Polysaccharides as Precipitants in Protein Crystallization for X-Ray

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Polysaccharides as Precipitants in Protein Crystallization for X-Ray Diffraction Studies Published as part of the Crystal Growth & Design virtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13). Laura Veps€al€ainen,† Katja Palmunen,‡ Sinikka Uotila,‡ Kalevi Visuri,‡ Juha Rouvinen,† and Johanna M. Kallio*,†,§ †

Department of Chemistry, University of Eastern Finland, P.O. Box 111, 80101 Joensuu, Finland Macrocrystal Oy, Tillinm€aentie 1 F, 02330 Espoo, Finland § EMBL Hamburg c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany ‡

ABSTRACT: A novel polysaccharide-based method for protein crystallization has been developed for industrial purposes. In this paper, we describe the application of this new method in crystal growth for X-ray diffraction structural studies of proteins. We have tested the crystallization of three commercially available proteins—lysozyme, xylose isomerase, and xylanase—and of two research targets—the amphiphilic protein hydrophobins HFBI and HFBII—on the polysaccharides alginate, chitosan, pectin, and dextrin, and enzymatic hydrolysates of alginate and pectin. With the use of polysaccharides, we managed to grow crystals of each protein. Crystals of lysozyme, xylose isomerase, and xylanase were suitable for X-ray diffraction studies on the basis of size, shape, and diffraction tests. Data were collected on xylanase crystals, and the structure was refined to ensure that the polysaccharide has no effect on structure determination. Both hydrophobins also yielded crystals suitable for X-ray analysis, once a suitable additive was used in combination with the polysaccharide. Thus, we conclude that this novel method is applicable also in protein structural studies. We suggest that the underlying phenomena in crystallization with polysaccharides is the formation of a polysaccharide
protein phase where the phase boundary acts as the initiation point for crystal formation.

’ INTRODUCTION X-ray diffraction analysis allows the structural characterization of ordered material. This is why proteins must be crystallized prior to data collection. Various methods have been developed to establish crystalline protein, since the crystallization cannot be considered a trivial task.1
3 Based on the previous experiments and research, many crystallization screens have been designed either to probe for preliminary crystallization or to assist in the optimization of the crystallization conditions.4
7 Most of these screens are based on the presence of a precipitant, which could be a salt, a polymer, or an organic solvent and a buffer. Some screens are commercially available; some are the heritage of the laboratory and mostly in internal use. Several methods8 are also applied in protein crystallization, such as dialysis, vapor diffusion, the batch method, and crystallization under oil or in a gel.9
11 Several variations of these techniques also exist, such as hanging and sitting drop experiments, the counter-diffusion approach,12 and microscaled13
15 procedures. Protein crystallization may be used as a purification procedure or for a specific industrial purpose, in addition to the r 2011 American Chemical Society

aims of structural biology. Hence, the convenient crystallization method may be selected according to the purpose and desired quality of the crystals. To facilitate the extensive screening of a large amount of conditions, high-throughput pipelines have been developed for the use of structural biology. These pipelines make use of automation in the form of robots mixing and pipetting the crystallization solution and detection of crystals by combination of cameras and software. A novel approach in the crystallization of proteins has been described in a patent16,17 for large-scale protein crystallization processes. This innovation concerns the use of polysaccharides as principal precipitants in the crystallization. Simple sugars and their derivatives are often used as additives in the crystallization or soaked into the crystal to probe the substrate binding. Sugars, such as glucose, xylitol, or sucrose, have also been successfully used as cryoprotectants in X-ray diffraction studies. However, to Received: October 14, 2010 Revised: February 24, 2011 Published: March 21, 2011 1152

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Crystal Growth & Design our knowledge and according to the biological macromolecule crystallization database,18 the use of polysaccharides as the main precipitating agent has not been described earlier. Many screens exist, however, that are based on use of other polymers, such as polyethylene glycols and polyglutamic acid.19 The polysaccharide agarose, along with other gels of natural or synthetic polymers, such gelatin, polyacryl amide, and silica, has to be used in crystallization in the role of the gel-forming agent, however, always in combination with a distinct precipitant. The polysaccharides described in the patent are alginate, chitosan, pectin, and dextrin. Alginate is produced by brown algae, and it is a linear, unbranched polymer of (1
4)-linked βD-mannuronate and R-L-guluronate. Chitosan is a linear polysaccharide with randomly distributed β-(1
4)-linked units of D-glucosamine and N-acetyl-D-glucosamine and produced commercially by deacetylation of chitin. Pectin is a heteropolysaccharide found in fruits, vegetables, and berries, consisting of an R-(1
4)-linked D-galacturonic acid backbone with various branches of sugars, such as D-xylose, L-fructose, and D-glucuronic acid. Dextrin is a low-molecular-weight carbohydrate produced by the hydrolysis of starch. For crystallization, the aqueous solutions of the above-mentioned polysaccharides are mixed with the protein solution with the result of protein crystals being produced. Usually a buffer is present, in order to keep the pH optimal, and sometimes a protein specific additive may also be used. The concentrations of the polysaccharide solutions used are low, especially for alginate, chitosan, and pectin, typically in the range of less than 5 mass %. Some of the solutions are viscous and stiff, and the mixtures can also be gelated, if desired. However, gel-formation is not a requirement for crystallization. The crystallization method with polysaccharides described in the patent of Macrocrystal Oy is designed to be used for largescale batch precipitation or crystallization of proteins. The batch volume in such a process may vary from hundreds of milliliters up to liters, and the primary objective is the purification of protein. Advantages of the polysaccharide method include very low reagent consumption when compared to salting out or PEG methods, and it does not require high protein concentration, like many traditional precipitation and crystallization methods. Usually proteins separate from the polysaccharide solutions as highly concentrated liquid droplets. Under optimized conditions, the crystals start to grow from these droplets. The aim is not in producing large single crystals as in crystallography but rather in manufacturing homogeneous crystalline material. The method has been applied to plasma proteins, peptide hormones, and microbial enzymes, as well as to polyclonal and monoclonal antibodies. We have now applied the polysaccharide crystallization also to production of diffraction quality crystals to be used in X-ray crystallographic studies and described our structural biology approach in this paper.

’ MATERIALS AND METHODS Polysaccharides. The polysaccharides were purchased from Sigma Aldrich for crystallization trials. The polysaccharides were alginic acid sodium salt from brown algae (CAS-number 9005-38-3), chitosan from crab shells, practical grade (CAS-number 9012-76-4), pectin from citrus peel (CAS-number 9000-69-5), and Dextrin 15 from maize starch (CASnumber 9050-36-6). These polysaccharides were then dissolved in MilliQ water or, in the case of chitosan, to 0.1 M sodium acetate buffer at the pH values 4.6 and 5.5. Dextrin was found readily soluble and dissolved to a final concentration of 30 mass %. However, most likely due to larger

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chain length, alginate, pectin, and chitosan were found poorly soluble and dissolved to final concentrations of 2.5, 2.5, and 0.8 mass %, respectively. The solutions of alginate, pectin, and chitosan were stiff and viscous, whereas dextrin appeared to be a water-like solution. Products of limited enzymatic hydrolysis of alginate and pectin were also used in the crystallization trials. Alginate was hydrolyzed by alginate lyase for a minimum of 24 h with the ratio of 5 mg of enzyme per 1 g of alginate at 40 °C. Pectin was hydrolyzed by pectinase for a minimum of 21 h with the ratio of 0.01
0.1 mg of enzyme per 1 g of pectin at 40 °C. The hydrolyses were terminated by boiling, the solutions were filtered and lyophilized. Hydrolysates proved to be more soluble than the intact polysaccharides, and thus, the stock solutions were made by dissolving alginate hydrolysate and pectin hydrolysate into Milli-Q water to final concentrations of 9.4 mass % and 6.0 mass %, respectively. Protein Materials. The crystallization potential of polysaccharides was tested with commercially available proteins: lysozyme from hen egg white (HEWL), xylose isomerase from Strepromyces rubiginosus (XI), and xylanase from Trichoderma reesei (XYNII). Lysozyme was purchased from Sigma Aldrich (product number L-6876, lot 57H7045), and xylose isomerase (H 301-303) and xylanase (GC-140, lot 301-99136-098) were prepared at Macrocrystal Oy. The lyophilized protein material of HEWL was dissolved in Milli-Q water to a concentration of 40 mg/mL. The stock solution (crystalline suspension) of XI was dialyzed against Milli-Q water for 48 h in order to solubilize the protein material. After dialysis, the protein solution was filtered with a 0.22 μm Ultrafree filter unit and the concentration was determined to be 15 mg/mL by A280. XYNII was also provided as a crystalline suspension. The suspension was centrifuged to the bottom of a container, the supernatant discarded, and the pellet dialyzed against Milli-Q water. The protein concentration was determined to be 10 mg/mL (A280). In addition to the commercially available proteins, the polysaccharide crystallization method was also applied on two research target proteins: hydrophobins HFBI and HFBII from Trichoderma reesei. Hydrophobins were included in the study to expand the testing of crystallization power of polysaccharides on research targets in addition to commercially available proteins. Hydrophobins HFBI and HFBII are amphiphiles, which are generally regarded as challenging to crystallize. Hydrophobin materials20,21 for crystallization trials were provided by Prof. Markus Linder at VTT Technical Research Center, Finland. The lyophilized protein material was dissolved in Milli-Q water to a concentration of 8 mg/mL for both hydrophobins HFBI and HFBII. Initial Crystallization Screen. The initial screen of polysaccharide solutions (Table S1, Supporting Information) was constructed by forming a dilution series of each polysaccharide solution and adding a buffer. For alginate and pectin, the 2.5 mass % stock solutions were diluted to 2%, 1%, and 0.5%. For Dextrin, the 30 mass % stock solution was diluted to 20%, 10%, and 5%. For chitosan, the 0.8 mass % stock solutions in 0.1 M sodium acetate buffer (pH 4.6 and 5.5) were diluted to 0.4% and 0.2% with 0.1 M sodium acetate buffer at corresponding pH values. The diluted solutions of alginate, pectin, and dextrin were prepared so that the final concentration of the buffer in solution was 0.1 M. The buffers used were sodium acetate at pH 4.6, Na-HEPES at pH 7.0, and Tris-HCl at pH 8.5. Similarly, with the use of these buffers, the stock solutions of alginate hydrolysate and pectin hydrolysate were diluted to 8%, 4%, and 2% and 4%, 2%, and 1%, respectively. Including the nonbuffered stock solutions of alginate, pectin, and dextrin, the initial crystallization screen consisted of the 48 polysaccharide solutions mentioned above. Crystallization Procedure. The crystallization trials were conducted mostly using the hanging-drop vapor-diffusion method at room temperature. Some trials were done at þ4 °C to evaluate the effect of the temperature, and the sitting-drop vapor-diffusion method was also tested. Crystallization was also tested as a high-throughput application with a microbatch method using crystallization robot IMPAX I-6 by Douglas Instruments. 1153

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Figure 1. Crystals of lysozyme, xylose isomerase, and xylanase, respectively, produced by the polysaccharide method. The manual crystallization experiments were performed on a 24-well Cell-star plate using siliconized cover slides. Equal amounts of protein and polysaccharide solution were pipetted on the cover slide. The drop size varied from 4 to 10 μL; however, it was generally advisable to use a large droplet volume, since pipetting small, exact amounts of viscous polysaccharide solutions was challenging. Especially the stock solutions of alginate, pectin, and chitosan and the hydrolysates presented problems when pipetted in small volumes. The diffusion was created in the experiment by pipetting 0.5 mL of 0.1
0.2 M sodium chloride solution into the well prior to inverting the cover slide containing the crystallization drop onto the well. However, the reservoir solution was not added to the crystallization drop (as is typically done in the vapordiffusion experiment) and the well solution did not contain polysaccharide solution or protein. The protein concentrations used in the initial screening experiments were 4 mg/mL, 4 mg/mL, 20 mg/mL, 10 mg/mL, and 15 mg/mL for HFBI, HFBII, HEWL, XYNII, and XI, respectively. Data Collection. The crystals originating from the polysaccharidebased screens were tested and the data collected with the home laboratory X-ray source. The X-ray source is a FR591 rotating anode by Bruker/Nonius, and the setup is equipped with Osmic confocal optics, a MarResearch Desktop Beamline goniometer, an Oxford cryosystems Cryostream 700, and a Mar345 Image Plate detector. The X-ray diffraction tests were performed at 100 K using dry paraffin oil or 35% glycerol as cryoprotectant.

’ RESULTS AND DISCUSSION Lysozyme. Initial screening produced small, needle-like crystals or bigger, irregular-shape crystals of lysozyme from droplets containing 0.5
2% alginate in 0.1 M Tris-HCl at pH 8.5. The crystallization conditions were optimized, and single crystals of about 300 μm from the largest dimension were obtained from conditions of 0.5% alginate in 0.1 M Tris-HCl at pH 8 (Figure 1). The protein concentration was lowered to 10 mg/mL, and the 10 μL droplet contained equal amounts of protein and polysaccharide solutions. The reservoir solution was 0.5 mL of 0.1 M sodium chloride. The crystals, on average of size 0.3 mm  0.2 mm  0.2 mm, grew within a few days. For HEWL, there are about 250 entries in the PDB, and the crystallization conditions in these entries mainly correspond to sodium chloride (0.6
1.1 M) at low pH, typically using sodium acetate at pH 4.2
4.8. In spite of the preference for low pH crystallization conditions, lysozyme can be crystallized with

various buffers (MES, HEPES, Tris, citrate) in the pH range 4.2
9.6, as was also the case in the polysaccharide crystallization. Sodium chloride was also present in the polysaccharide experiment: even though it was not added to the crystallization droplet, sodium chloride was used as well as solution to create vapordiffusion; that is, the concentration difference between the wells and the droplet. However, the concentration of sodium chloride in the well was considerably less (0.1 M) than what is typically used in lysozyme crystallization, and we were also able to produce lysozyme crystals by replacing NaCl in the well with another salt, e.g. ammonium sulfate. When tested with X-ray radiation, the crystals of HEWL produced by the polysaccharide method diffracted to a maximum of about 2.0 Å. The diffraction power of the crystals could likely be enhanced by further optimization of the crystallization conditions and by developing a suitable cryoprotection protocol. The processing indicated a primitive tetragonal space group with unit cell parameters a = 77 Å, b = 77 Å, c = 38 Å. The space group P43212 with similar unit cell parameters is frequently found for lysozyme in the Protein Data Bank. Xylose Isomerase. Initial screening produced tiny xylose isomerase crystals from several conditions: the stock solutions of chitosan and alginate with no buffer, 1% pectin hydrolysate in 0.1 M Na-HEPES at pH 7.0, and 2% pectin in 0.1 M Tris-buffer at pH 8.5. The crystallization conditions were further optimized, and the largest single crystals (Figure 1) were produced from a solution containing 1% pectin hydrolysate in 0.1 M Na-HEPES at pH 7.0. The protein concentration was 15 mg/mL, and the 10
14 μL sitting drops contained equal amounts of protein and polysaccharide solutions. The reservoir solution was 0.5 mL of 0.1 M sodium chloride, and the crystallization took place at þ4 °C. The time taken up by crystal formation varied from one week to three months, and the crystal size was 0.2 mm  0.2 mm  0.04 mm on average. In the 33 PDB entries for XI, the most common crystallization condition was 1.5
2.5 M ammonium sulfate with pH 6.0
9.0. An alternative precipitant was 10
20% MPD in combination with a salt, such as ammonium sulfate or magnesium chloride. The condition in which the XI crystals grew in using the polysaccharide method is with no salt in the previously observed pH range. The crystals diffracted to a maximum of 3 Å. As in the case of lysozyme, the diffraction limit could likely be improved by 1154

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optimization. In some cases, the xylose isomerase crystals aged very fast and started to visually degrade after a few days. However, this behavior was not observed with any other protein or with all of the xylose isomerase crystals. Xylanase. Initial screening produced no crystals of xylanase. Some precipitant was formed in the droplets of pectin and pectin hydrolysate at higher concentrations of the polysaccharide. To probe a larger scale of pH, a polysaccharide screen to be used with the crystallization robot was constructed. This screen (Table S2, Supporting Information) contained the solutions of the initial screen and solutions of alginate, pectin, and dextrin with a varying pH from 3.5 to 9.0 in diverse buffers, altogether 88 conditions. This larger screen was pipetted using the crystallization robot IMPAXI-6 and xylanase in a concentration of 10 mg/mL. The droplet size was 1 μL and contained a 50% protein solution. The hydrophilic vapor batch plates were used for screening, and the crystallization drop was covered by 7 μL of paraffin oil immediately after pipetting. Finally, with all the pipetting done, the Table 1. Data Collection and Refinement Statistics of Xylanase a (Å)

45.75

b (Å)

38.73

c (Å)

56.70

β (deg) space group

107.40 P21

source

Copper rotating anode

wavelength (Å)

1.5418

resolution range (Å)

20
1.6 (1.7
1.6)

no. of observations

83566

no. of unique reflections

24344 (3364)

completeness (%)

96.2 (80.5)

Rmeas (%) I/σ(I)

6.5 (33.2) 16.29 (5.33)

R (%)

15.4

Rfree (%)

18.7

rmsd bond length (Å)

0.006

rmsd bond angle (deg)

1.139

no. protein atoms

1486

no. of water molecules

221

no. of other atoms average B-factor (Å2)

12 17.4

crystal tray was filled with 5 mL of Al’s Oil. Small crystals of irregular shape formed from conditions of 1% pectin in 0.1 M sodium citrate at pH 4.0. Optimizing crystallization conditions finally produced a large crystal of about 0.7 mm  0.15 mm  0.1 mm (Figure 1) from 5% alginate hydrolysate at pH 9.0 using Tris-HCl as a buffer. The crystal grew in about three weeks. The data were collected on large crystals, crystallized with alginate hydrolysate, to maximum resolution of 1.6 Å (maximum diffraction for the setup). The data collection statistics are presented in Table 1. The structure determination was completed in order to ensure the crystallization method had no effect on the data quality, the structure, or the process of structure determination (for PDB and CIF files, see the Supporting Information). The structure consisted of 1486 protein atoms, equal to one xylanase molecule (190 residues), 221 water molecules, and two glycerol molecules (originating from the cryoprotectant solution) in the asymmetric unit (Figure 2). The Matthew’s coefficient was calculated to 2.28, and the solvent content was 46%. The space group was determined to be P21 with unit cell parameters a = 45.75 Å, b = 38.73 Å, c = 56.70 Å, and β = 107.40°. The data were processed with XDS22 and the structure determined and refined with Phenix23 using Coot24 to inspect the model. The structure was determined using PDB-ID 2DFB as search model in molecular replacement. There are nine previous PDB-entries of xylanase II from Trichoderma reesei: 2D97,25 2DFB, 2DFC,26 1RED, 1REE, 1REF,27 1ENX, 1XYO, and 1XYP.28 All of the previous crystals have been grown with the use of ammonium acetate as the precipitant. Entries 2DFB and 2DFC describe and orthorhombic crystal form, while the remaining seven entries are monoclinic P21, similarly to crystals produced with the polysaccharide method. However, the unit cell parameters differ in such a way that a = 82 Å, b = 61 Å, c = 38 Å, and β = 94° for monoclinic PDB-entries (excluding entry 2D97), and the asymmetric unit contains two molecules. Entry 2D97 has unit cell parameters a = 40.35 Å, b = 38.58 Å, c = 57.16 Å, and β = 110.31° and, alike the data from the polysaccharide method, contains one molecule in the asymmetric unit. 2DFB and 2DFC have been crystallized at pH 9.0, similarly to the polysaccharide method, while the pH ranges from 4.3 to 6.5 for the monoclinic PDB entries. The resolution of the data is 1.1 Å for entries 2DFB and 2DFC, which have been collected with a synchrotron source, and in the range 1.5
1.8 Å for all the other entries collected with the home source, except for entry 2D97, with resolution 2.0 Å with the home source.

Figure 2. (a) Cartoon representation of the structure of XYNII determined after crystallization by polysaccharide methods. Attached glycerols shown in yellow and red. (b) Superimposition of all XYNII PDB-entries on (a) ribbon representation. Entries 1ENXa, 1XYOa, and 1XYOa in red; 1REDa and 1REEa in pink; 1ENXb, 1XYOb, 1XYPb, and 1REFa in yellow; 2DFB and 2DFC in green; 1REDb, 1REEb, 1REFb, and 2D97 in cyan; and (a) in blue. 1155

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Figure 3. Crystals of HFBI in combination with zinc ions, HFBII, and HFBII in combination with manganese ions, respectively, produced by the polysaccharide method.

The conformational states are slightly different in the XYNII structures, with a similar overall fold of 14 β-strands and an R-helix. The β-strands are organized in two twisted sheets. Entries 1RED, 1REE, and 1REF contain an inhibitor molecule at the substrate binding site of molecules B in the asymmetric unit. Due to cryosoak, also the crystals produced with the polysaccharide method host additional molecules, glycerols, in the active site. Possibly for this reason, the conformational state of xylanase is closest to those of entries 1REDb, 1REEb, and 1REFb. Also, the conformation in entry 2D97 is very alike. However, the overall conformation is very similar in all the determined xylanase structures. Hydrophobin HFBI. HFBI had been previously crystallized with 0.1 M zinc sulfate and 0.1 M sodium cacodylate at pH 6.5.29 Initial screening of HFBI with polysaccharides produced no crystals. However, the presence of zinc ions had previously been recognized to be critical to crystal formation. Bearing this in mind, 0.5 μL of 0.5 M zinc sulfate was added to the conditions of the initial screen and the volume of the polysaccharide solution in the 5 μL crystallization drop was reduced to 2 μL. In this way, using zinc ions as additive, crystals formed in all the droplets. They were of similar appearance (Figure 3), as previously observed for HFBI, rectangular, and fairly large (0.2 mm  0.05 mm  0.05 mm on average). However, it could be concluded that, most likely, the crystals formed in spite of, rather than because of, the polysaccharide solutions and simply due to the presence of zinc ions. Zinc ions also caused the polysaccharide solutions to gelate, forming a very strong gel, hardly permeable with a cryo loop. Gelation appeared similarly with all the polysaccharides, excluding dextrin. The crystals produced with zinc were analyzed with X-ray diffraction. The crystals diffracted X-rays poorly (8 Å), as is typical of the crystal form of HFBI in the presence of zinc ions.

Hydrophobin HFBII. HFBII has been crystallized from a solution containing 0.2 M lithium sulfate, 25% polyethylene glycol (MW 2000), and 0.1 M Na-HEPES at pH 7.5.30 Different crystal forms of HFBII have been produced by addition of manganese ions or the detergent heptyl-β-D-thioglucoside to the crystallization solution.31 Initial screening of HFBII produced needle-like crystals with the stock solution of alginate (pH 6.3) and with 2% pectin hydrolysate in 0.1 M Na-HEPES at pH 7.0. The crystallization condition with alginate was further optimized, and bigger, needle-like crystals (Figure 3) formed with 2% alginate without any buffer and with a protein concentration of 4 mg/mL. The length of the needles varied from 0.2 to 2 mm. The droplet size was 10 μL, containing equal volumes of protein and polysaccharide solutions. The reservoir solution was 0.1 M sodium chloride. As the addition of manganese ions or a detergent had previously influenced the crystallization of HFBII, these were also tested in combination with the stock solution of alginate as precipitant. The 5 μL droplet was formulated by 2.5 μL of HFBII, 2 μL of 2.5% alginate, and 0.5 μL of either 0.5 M manganese chloride or 300 mM heptyl-β-D-thioglucoside (HSG). Crystals grew in the presence of both the metal ion and the detergent. Crystals formed in the presence of manganese chloride (Figure 3) were rectangular and visually resembled the previously described crystals of HFBII in combination with MnCl2, salt, and buffer. The crystal size was very small, with the largest edge of approximately 0.05 mm. However, the crystals grown in the presence of the detergent appeared very similar to crystals grown with alginate only; that is, they were needle-like. The crystals grown with the detergent in combination with salt and buffer were usually thin plates. The diffraction testing of HFBII crystals with a home source was not feasible due to the crystal size. 1156

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Table 2. Summary of the Success Rate of Polysaccharides as Precipitantsa alginate LYSO

0.5%

XI

stock

XYN

a

pectin

2%

chitosan

dextrin

alginate hydrolysate

stock

1%

1%

HFBI

þ Zn

HFBII

2%, þMn, þHSG

þ Zn

pectin hydrolysate

5% þ Zn

þ Zn

þ Zn 2%

The concentration of the polysaccharide or the additive present that produced crystals of the protein is given. Empty slot indicates no crystals.

Comparison of the Polysaccharides as Precipitants. Table 2 summarizes the success rate of each polysaccharide as precipitant. Alginate was the most successful precipitant, producing crystals of four different proteins, while dextrin was the least favored precipitant, only producing crystals of HFBI, which crystallized irrespective of the precipitant in the presence of zinc ions. The pH ranged from 4.0 to 9.0 in successful crystallization experiments. The principles underlying the crystallization are not easy to comprehend, as the variables in the process are so numerous. However, during the crystallization experiments, some phase separation was often observed, which has lead us to believe that the underlying principle of the polysaccharide-based crystallization might be the formation of a two-phase system. A phenomenon termed coaservation32 describes an associative phase separation of protein and polysaccharide in water, leading to formation of protein
polysaccharide containing droplets within a liquid phase. As the viscosity of polysaccharide solutions increases with increasing concentration, it is unclear whether the protein
polysaccharide phase resembles more of a gel or liquid in our crystallization experiments. Possibly due to high local concentration of the protein in the protein
polysaccharide phase, crystallization is favored and the phase-interface might act as the starting point of the crystallization. The phase separation also seems to vanish during the crystal formation, which would indicate the resolubilization of the polysaccharide after crystal growth has consumed the protein from the associative droplets.

’ CONCLUSIONS Based on the initial testing presented here, we conclude that the polysaccharide crystallization method is applicable and feasible for use in crystallization X-ray structure determination. High-throughput crystallization (using a crystallization robot) proved to be possible in spite of the viscosity of the polysaccharide solutions. Alginate was found to be the most effective precipitant of the polysaccharides tested in this study.The benefits of the method include the use of nontoxic chemicals and salt-free crystallization conditions, which reduce the number of false positives. The method is also very suitable to probe a preferred additive or to find a new crystal form. The method produces no attaching ions or small molecules to the structure, unless desired. The xylanase diffraction data shows that the structure determined with this crystallization method is of good quality. ’ ASSOCIATED CONTENT

bS

Supporting Information. Polysaccharide screen for initial screening, enlarged polysaccharide screen used with the crystallization robot, the coordinate file (PDB) of the xylanase

XYNII structure determined by the polysaccharide crystallization method, and the structure factor file (CIF) of the xylanase XYNII structure determined by the polysaccharide crystallization method. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: þ49 40 89 902 176. Fax þ49 40 89 902 149. E-mail address: [email protected].

’ ACKNOWLEDGMENT Funding from the MABIO-project (European Union Social Fund, State provincial office of Eastern Finland) and the Academy of Finland (J.M.K.) is gratefully acknowledged. The authors thank Prof. Markus Linder at VTT Technical Research Centre, Finland, for providing the hydrophobin material and Mrs. Reetta Kallio-Ratilainen for skilled technical assistance. ’ REFERENCES (1) McPherson, A. Crystallization of biological macromolecules; Cold Spring Harbor Laboratory Press: New York, 1999. (2) Bergfors, T., Ed. Protein Crystallization: Techniques, Strategies, and Tips; International University Line: La Jolla, CA, 1999. (3) Ducruix, A., Giege, R., Eds. Crystallization of Nucleic Acids and Proteins a practical approach, 2nd ed.; Oxford University Press: New York, 1999. (4) Jancarik, J.; Kim, S.-H. J. Appl. Crystallogr. 1991, 24, 409–411. (5) Bzozowski, A. M.; Walton, J. J. Appl. Crystallogr. 2001, 34, 97–101. (6) Gilliland, G. L.; Tung, M.; Ladner, J. E. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 916–920. (7) McPherson, A.; Cudney, B. J. Struct. Biol. 2006, 156, 387–406. (8) Bolanos-Garcia, V. M.; Chayen, N. E. Prog. Biochem. Biophys. 2009, 101, 3–12. (9) Chayen, N. J. Cryst. Growth 1999, 196, 434–441. (10) Robert, M. C.; Lefaucheux, F. J. Cryst. Growth 1988, 90, 358–367. (11) Lorber, B.; Sauter, C.; Theobald-Dietrich, A.; Moreno, A.; Schellenberger, P.; Robert, M. C.; Capelle, B.; Sanglier, S.; Potier, N.; Giege, R. Prog. Biochem. Biophys. 2009, 101, 13–25. (12) Garcia-Ruiz, J. M.; Gonzalez-Ramirez, L. A.; Gavira, J. A.; Otalora, F. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 1638–1642. (13) Korczynska, J.; Hu, T.-C.; Smith, D. K.; Jenkins, J.; Lewis, R.; Edwards, T.; Brzozowski, A. M. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2007, 63, 1009–1015. (14) Chayen, N. E.; Shaw Stewart, P. D.; Maeder, D. L.; Blow, D. M. J. Appl. Crystallogr. 1990, 23, 297–302. (15) D’Arcy, A.; Mac Sweeny, A.; Stihle, M.; Haber, A. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 396–399. 1157

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Crystal Growth & Design

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dx.doi.org/10.1021/cg101359m |Cryst. Growth Des. 2011, 11, 1152–1158