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GLUCOSE ISOMERASE POLYMORPHS OBTAINED USING AN AD HOC PROTEIN CRYSTALLIZATION TEMPERATURE DEVICE AND A GROWTH CELL APPLYING AN ELECTRIC FIELD Siseth Martínez-Caballero, Mayra Cuéllar-Cruz, Nicola Demitri, Maurizio Polentarutti, Adela Rodriguez-Romero, and Abel Moreno Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.5b01755 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 4, 2016

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Crystal Growth & Design

GLUCOSE ISOMERASE POLYMORPHS OBTAINED USING AN AD HOC PROTEIN CRYSTALLIZATION TEMPERATURE DEVICE AND A GROWTH CELL APPLYING AN ELECTRIC FIELD

Siseth Martínez-Caballero1, Mayra Cuéllar-Cruz2, Nicola Demitri3, Maurizio Polentarutti3, Adela Rodríguez-Romero1,* and Abel Moreno1,* 1

Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad

Nacional Autónoma de México, Av. Universidad 3000, Ciudad Universitaria, C.P. 04510, México, D.F., México. 2

Departamento de Biología, División de Ciencias Naturales y Exactas, Campus

Guanajuato. Universidad de Guanajuato. Noria Alta S/N. C.P. 36050 Guanajuato Gto., México. 3

Elettra – Sincrotrone Trieste, S.S. 14 km 163.5 in Area Science Park, 34149 Basovizza

– Trieste, Italy.

Abstract

In this work, we investigated the effects of the temperature on the crystal growth of a model protein: glucose isomerase. The use and design of two temperature devices on the crystallization process of the protein glucose isomerase allowed us to obtain different polymorphs. Additionally, in order to obtain insights into the concomitant crystallization of different polymorphs of this enzyme, the solubility diagram of the protein was obtained. After fixing the maximum solubility at the optimal temperature, we tested how to separate nucleation and crystal growth by using an ad hoc crystal growth cell applying a direct current. We succeeded in controlling the nucleation first and latter the growth process moved forward by vapor diffusion. Finally, the 3D structures of glucose isomerase obtained at different temperatures were assessed by Xray diffraction, and crystallographic methods. Keywords: Glucose Isomerase, Temperature Devices, Crystal Growth, Polymorphs, Crystal Structure.

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1. INTRODUCTION Protein crystals of size large enough for high quality X-ray diffraction data collection and subsequent 3D structure determination are still a difficult task to achieve. In spite of all the advanced techniques for obtaining biomacromolecular crystals in the searching of the crystallization conditions by means of robotics, not many of them are focused on investigating systematic procedures to obtain different polymorphs of protein crystals, based on solubility plots. Furthermore, they have been mostly focused on pharmaceutical compounds.1,2 Nevertheless, some of the efforts have been concentrated on either understanding the kinetics of protein crystallization or the physicochemical parameters that affect the protein crystallization behavior.3 Only a few have undergone a systematic study to understand how these chemical (concentrations, pH, ionic strength) and physical variables (temperature, pressure) influence the outcome in terms of crystal type (polymorph), size and diffraction limit (which is related to final structural model quality).4,5 In the last decade, most of the researches were devoted to investigate the crystal growth of biological macromolecules to increase the crystal quality for high resolution X-ray crystallographic research. Particularly, model proteins like glucose isomerase (MW 43.0 kDa) amongst others have been extensively studied in terms of getting new different crystalline forms.6, 7 However, none of these studies were focused on understanding the aspects on how to obtain polymorphs or their plausible parameters for the obtainment any specific crystal form. To achieve this task it is necessary not only to obtain the protein crystal but also to characterize the crystal morphology based on solubility diagrams where some polymorphism could appear.

During the crystallization of biological macromolecules from solution we faced at least two successive problems that still need to be sorted out. One is about the crystallization conditions, and the second is about the nucleation rate. Thus, the challenging issue for crystal growers is to separate nucleation and growth processes. To avoid uncontrolled nucleation and because primary nucleation is a stochastic phenomenon, seeding techniques are often used.8,9 An alternative solution is to induce crystallization from metastable solutions, using external fields: magnetic,10-12 electric,13,14 ultrasonic15 or electromagnetic.16 From these different methods, a powerful and practical procedure to grow high quality crystals is either to look for higher quality crystals by combined methods of internal electric fields16,17 and strong magnetic fields during the crystal growth process of model proteins,18,19 or by electro-migration phenomena when


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applying electric fields as electrochemically-assisted protein crystallization process.20 In this work, we focused our interest in understanding the combined role of the temperature and electric fields on the solubility plot during the crystallization process of a model protein glucose isomerase. We analyzed the influence of temperature to obtain different polymorphs, as well as the effect of electric fields on the nucleation stage of glucose isomerase. This enzyme has two structural domains, the first one consists of an [α/β]8 barrel with a core of eight β-strands separated by α-helices, and the C-terminal domain comprising a small loop of 65 residues that embraces the first domain of another subunit in the dimer.21 We also obtained different crystal habits at different temperatures contained in two orthorhombic glucose isomerase polymorphs, which exhibit slightly different contacts at the interface of the chains that are closed in the crystal lattice. Finally, the 3D structures of these polymorphs were assessed using X-ray diffraction techniques and a comparison of these structures was performed.

2. EXPERIMENTAL SECTION

2.1 Model protein and crystal conditions The protein used for the experiments was Glucose Isomerase (GI) (Hampton Research Corporation Cat. No. HR7-102). The crystallization conditions consisted of a typical protein concentration of 30 mg/mL and, as precipitating agent we used 200 mM Naacetate buffer pH 7.0, 30% PEG 6000.

2.2 Crystallization of GI at different temperatures based on two devices, and determination of its solubility plot The crystallization assays were performed using temperatures from 4 to 26 °C, with an interval of 4 °C, in a TG40 temperature controller from Centeo Biosciences (Blacktrace Holdings, UK) (Figure 1a). The classic vapor diffusion method in a sitting-drop set up was used with 40 µL of the mother liquid and drops of 1 µL of protein plus the same amount of precipitating agent. At temperatures lower than 10 °C or higher than 24 °C the plastic cover sealant of the TG40 lanes was dimmed by condensed water vapor. Then a second device was alternatively used as that shown in Figure 1b and 1c. A fluid cell commonly used for electrochemical research on the atomic force microscope (EC-



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AFM) was adapted as a crystal growth cell.22,23 This cell was supported on a highly oriented pyrolytic graphite (HOPG). This allowed us to perform the crystal growth at different temperatures, without dimming the plastic cover, in order to record the crystal growth process properly. For this EC-AFM fluid cell, the crystal growth was performed using the batch method by mixing 40 µL of the protein solution with the same amount of precipitating agent. A layer of light oil (paraffin) was put on top of the droplet to allow the video camera to record the crystal growth process at temperatures that could go higher than 24 °C or lower, such as 4 °C. In order to guarantee a proper temperature control, the laboratory (where the crystal growth proceeded) was fixed at the same temperature of the experiment in the EC-AFM fluid cell. We also adapted a Peltier element (temperature controller as shown in Figure 1c) to get a strict control of the temperature at the bottom of the growth cell. After finishing the crystal growth process, the system was set still for a week thus allowing it to reach the thermodynamic equilibrium. To obtain the solubility plot, eight droplets set at the same temperature were collected and centrifuged to measure the protein concentration at the equilibrium. This procedure was repeated twice for each tested temperature.


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Figure 1. Temperature devices for protein crystallization: a) based on the use of TG40, b) an ad hoc growth cell based on the fluid cell of electrochemistry of the atomic force microscope (EC-AFM), c) full experimental setup by using a heater controller of the EC-AFM equipment. The temperature of the laboratory was fixed at the same temperature of the Peltier. 2.3 Crystallization of GI using an ad hoc crystal growth cell combining electric field and vapor diffusion set up The electrochemically assisted growth cell consisted of two polished ITO (Indium Tin Oxide) electrode glasses, whose conductive-coated surfaces were placed inward and facing parallel to each other, as has already been published elsewhere.17 Figure 2 shows the front dimensions of the reengineered electrically-assisted crystal growth cell. The main difference is that it contains two small reservoirs for the mixture of protein and precipitating agent (Figure 2a). The larger reservoir was filled with precipitating agent only. One of the walls of the small reservoir (containing the control) was internally covered with nail varnish in order to avoid the electric field effect, while allowing the other to show the effect of the electric field. Figure 2b shows the lateral view and dimensions of this new crystal growth cell. During nucleation, the device was connected



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to a galvanostat programmed to apply a direct current of 2 µA at a constant temperature. The current was switched off after 48 h leaving the crystal growth process to proceed by vapor diffusion. The time is arbitrary. However, we had previously tested the induction time for nucleation of glucose isomerase, and realized that it ranged from 24 h to 36 h in our current crystallization conditions. Crystal growth was monitored for 1-6 weeks. GI control crystals were grown in a sitting-drop vapor diffusion set up inside a similar crystal growth cell but in the absence of electric current. We observed that temperatures between 22 °C and 26 °C showed different crystal habits. Subsequently, we decided to grow crystals of this enzyme in situ transporting the TG40 apparatus to the synchrotron by collecting the data in the same environment where the data collection was performed.

Figure 2. The reengineered electrically assisted growth cell contains: a) two small reservoirs one for the control experiment and one for testing the electric field effect on protein crystallization, locating, in the central part, only the large reservoir for precipitating agent, b) it shows the lateral view dimensions of the new growth cell.

2.4 X-ray diffraction analysis X-ray diffraction data sets were collected for single crystals of similar size using the inhouse Rigaku/MSC Micromax-007 with a rotating-anode generator (copper anode) and a Raxis-IV++ detector. The crystals were cryo-protected using the reservoir solution and were plunged into liquid nitrogen prior irradiation. All data sets were processed using XDS,24 combined with POINTLESS25 and scaled, using SCALA from the CCP4 suite v.6.1.2 (Collaborative Computational Project 4, Number 4, 1994).26 The analyses in situ


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of the polymorphs were performed at the XRD1 beamline of the Elettra Synchrotron (Trieste, Italy).27 The electrocrystallization growth cell has been fitted on a standard goniometer head with a custom built frame. Data were collected at room temperature, using a monochromatic wavelength of 0.700 Å to limit absorption due to ITO glasses. The structures were solved by the molecular replacement method, using the coordinates of the PDB entry 1AOD,21 with PHASER-MR28 in PHENIX29 and map inspection was performed with COOT.30 Subsequent rounds of model building and refinement were performed using PHENIX-REFINE.31 Data collection and refinement statistics of the different data sets are summarized in Table 1.

3. RESULTS AND DISCUSSION In general, within a phase diagram, there are distinct thermodynamically defined zones where the physicochemical conditions of the solution determine either the protein crystal phase or the specific polymorph to be obtained. These zones are defined as undersaturated, saturated, metastable, labile, and precipitation. All undersaturated solutions will remain a single liquid phase. It is important to mention that the upper limit of the metastable zone, called supersolubility curve, has a kinetic origin. Nucleation phenomena appear at high supersaturation, while crystal growth proceeds at low supersaturation values. The phase diagram is typically presented as a twodimensional plot with the major factors influencing outcome as axes. In reality this is a multidimensional sampling of phase space whereas the two-dimensional representation is a slice through this space that can be found at constant temperature or at constant pressure. Consequently, there can be a considerable variability due to the apparently small changes in either the concentration or the temperature of the crystallization process. It is therefore capital to obtain a solubility plot of the studied protein in order to determine whether the areas in which the different polymorphs appear are at a certain temperature and/or pressure.



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Figure 3. Solubility plot of glucose isomerase obtained by combining the TG40 apparatus and an EC-AFM fluid cell as a novel device for temperature control.

The graphical representation of the solubility plot for glucose isomerase, as shown in Figure 3, offered by the phase diagram, was obtained by concentration measurements sets boundaries for chemically defined regions with a higher probability to produce different types of crystals (polymorphs). This phase diagram was determined by preparing samples at the same given crystallization conditions and protein concentration though at different temperatures. The first region goes from 4 °C up to the minimum of the solubility plot located between 18 and 20 °C. Then the second region goes from temperatures from 18-20 °C to more than 22 °C. We have determined by X-ray crystallographic methods (further comments on this will be made along the paper) that one polymorph (P21212) grown at temperatures between 4-22 °C contained two chains in closely packed position in the asymmetric unit (ASU), while above 22 °C a different polymorph was obtained (I222) containing one monomer in the ASU. This polymorph (I222) occasionally appeared also at 4 °C or lower temperatures when the electric field was applied (we will comment further on this later). These two polymorphs have


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already been obtained for this protein in previous works by using different precipitating agent, by changing the pH or by modifying the temperature.6,32 However, no study on the variation of temperature during the crystal growth had been performed so far. Figure 4 shows a record of a temperature screening where the effect of the minimum of solubility shown in Figure 3 is observed for GI. The majority of crystals did not display well-defined crystal morphology at this temperature (20 °C as the minimum of the curve of Figure 3). This was due to the low solubility value of glucose isomerase. On the other hand, temperature values on both the left and right hand-side of the solubility minima produced crystals with normal and regular shapes. Nonetheless, those grown at low temperature (4-18 °C), under the conditions described above, presented higher B Wilson and Rmerge values in data reduction. The crystals diffracted at lower resolutions (2.2 to 2.7 Å), which affected the refinement of the structures that gave bad R/Rfree values 1819/26-28 (Table 1). The only difference is that the crystal quality was obtained either very close or far away from the minimum of the solubility. The best crystals, according to Table 1, were those grown at 22 °C (P21212) and 24 °C (I222). Interestingly enough, and within a ranging temperature from 22 to 26 °C, we observed a possible third orthorhombic polymorph P21221 with cell dimensions: a=123.0 Å, b=99.5 Å, c =115.2 Å. Nonetheless, the refinement behaved oddly, since the difference in the R and Rfree values increased in each cycle. This result suggests that defects in the crystal make the determination of the space group difficult. It is worth mentioning that three to four different morphologies (habits) coexisted in the drops grown at 22 °C, belonging to the two different orthorhombic crystal forms primitive and body centered - mentioned previously. A possible explanation could be that as protein diffuses across temperature gradients in the drop, different crystal polymorphs could be formed, though with different qualities. This happened below the curve of Figure 3 on the over cross of the two solubility trends in the conditions highlighted by the red arrow. The best crystals were obtained when the crystallization condition was fixed at a temperature far away from the minimum of solubility of both plots.

Recent publications have demonstrated that the application of an electric field during crystal growth improved crystal quality.13-17 This improvement is observed as a higher



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resolution limit, as well as a better crystallographic statistics (data-reduction and refinement).14 The next obvious step was to combine both procedures (temperature and electric fields) and to evaluate the effect of temperature on the crystal quality and growth. A combination of electric field and crystal growth in vapor diffusion set-up had a remarkable effect on the overall crystal quality as judged by the quality of the dataset and the diffraction data shown in Table 1 and Table 2. Even though the crystals grown at 26 °C look bigger and well defined, diffraction limit was poor and data reduction was not possible in any space group, so crystal quality was bad (Figure 4).

Figure 4. GI crystals grown at different temperatures using a TG40 apparatus. Qualitatively, GI crystals grown using different temperatures (outside of the minimum of solubility) had a well-defined habit, and were homogeneous in size compared to those grown in the absence of temperature control or grown in the minimum of the plot shown in Figure 3. However, when combining electric field and crystal growth in a vapor diffusion set-up (Figure 2), the electric force had a more pronounced effect when combining nucleation and vapor diffusion as follows: 48 h for nucleation and then vapor diffusion for crystal growth, for some weeks as it is shown in Figure 5.


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Figure 5. Combining the electrocrystallization cell and vapor diffusion set-up at 4 ºC.

Under batch or vapor diffusion conditions, the electric field favors the formation of crystallites perfectly fixed at the electrode (ITO) in one side of the growth cell. Additionally, the use of the both direct current and vapor diffusion together with the transport phenomena resulted in individual larger crystals at the maximum solubility and the lower temperature, 4 °C for instance (Figure 5). Diffraction images have been collected in-situ, mounting the electrocrystallization growth cell directly on the synchrotron diffractometer. Patterns were successfully indexed from bigger crystals grown on the cathode, demonstrating improvement on the crystals grown in the presence of coupled methods (temperature and electric field) as shown in this contribution.



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Table 1. Crystallographic X-ray data statistics for glucose isomerase 4 °C

12 °C

18 °C

22 °C

24 °C

P21212 a= 82.63, b= 93.43, c= 98.60 100661/26838 43.6-2.5 99.13 (100.0) 0.12 (0.46) 10.72 (2.97) 3.8 (3.7) 25.44

P21212 a= 86.12, b= 93.22, c= 98.95 149586/40941 39.49-2.2 99.52 (100.0) 0.10 (0.49) 11.78 (2.93) 3.7 (3.6) 22.29

P21212 a= 82.06, b= 93.36, c= 98.81 66916/6354 42.33-2.7 98.07 (97.76) 0.12 (0.31) 10.74 (3.93) 3.2 (3.1) 22.88

P21212 a= 82.69, b= 93.22, c= 97.94 271227/83408 38.40-1.70 99.8 (98.7) 0.07 (0.27) 11.7 (3.8) 3.3 (3.1) 14.38

I222 a= 93.05, b= 98.28, c= 102.13 267071/51417 35.41-1.70 99.8 (98.7) 0.08 (0.37) 11.1 (3.1) 5.2 (4.8) 18.70

0.19/0.27 6084 14 4

0.20/0.26 6076 118 4

0.19/0.28 6072 8 4

0.16/0.19 6180 759 12

0.15/0.18 3135 467 23

0.009 1.26 95/1

0.009 1.17 95/0.52

0.009 1.36 92/1

0.006 0.97 97/0.25

0.007 1.07 97/0.25

6.88 25.10

5.71 22.50

10.43 22.70

2.79 16.10

3.69 19.0

Data collection and processing statistics Wavelength (Å) Space group Unit-cell parameters (Å, °) Reflection (total/unique) Resolution limits (Å) Completeness (%) Rmerge I/σ(I) Average multiplicity B factor from Wilson plot (Å2) Refinement statistics R/Rfree No. of protein atoms No. of water molecules No. of ligand molecules R.m.s. deviations Bonds lengths (Å) Bonds angles (°) Ramachandran favoured/outliers (%) Clashcore from Molprobity Average B-factor

In a previous publication we reported the design of a crystallization cell suitable to grow protein crystals under the influence of a direct electrical current in batch method;22 improved recently for the vapor-diffusion set-up.17 However, batch crystallization conditions are not available for all proteins, nor are they easy to interconvert between different crystallization methods. We reengineered the electrically assisted crystal growth device compared to the previous approaches17,20,22 (Figure 2) so that it could be used in a sitting-drop and vapor diffusion configuration, and thus impact a wider range of protein crystallization experiments.17 Besides controlling crystal nucleation, this cell has the additional advantage that once the electric current is switched-off, the sealed cell can be kept for several days allowing the protein crystals to grow by vapor diffusion until they reach their maximum size (as shown in Figure 5). The presence of electric current influenced the resulting number of crystals. A large number of crystals were observed in the absence of electric field, but there were at least three times fewer crystals when grown in the presence of electric current. GI molecules were positively charged at the pH of the experiment because the pH in the crystallization conditions used is lower than the pI of the protein. In agreement with the previous results, the electrical field had a profound impact on GI nucleation and crystals growth in the


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cathode of the ITO growth cell, but not in the anode probably due to charge repulsion. Few new crystals appeared once the electrical current was turned off suggesting that the nucleation process had almost ceased by that time. However, the 48 h time previously mentioned is an arbitrary time, although we usually tested in advance (before fixing the crystal growth experiment under the influence of the electric field) the induction time for the nucleation at the crystallization conditions used for glucose isomerase crystallization. Once the electric current was turned off, the growth process proceeded by vapor diffusion. The crystals already formed remained stable in the solution and grew further until the experiment reached the end of the process. GI crystals grew up to 300 µm in 28 days.

Crystals obtained at temperatures between 18-20 °C did not present well defined morphologies (Figure 4) and they all belonged to the orthorhombic space group P21212; nonetheless, the crystallographic data sets presented poor statistics, as shown in Table 1. The best P21212 polymorph was grown at 22 °C. All these crystals presented a Matthews coefficient33 (VM) value of 2.16 Å3/Da, compatible with two monomers in the asymmetric unit. The crystals obtained at 24 °C were the best according to the data presented in Table 1 these crystals contained a monomer in the ASU with a VM of 2.67 Å3/Da. However, in order to prove the positive effect of the electric field combined with temperature control, we run experiments in duplicates at two different temperatures (4 ºC and 24 ºC), where the worst and the best crystals appeared, respectively. These crystals were compared to those grown at the same temperature under the influence of electric field in terms of size (Figure 6), we measured the longest distance of parallel faces to measure the growth for at least two weeks after applying the direct current for 48h. Additionally, the crystal quality was analyzed in duplicates by X-ray diffraction, and crystals of similar size were diffracted and processed to obtain the crystallographic X-ray data statistics as shown in Table 2.



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Figure 6. It shows the comparison of crystal growth of glucose isomerase at two temperatures. Control crystals were grown in the same crystal growth cell made of ITO transparent glass electrodes without applying the electric field (E-field).

Table 2. It shows the crystallographic X-ray data statistics for the crystal quality analysis at two temperatures. Absence of electrid field 4°C 24°C Data collection and processing statistics Space group Unit-cell parameters (Å) Reflection (total/unique) Resolution limits (Å) Completeness (%) Rmerge I/σ(I) Average multiplicity B factor from Wilson plot (Å2)

Presence of electrid field 4°C 24°C

P21212 a= 82.63, b= 93.43, c= 98.60 100661/26838

I222 a= 93.05, b= 98.28, c= 102.13 267071/51417

I222 a= 92.99, b= 97.49, c= 102.39 157159/51901

I222 a= 93.37, b= 98.59, c= 102.07 119914/39831

43.6-2.5

35.41-1.70

40.74-1.68

46.69-1.85

99.13 (100.0) 0.12 (0.46) 10.72 (2.97) 3.8 (3.7)

99.8 (98.7) 0.08 (0.37) 11.1 (3.1) 5.2 (4.8)

97.9 (97.1) 0.08 (0.43) 8.6 (2.2) 3.0 (2.9)

98.6 (94.9) 0.07 (0.43) 9.1 (2.1) 3.0 (2.9)

25.44

18.70

10.35

16.9

Basically, it can be appreciated from this X-ray data that crystals obtained at 4 ºC and 24 ºC (under the influence of this electric field) were statistically better in terms of crystal quality compared to control crystals. The lower values of B factor from the Wilson plot of the analyzed crystals, grown under the influence of the electric field, are a good evidence of higher quality crystal in general into the crystal packing, not to


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mention that they were more homogeneous and bigger in size. Additionally, the resolution limit in general was much better even at lower temperatures (4 ºC), where the worst crystals were initially obtained without the electric field influence.

A Cα superposition of the monomer in space group I222, and chain either A or B of the P21212 crystals (22 °C) gave an r.m.s.d. value of 0.127 Å. Therefore, there are no significant differences in the conformation of the chains between the two crystal forms. According to the PISA interface analysis34 for the two molecules present in the ASU of the crystal in the primitive space group form, there are 13 salt bridges established by the interaction of residues His96, Asp108, Asp110, Glu204, Arg205, Arg368 and Asp363 (Figure 7A) of both chains. These interactions are absent in the equivalent arrangement observed in crystalline body centered form, considering a neighbor symmetry related molecule (Figure 7B), since Arg205 and Arg368 exhibit different conformations that prevent the formation of the salt bridge. In addition, the B average factor of these amino acids is slightly higher in the I222 polymorph obtained at 24 °C, which could explain the side chain rearrangement at this temperature compared to the conformation preferred at lower temperatures.



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Figure 7. Molecules A and B in the asymmetric unit of Glucose Isomerase obtained at Temperatures up to 22 °C in the space group P21212. A) The residues involved in interactions at the interface are highlighted. B) Details of the interactions between chains A (blue) and B (green). For comparison a superposition of the monomer obtained in the space group I222 (red) with A (r.m.s.d. 0.127 Å) is shown. For this monomer (red) it is also shown a symmetry related molecule that establishes similar interactions as those present in between chains A and B in the ASU of the polymorph P21212. As it has been previously established the overall fold of the protein is analogous to a TIM barrel: an eightfold α/β-barrel core structure with a long C terminal arm. There is also a tight tetrameric association of the enzyme in both crystal forms, as shown in Figure 8. The packing in the crystalline forms P21212 and I222 is tetrameric, which is common in GI of different sources of Streptomyces (Figure 8).21 However, the interaction between different tetramers is more compact and there are more intermolecular contacts in space group P21212 (Figure 7B). The lower left and right hand-side of Figure 8A and B show a zoom-in of the crystal lattice contacts for both polymorphs.


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A

B

ab diagonal b c

a

c

Asp336 Arg340 Arg340

Gly83 Lys85

Glu8

His49 Arg109

Asp80

Figure 8. Packing in the two crystal polymorphs: A) P21212 obtained at 22 °C, B) I222 obtained at 24 °C. Lower parts show a zoom-in of the crystal lattice contacts for both polymorphs respectively.

4. CONCLUSIONS According to our results, the best crystals were obtained at an optimal temperature base on a solubility curve, being the nucleation fixed firstly by applying an electric field, then leaving the crystal growth process to proceed by vapor diffusion setup. The obtaining of two different polymorphs into two different areas of the phase diagram was studied based on the solubility plot. Higher temperature (22 ºC) promotes better diffracting crystals with only one GI monomer in the asymmetric unit, while at lower temperature 4-20 ºC less ordered crystals, with two independent monomers in the ASU, have been found. The two crystal forms differ only in the packing arrangement of the tetramers, which is illustrated in Figures 8A and B for both crystal forms. In the I222 form each tetramer forms eight equivalent contacts with eight other tetramers, related by the I-centering of the cell. The packing of tetramers in the P21212 form is analogous, but



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lies at the two fold axes, as previously observed for these two GI polymorphs. These results are particularly interesting and will open a new trend in research focused on polymorphism in proteins and advanced methods for protein crystallization research (combining different crystal growth approaches).

ACKNOWLEDGMENTS This work was supported by CONACYT project Number 82947 (assigned to A.R.R) and DGAPA-UNAM Project Number PAPIIT IT200215 (assigned to A.M). We thank LANEM–IQ-UNAM and Georgina Espinosa for the technical support. Two of the authors (A.M. and M.C.C) acknowledge the XRD1-Hard X-ray Diffraction Beamline and Structural Biology Laboratory of the Elettra Synchrotron, in Italy for the support and beamtime awarded to collect data of different polymorphs. A.M. and M.C.C. are thankful for the financial support provided by Red Temática de Usuarios de Luz Sincrotrón (REDTULS, CONACyT) Mexico.

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GLUCOSE ISOMERASE POLYMORPHS OBTAINED USING AN AD HOC PROTEIN CRYSTALLIZATION TEMPERATURE DEVICE AND A GROWTH CELL APPLYING AN ELECTRIC FIELD

Siseth Martínez-Caballero, Mayra Cuéllar-Cruz, Nicola Demitri, Maurizio Polentarutti, Adela Rodríguez-Romero* and Abel Moreno*

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Synopsis. Searching for polymorphs of glucose isomerase by using two temperature devices and an electrochemically-assisted crystallization growth cell.


Glucose Isomerase Polymorphs Obtained Using ... - ACS Publications

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