized in Figure 5a,aa. The crystal was sectioned quite well vertical to the c-axis, since the slices at both ends were obtained as shown in Figure 4. The crystal was slightly deformed as seen in Figure 5g-j, in which the surface of the crystal was broken. Figure 6 also presents a successful result. The crystal center was included in Figure 6n, which corresponded to Figure 4b1. Areas above and below the center were visualized as the same as Figure 4b2-b4. Next, we analyzed the fluorescence intensity distribution along the line crossing the center in Figures 5n and 6n. Figure 7 demonstrates that the fluorescence intensity decreased from the center to the surface in both slices. The impurity distribution coefficient at the crystallization condition (K ) 1.2) is represented. We confirmed that processing by the embedding method was possible without disturbing the impurity distribution inside crystals.
Figure 7. Fluorescence intensity distributions along the line crossing the crystal center. (a) Fluorescence intensity distribution of Figure 5n. (b) Fluorescence intensity distribution of Figure 6n.
4. Summary The embedding method was developed as a processing technique to qualitatively visualize the impurity distribution inside protein crystals. The method consists of three procedures: (i) chemical fixation by glutaraldehyde, (ii) embedding by resin, and (iii) slicing by a
300
Crystal Growth & Design, Vol. 5, No. 1, 2005
microtome. The fixation step was examined in detail, and the optimal fixation state was attained by using CRG of glutaraldehyde, which contains oligomers. The embedding method was applied to tetragonal lysozyme crystals contaminated with FL-dimer. Continuous slices of the crystals were processed. We used CLSM and succeeded in visualizing the sector structure and the tendency of impurity distribution according to the distribution coefficient as expected, confirming that the crystal inner structure was maintained after processing. We believe that the embedding method can be widely applied to almost all protein crystals that include lysine residues because glutaraldehyde mainly reacts with lysine residue.3 When the target protein has no lysine residues, another cross-linking reagent might be able to fix the crystals. Also, this method can be applied for other purposes, such as successive defect observation inside the crystal or electron microscopy observation of the crystal surface. Further applications are currently under investigation. References (1) Quiocho, F. A.; Richards, F. M. Proc. Natl. Acad. Sci. 1964, 52, 833-839. (2) Quiocho, F. A. Insolubilized Enzymes 1974, 113-122. (3) Yonath, A.; Sielecki, A.; Moult, J.; Podjarny, A.; Traub, W. Biochemistry 1977, 16, 1413-1417. (4) Ha¨ring, D.; Schreier, P. Curr. Opin. Chem. Biol. 1999, 3, 35-38. (5) Morozov, V. N.; Morozova, T. Y. Biopolymers 1981, 20, 451467. (6) Iimura, Y.; Yoshizaki, I.; Nakamura, H.; Yoda, S.; Komatsu, H. Cryst. Growth Des. Submitted for publication.
Iimura et al. (7) Iimura, Y.; Yoshizaki, I.; Nakamura, H.; Yoda, S.; Komatsu, H. Jpn. J. Appl. Phys. 2003, 42, 5831-5836. (8) Hell, S.; Reiner, G.; Cremer, C.; Stelzer, E. H. K. J. Microsc. 1993, 169, 391-405. (9) Pender, M. P. J. Neurosci. Methods 1985, 15, 213-218. (10) Sabatini, D. D.; Bensch, K. G.; Barrnett, R. J. Anat. Rec. 1962, 142, 274. (11) Sabatini, D. D.; Bensch, K. G.; Barrnett, R. J. J. Cell Biol. 1963, 17, 19-58. (12) Anderson, P. J. J. Histochem. Cytochem. 1967, 15, 652661. (13) Prento, P. Histochem. J. 1995, 27, 906-913. (14) Monsan, P.; Puzo, G.; Mazarguil, H. Biochimie 1975, 57, 1281-1292. (15) Peters, K.; Richards, F. M. Annu. Rev. Biochem. 1977, 46, 523-551. (16) Lusty, C. J. J. Appl. Crystallogr. 1999, 32, 106-112. (17) Haas, D. J. Biophys. J. 1968, 8, 549-555. (18) Trelstad, R. L. J. Histochem. Cytochem. 1969, 17, 756-757. (19) Korn, A. H.; Feairheller, S. H.; Filachione, E. M. J. Mol. Biol. 1972, 65, 525-529. (20) Maser, M. D.; Powell, T. E.; Philpott, C. W., III. Stain Technol. 1967, 42, 175-182. (21) Cacioppo, E.; Pusey, M. L. J. Cryst. Growth 1991, 114, 286292. (22) Monaco, L. A.; Rosenberger, F. J. Cryst. Growth 1993, 129, 465-484. (23) Caylor, C. L.; Dobrianov, I.; Lemay, S. G.; Kimmer, C.; Kriminski, S.; Finkelstein, K. D.; Zipfel, W.; Webb, W. W.; Thomas, B. R.; Chernov, A. A.; Thorne, R. E. Proteins 1999, 36, 270-281. (24) Caylor, C. L.; Dobrianov, I.; Kimmer, C.; Thorne, E. Phys. Rev. E 1999, 59, R3831-R3834. (25) Kurihara, K.; Miyashita, S.; Sazaki, G.; Nakada, T.; Durbin, S. D.; Komatsu, H.; Ohba, T.; Ohki, K. J. Cryst. Growth 1999, 196, 285-290.
CG049918I
