Evaluating Protic Ionic Liquids as Protein Crystallization Additives

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Evaluating Protic Ionic Liquids as Protein Crystallization Additives Danielle F. Kennedy,*,† Calum J. Drummond,† Thomas S. Peat,‡ and Janet Newman‡ †

Commonwealth Scientific and Industrial Research Organisation (CSIRO), Materials Science and Engineering, Bag 10, Clayton MDC, Victoria 3169, Australia ‡ Commonwealth Scientific and Industrial Research Organisation (CSIRO), Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052, Australia

bS Supporting Information ABSTRACT: Protic ionic liquids (PILs) have been evaluated as additives in protein crystallization trials. Ten ionic liquids were incorporated as additives along with the JCSGþ sparse matrix screen for the crystallization of lysozyme, glucose isomerase, and trypsin. When used as additives, the PILs were identified to influence the crystallization of these globular proteins. Crystal structures obtained for lysozyme with each of the 10 PILs under identical conditions showed no change in the crystal structure of the protein; however, in three of the ten cases fragments of the ionic liquids were observed to be incorporated within the protein.

’ INTRODUCTION Ionic liquids are commonly described as materials comprised entirely of ions which are liquid at or below 100 °C.1 Aqueous solutions of ionic liquids have generally been thought of as salt solutions or at best designer buffers, as they fall outside the strict definition of ionic liquids.2 For simplicity however, in this work we will use the term “ionic liquid” to include both the pure salt and aqueous solutions of the salt. Empirically ionic liquids have a markedly different effect than standard buffers and salt solutions when used as additives for in vitro biological applications such as enzyme catalysis,3,4 whole cell biocatalysis,58 and macromolecular crystallization.913 Perturbations of the macromolecular environment induced by ionic liquid additives are only just beginning to be examined, and there are numerous reports of how these perturbations may be beneficial.1421 The mechanism of the ionic liquids effects are unclear; arguments based around pH effects or anion and cation addition in the absence of Naþ or Cl ions seem insufficient and more research is warranted. In addition, there is a need for studies which can help identify optimal ionic liquid additives empirically for various types of biological applications and to develop real world applications of this technology. Protic ionic liquids (PILs) are a subclass of ionic liquids which were discovered over 100 years ago. These ionic liquids remained largely unstudied until the 1980s research of Evans et al.22 PILs are synthesized through the neutralization of a Brønsted acid with a Brønsted base generating an ion pair. Unlike other ionic liquids, the ionicity of these ionic liquids depends on the equilibrium position of the acidbase neutralization.2,23 The available proton in PILs is capable of generating a hydrogen Published 2011 by the American Chemical Society

bonding network which leads to physicochemical properties particularly useful in protein applications including the ability to buffer the pH of the media. Recently, PILs have attracted increasing attention, largely due to their water-like physicochemical properties.24,25 Like water, PILs have been found to support the self-assembly of amphiphiles and lipids;26,27 additionally they may also act as cryo-protectants, exhibiting glass transitions28,29 on cooling and are predominantly water-soluble. Unlike aprotic ionic liquids, many PILs are volatile due to the equilibrium between their ionic (involatile) and neutral (volatile) components.24 The formation of well-ordered crystals of proteins and other macromolecules is currently a necessary step in the production of structural information by diffraction methods. This step has become one of the major bottlenecks in structural biology, and a lot of effort has been expended in the development of new technologies — for example, robotics, reagent development, protein engineering — to try to make this process more robust. Ionic liquids possess the ability to either improve the solubility or alternatively to “salt out” proteins depending on physicochemical properties.14,18,30,31 These properties make their use in crystallization trials an interesting prospect. Recently, there have been several reports where ionic liquids have been utilized as precipitants or additives in protein crystallization trials.9,10,12 The protic ionic liquid ethylammonium nitrate (EAN) was first reported by Garlitz et al. in 1999 to be a precipitating agent able to crystallize lysozyme.9 Lysozyme is well-known to adopt a Received: December 23, 2010 Revised: March 12, 2011 Published: March 30, 2011 1777

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Crystal Growth & Design

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Figure 1. Chemical structure of the protic ionic liquids employed in the study and their abbreviations.

number of different crystal morphologies, with the tetragonal form (P43212, a = b = 78 Å, c = 38 Å) being most common.32 Garlitz et al. found lysozyme crystallized from EAN solutions in two previously identified crystal forms. The crystal form obtained was dependent on the pH of the crystallization solution with monoclinic crystals obtained around pH 4.5 and tetragonal crystals obtained around pH 5.5. Both crystal forms were seen with precipitant concentrations of 300500 mM. Refinement of the crystal structures indicated that lysozyme in these EAN crystals was in the same conformation as lysozyme found in more conventionally grown crystals. Pusey et al. conducted a study of aqueous solutions of the ionic liquids 1-butyl-3-methylimidizolium chloride ([BMim]Cl), 1-butyl-3-methylimidizolium 2(2-methoxyethoxy)ethylsulfate ([C4mim][MDEGSO4]), and 1-butyl-1-methylpyrollidinium dihydrogenphosphate ([p1,4] [DHP]) investigating their effect on the crystallization of the standard test proteins canavalin, β-lactoglobulin B, xylanase, and glucose isomerase using the crystallization screen Crystal Screen HT (Hampton Research).12 Crystals were obtained for all four proteins under a number of crystallization conditions where they were not obtained from the ionic liquid-free control experiment, and vice versa — crystals did not grow under some conditions where the protein had crystallized in the control experiment. It was suggested that a common cause for the negative outcomes was the increased solubilization of the protein by the ionic liquid. Judge et al. conducted a study of 16 aprotic ionic liquids, predominantly imidazolium salts, which were investigated as precipitants and additives at up to 30% w/v in the crystallization of several proteins including lysozyme, trypsin, and a Fab complex.13 This study had findings similar to Pusey et al.,12 in that Judge et al. identified that the ionic liquids were weak precipitating agents due to the increased solubilization of the protein by the ionic liquid. They determined that when the ionic liquids studied were used as additives there were changes in the crystal habit and an improvement in the diffraction resolution of the Fab complex. Judge et al. concluded that the best method to use ionic liquids was as additives generating crystals with diffraction resolution comparable to or better than that obtained without their use. Recently, the use of the ionic liquid [BMim]Cl as an additive has successfully resulted in the crystallization and volume scale up of a difficult to crystallize protein, C. necator NapAB, a nitrate reductase.33 This case was interesting as it was the reproducibility of crystal growth that was improved by the inclusion of the ionic liquid.

Table 1. Comparison of the pH of 2 M Solutions of the Different Protic Ionic Liquids (PILs) and Controls Used in This Study additive EAF

pH (2 M solution) 4.83

EAP

7.46

EAMs

1.83

EAPv

7.29

EATfA

2.7

EAA

7.63

EAN EOAN

5.54 3.24

TEOAN

4.14

DEOAN

5.38

It seems unlikely that there will be a universal “most suitable” ionic liquid for any given protein in any given application. For this reason, further investigation of their use as crystallization additives is needed. Improved understanding of the structure property relationship between the nature of the ionic liquid and the stability and crystallization of proteins will lead to the possibility of the design of application specific ionic liquids for protein crystallization. Herein, we report the study of 10 protic ionic liquids (PILs) in crystallization trials. The effects of the PILs on three proteins (lysozyme, trypsin, and glucose isomerase) used in conjunction with 96 crystallization conditions (the JCSGþ screen) were studied. The JCSGþ screen is a sparse matrix screen; it is most frequently used as a first trial for proteins when crystallization conditions are not known. It was hypothesized that using the protic ionic liquid additives in conjuction with a sparse matrix screen would be a practical method for the use of ionic liquids in macromolecular crystallization which can also be easily integrated with current methodologies.

’ EXPERIMENTAL SECTION Materials. Protic ionic liquids: The acid and base reagents were all used as received. The amine and bases used were ethylamine (70% in water, Fluka Chemika), diethanolamine (MERCK, was distilled, fraction collected at 122 °C @ 6.8 101 mm Hg), and triethanolamine (BDH, was distilled, fraction collected at 152 °C @ 2 101 mm Hg). The organic acids used were formic acid (98%, Ajax Chemicals), acetic acid (99.8%, BDH), propionic acid (>99% Aldrich), pivalic acid (puriss, Fluka), and methylsulfonic acid (99%, BDH). The inorganic acid used 1778

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Crystal Growth & Design was nitric acid (69% in water, Merck). The reaction with strong inorganic acids is extremely aggressive and care should be taken during synthesis. The series of 10 PILs, ethylammonium formate (EAF), ethylammonium propionate (EAP), ethylammonium methanesulfonate (EAMs), ethylammonium pivalate (EAPv), ethylammonium trifluoroacetate (EATfA), ethylammonium acetate (EAA), ethylammonium nitrate (EAN), ethanolammonium nitrate (EOAN), triethanolammonium nitrate (TEOAN), and diethanolammonium nitrate (DEAON), were synthesized by a stoichiometric neutralization of the corresponding amine and acid via previously reported methods (see Figure 1);34,35 their purity was determined using a combination of NMR, Karl Fischer (KF) titration, and thermogravimetric analysis (TGA). All PILs prepared were found to be >99% pure. All ionic liquid stock solutions for crystallization trials were prepared at 4 and 0.4 M concentration with deionized water. The protein solutions were prepared from commercially sourced protein without further purification; hen egg white lysozyme (HEWL, Sigma L6876) was prepared for trials by dissolving in water at 20 mg/mL (Trial 1) and 30 mg/mL (Trial 2). Trypsin (Sigma T1426) was prepared by dissolving at 47 mg/mL (2 mM) in 10 mM CaCl2 and 4 mM benzylamine. Glucose isomerase (Hampton Research HR7-100) was prepared by dialyzing against 10 mM HEPES pH 7.0, 1 mM MgCl2, with the final concentration of protein being 30 mg/mL. The 96 condition JCSGþ crystallization screen (Qiagen) was modified by the addition of 20%(w/v) sodium azide to a final concentration of 0.02%(w/v) to each condition. Sitting-drop vapor-diffusion crystallization screens were undertaken at the Collaborative Crystallization Centre (C3) at 281 or 293 K as has been previously described.36 Crystallization droplets of 400 nL total initial volume and a reservoir volume of 50 μL were set up in Innovaplate SD-2 crystallization plates (IDEX Corp, California). The plates were incubated in a Gallery 700 incubator for two months and imaged with a Minstrel III imaging system (Rigaku Automation, California), and images were observed and scored by hand using the CTweb or CrystalTrak applications (Rigaku Automation, California). As crystal quality varies widely from protein to protein, we have found it necessary to vary the scoring system accordingly between proteins. Exemplars of the scoring system used for each protein can be found in Figure 2. The simple scoring system employed ranged from a score of 0-white, c-pale yellow, 4-yellow, 5-orange, to a score of 6-red depending on visual crystal quality. Crystallization Trial 1: Determining if the PILs Had Any Effect on Crystallization. Two crystallization plates (plates 1-1 and 1-2) were prepared with two proteins, trypsin and HEWL in the top and bottom subwells, respectively. The 400 nL crystallization droplets consisted of 200 nL of protein solution, 180 nL of the crystallant solution, and 20 nL of the PIL additive. All 96 reservoir wells contained the same crystallant, which consisted of 0.02 w/v sodium azide, 200 mM ammonium sulfate, 100 mM bis-tris pH 5.5, and 25% w/v polyethylene glycol (PEG) 3350. Each column of the two plates used a different PIL additive, resulting in 8-fold duplication of each experiment, Table S1, Supporting Information. Plate 1-1 used 0.4 M PIL stock solutions, and plate 1-2 used 4 M PIL stock solutions, respectively. Control experiments were also performed with column 11 of each plate containing 20 nL of sodium chloride solution of equivalent concentration to the PIL additive (0.4 and 4 M, respectively) and column 12 containing 20 nL of deionized water. So, for example, droplet A1.1 of Plate 1-1 contained at setup: 1 mM trypsin, 5 mM CaCl2, 2 mM benzylamine, 90 mM ammonium sulfate, 45 mM bis-tris pH 5.5, 11.25% w/v PEG 3350, 20 mM ethylammonium formate. The HEWL solution utilized in this experiment proved not to crystallize under these conditions, and no crystallization was observed after 67 days for lysozyme even in the aqueous control samples. Crystallization Trials 2, 3, and 4. Crystallization of three proteins; trypsin (Trial 2), HEWL (Trial 3), and glucose isomerase (Trial 4) were

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Figure 2. Representative examples of the scoring system employed for each of the three proteins used; Trypsin, lysozyme, and glucose isomerase. investigated with the PILs used as additives in addition to the JCSGþ crystallization screen incubated at 293 K. Each PIL was used in combination with every condition of the JCSGþ screen for each of the proteins. Eleven plates were prepared for each of the proteins lysozyme, trypsin, and glucose isomerase, one for each of the PIL additives and one water control, giving a total of 33 crystallization plates. As the plates contain two subwells, each experiment was conducted in duplicate. Each plate consisted of a standard 96 condition JCSGþ crystallization screen with one PIL additive (1 M) or control added to every well in the entire plate. The PIL additive was only added to the droplet, and the reservoir contained only the condition found in the JCSGþ screen. The 400 nL crystallization droplets consisted of 200 nL of protein solution, 180 nL of the crystallant solution (JCSGþ screen), and 20 nL of the PIL additive (0.05 M in the crystallization drop). X-ray Diffraction Data. Data sets were obtained from the MX-1 beamline at Australian Synchrotron. Crystals were cooled in the nitrogen stream after being carefully removed from the crystallization drop with a MiTeGen loop. In all cases, the 181 frames were collected with a one degree oscillation per frame and one second total exposure time per frame. Data sets were indexed using MOSFLM, scaled using SCALA (CCP4), molecular replacement was done using Phaser (CCP4), and refinement was accomplished iteratively using Coot and Refmac (CCP4).37 Each data set and model was treated independently, and all waters, ions, and other compounds were added based on the electron density maps and were not a part of the original model which only contained the lysozyme protein model.

’ RESULTS The proteins used in this study were selected due to their common use as model proteins for crystallization method development. A series of 10 protic ionic liquids were selected containing four cations and seven different anions. This series of ionic liquids was chosen primarily to investigate the effect of the anion on the crystallization of the different proteins. In addition, the effects of hydroxyl substituents and varied substitution on the ammonium cation were also investigated. Trial 1. An initial crystallization trial was conducted with trypsin to investigate the ability of the PIL series to perturb the crystallization of the protein. Trypsin is often used as a model 1779



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Figure 4. Photographs of representative experiments for trypsin crystallized in Trial 1, Plate 1-2 from (a) water control well F12, (b) with 200 mM ethylammonium acetate (EAA) additive, well G6. Scale bar represents 0.25 mm. Well numbers F12 and G6 refer to grid references for the relevant experiments in the crystallization plates.

Figure 3. Graphical representation of the results from Crystallization Trial 1, (top: Plate 1-1 and bottom: Plate 1-2) trypsin after 67 days at 293 K. The PILs were varied in each column with NaCl and H2O used as controls. Eight repeats were conducted. Crystallization events are color coded according to their score, Figure 2; red scoring 6, orange scoring 5, dark yellow scoring 4, white indicates clear droplet or denatured protein, purple circle indicates a null experiment due to robotic error.

protein to investigate the effect that additives have on the crystallization of proteins. Two sitting drop crystallization plates were set up with trypsin in Trial 1; each PIL was added to a different column of the plate at two concentrations (20 mM Plate 1-1 and 200 mM Plate 1-2) with two controls, NaCl and water. This generated 192 experiments with 8-fold redundancy. The results of the trypsin crystallization trial are summarized in Figure 3; multiple successful crystallization events were noted and full experimental results can be found in Supplementary Tables S1 and S2, Supporting Information. The results of the crystallization trial 1, Figure 3 and Tables S1 and S2, Supporting Information, indicate that the series of PIL additives are effective at influencing the crystallization of trypsin. No crystallization was observed from any well of the NaCl control. Crystallization from the water control occurred as expected with the optimized conditions employed; crystallization events scoring 6 occurred with 85% reproducibility. The reproducibility obtained by the water control was better than what was observed for the majority of the experiments containing the PIL additives with the exception of ethylammonium acetate (EAA) at 200 mM which produced crystallization of trypsin scoring 6 with 88% reproducibility. In addition, the optical crystal quality was consistently better for the experiments containing 200 mM EAA, with larger crystals with fewer defects observed, Figure 4. The number of wells or experiments where

crystallization is observed was considered to be as the nucleation rate. The nucleation rate for trypsin grown in the presence of EAA was not affected and was similar to what was observed for the water control experiments, Figure 5a. The improved visual crystal quality, with fewer larger crystals per experiment, is due primarily to the slower crystal growth rates and fewer nucleation sites observed per well when EAA was used as an additive, Figure 5b,c. Trial 2-4. In addition to the investigation of the PILs as crystallization additives to an optimized growth condition for trypsin, the PILs were screened as “standard” additives in conjunction with the JCSGþ screen. The JCSGþ screen is an array of 96 crystallization conditions with a wide range of salts, buffers, and crowding agents (PEG polymers).38 Three crystallization trials were set up, one for each of the proteins, trypsin (Trial 2), lysozyme (Trial 3), and glucose isomerase (Trial 4). Each Trial consisted of 11 crystallization plates, one water control and one for each of the PILs in the study. These three trials were set up using the standard “additive” screen protocol in the Collaborative Crystallization Center, using the JCSGþ 96 condition screen with the same PIL additive added to every well in the entire plate, as described in the Experimental Section. The results of the crystallization Trials 2, 3, and 4 are tabulated in Tables 2, 3, and 4, respectively. An example of the results from two representative crystallization plates from Trial 4 with the crystallization additive EAA are graphically represented in Figure 6. The subwells of the sitting drop crystallization plate are colored according to the crystal scoring system employed, Figure 2. While the relative number of successful crystallization events, or nucleations, remains relatively similar between the control and the experiment when EAA is used as an additive, the conditions where crystallization occurs are different in the presence of the additive which is visually apparent by the change in location of the successful experiments in the plate. It is immediately obvious that the ionic liquid additives are affecting the crystallization environment of the protein and perturbing crystal growth. The perturbation of the crystal growth is highlighted in the graphical representations of the crystallization results, Figures 7 and S16 and S28, where the experiments are color coded according to whether or not crystallization occurred in the water control experiment and whether the crystallization was reproducible in both repeats. Predominantly, in Trial 2 Figure 7, there is no correlation between the conditions which produced quality crystals in the control experiment and the conditions which produced quality crystals with additional PIL. 1780



Figure 5. Graphs of experimental results from crystallization Plate 1-2 using 200 mM ethylammonium acetate (EAA) as an additive compared to the water control. (a) Number of experiments/wells where trypsin crystallization (nucleation) has occurred, (b) average length (μm) of the largest crystal dimension, (c) bar graph of the crystal nucleation events per well, that is, experiment count for each number of crystals.

For the JCSGþ conditions A7 (CHES 0.1 M, pH 9, 20 wt % PEG 8000) and A11 (ammonium hydrogen phosphate 0.2 M, TRIS 0.1 M, pH8.5, 50 v/v% MPD) crystallization occurs in the presence of almost all of the PIL additives in addition to generating at least a crystalline material scoring “c” in the control experiment, Tables S5S15, Supporting Information. In these instances, the buffer and crowding agents have a more significant effect on the crystallization of trypsin than the PIL additive. For Trial 2, trypsin Table 2, the control plate 2-1 contained five conditions where crystallization events scoring 4 and above occurred, and one of which scored 6. For all of the PIL additives investigated, at least two and up to seven different conditions produced crystals scoring g4. The plates containing the PIL additives EAF (Plate 2-2), EAP (Plate 2-3), EAPv (Plate 2-5),

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EATfA (Plate 2-6), and EAA (Plate 2-7) all contained at least one crystallization event scoring 6. In general, nucleation times were not affected by the presence of the PILs; however, the number of nucleation events was decreased, with fewer, larger crystals obtained for most of the wells where crystallization occurred in the presence of the PIL additives with the exception of EATfA. In Trial 3, the crystallization of lysozyme, all of the plates contained successful crystallization events scoring 6, Table 3. The control plate 3-1 contained eight conditions where crystallization events occurred, six of which scored 6. For all of the PIL additives investigated, at least 4 and up to 14 different conditions produced crystals scoring g4. All of the plates containing the PIL additives contained at least three crystallization events scoring 6. In general, the number of nucleation events (crystals) was not affected by the presence of the PILs; however, nucleation times were increased relative to the control plate. An increase in the reproducibility of the crystallization events scoring 5 or 6 was also observed, relative to the control plate, particularly with EAMs, EAA, EAPv, and EATfA where in both cases the crystallization events were reproduced 60% of the time. In Trial 4, the crystallization of glucose isomerase, all of the plates contained successful crystallization events scoring 6, Table 4. The control plate 4-1 contained 22 conditions where crystallization events occurred, six of which scored 6. For all of the PIL additives investigated, at least 14 and up to 22 different conditions produced crystals scoring g4. All of the plates containing the PIL additives contained at least three crystallization events scoring 6. In general, glucose isomerase nucleation times were decreased by the PIL additives, and the number of nucleation events fluctuated relative to the control plate. Fewer but larger crystals were obtained when EAA and EAN were used as additives; however, an average of 10 crystals per well for the wells scoring 6 were obtained for the PIL additives EAON, DEOAN, and TEOAN. An increase in the reproducibility of the crystallization events scoring 5 or 6 was also observed for all of the PIL additives, largely due to the poor reproducibility of glucose isomerase crystallization in the control. The best reproducibility was obtained with EOAN as additive, with 83% of crystallization events scoring 5 or 6 reproduced in both subwells. To test the diffraction quality of the protein crystals obtained in the presence of PIL additives, the crystal structure of lysozyme was solved 11 times, once for each ionic liquid additive and one control . X-ray quality single crystals (score 4.5 or 6) were obtained from the JCSGþ screen well F12 (30% w/v Jeffamine M-600, pH = 7.0; 0.1 M HEPES (2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid), pH = 7.0) in each of the plates 3-1 to 3-11. This was the ideal situation for the systematic study of crystallographic quality for crystals obtained with the 10 different PIL additives, as this was a condition that was already appropriate for cryocooling. Crystals were harvested and flashcooled in the cold nitrogen stream without additional cryoprotection. X-ray data for these crystals was obtained on the macromolecular crystallography beamline (MX-1) at the Australian Synchrotron. The crystals diffracted on average to at least 1.40 Å, and all but two data sets were processed to this resolution (data from 3 to 9 F12 with EOAN (1.42 Å) and 3-10 F12 with TEOAN (1.46 Å) were the exceptions). The following residues had more than one conformation, or weak density for part of the residue, and showed some slight differences between the 11 models: Lys13, Arg14, Asp18, Asp19, Arg21, Arg45, Thr47, Arg61, Pro70, Gly71, Ser72, Arg73, Asn77, Leu78, Ser85, Ser86, Lys97, Ile98, Val109, Arg112, Arg125, Arg128, Leu129. One HEPES buffer 1781



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Table 2. Summary of the Results of Crystallisation Trial 2; Trypsin/JCSGþ/PIL Additive after Two Months protic

number of

number of

relative

average

average

crystallization trial

ionic liquid additive

crystallization eventsa

crystallization conditions scoring 6

reproducibility of crystallization (%)b

number of crystalsc

nucleation time (days)c

2-1

water control

5

1

100

5

318

2-2

EAF

3

1

100

1

218

2-3

EAP

3

2

100

3

218

2-4

EAMs

2

0

N/A

N/A

N/A

2-5

EAPv

4

1

100

1

218

2-6 2-7

EATfA EAA

4 7

1 2

100 100

5 3

218 218

2-8

EAN

5

0

N/A

N/A

N/A

2-9

EOAN

4

0

N/A

N/A

N/A

2-10

TEOAN

3

0

N/A

N/A

N/A

2-11

DEOAN

5

0

N/A

N/A

N/A

Number of crystallization conditions with crystallization events scoring g4 occurring in at least one of the replicates. b Relative reproducibility = (number of events scoring 5 or 6 in both replicates)/(total number of conditions at which crystals scoring 5 or 6 occurred)*100%. c For crystallization events scoring 6, average range of days as monitoring was not performed every day. a

Table 3. Results of Crystallization Trial 3; Lysozyme/JCSGþ/PIL Additive after Two Months crystallization

protic ionic

number of

number of crystallization

relative reproducibility of

average number of

average nucleation

trial

liquid additive

crystallization eventsa

conditions scoring 6

crystallization (%)b

crystalsc

time (days)c

3-1

water control

8

6

43

2

2230

3-2

EAF

6

5

50

1

2234

3-3

EAP

8

5

50

1

2839

3-4

EAMs

6

3

60

1

3338

3-5 3-6

EAPv EATfA

9 6

5 5

71 100

1 1

2228 2026 1925

3-7

EAA

4

3

75

1

3-8

EAN

14

7

50

1

1320

3-9

EOAN

13

9

40

1

3339

3-10

TEOAN

13

9

40

1

3641

3-11

DEOAN

13

7

56

2

3744

a Number of crystallization conditions with crystallization events scoring g4 occurring in at least one of the replicates. b Relative reproducibility = (number of events scoring 5 or 6 in both replicates)/(total number of conditions at which crystals scoring 5 or 6 occurred)*100%. c For crystallization events scoring 6, average range of days as monitoring was not performed every day.

Table 4. Results of Crystallization Trial 4; Glucose Isomerase/JCSGþ/PIL Additive after Two Months crystallization trial

protic ionic liquid additive

number of crystallization eventsa

number of crystallization conditions scoring 6

relative reproducibility of crystallization (%)b

average number of crystalsc

average nucleation time (days)c

4-1

water control

22

6

14

8

921

4-2

EAF

19

4

50

7

714

4-3

EAP

17

6

50

8

1

4-4

EAMs

22

7

57

4

515

4-5

EAPv

14

6

67

4

34

4-6 4-7

EATfA EAA

17 16

8 5

50 45

5 2

45 34

4-8

EAN

14

3

60

3

23

4-9

EOAN

17

4

83

10

24

4-10

TEOAN

20

10

58

9

4-11

DEOAN

19

7

46

10

34 1

Number of crystallization conditions with crystallization events scoring g4 occurring in at least one of the replicates. b Relative reproducibility = (number of events scoring 5 or 6 in both replicates)/(total number of conditions at which crystals scoring 5 or 6 occurred)*100%. c For crystallization events scoring 6, average range of days as monitoring was not performed every day. a

1782



Figure 6. Graphical representation of the crystallization results for two plates from Crystallization Trial 4 after 67 days at 293 K; (a) glucose isomerase/JCSGþ/water control (top), (b) glucose isomerase/JCSGþ/ EAMs (bottom). The subwells of the sitting drop crystallization plate are colored according to the crystal scoring system employed; pale yellowmicrocrystalline, dark yellow-crystals*, orange-crystals**, and redcrystals***, Figure 2.

molecule was seen in each of the structures and one chloride ion was added to each of the structures; each of these was found in the same relative location when the 11 structures were compared. Crystals grown from 3 to 2 (EAF) and 34 (EAMs) were also found to contain one ethylammonium moiety incorporated into the lysozyme structure.

’ DISCUSSION PILs have been investigated both as additives to an optimized protein crystallant and also in conjunction with the JCSGþ crystallization screen. This was undertaken to determine the best use of the PILs in conjunction with frequently used protocols and methodology. When the 10 PILs were used as an additive to the optimized trypsin crystallization conditions, Trial 1, perturbation of trypsin crystal growth was observed at both concentrations investigated. At the lower concentration (20 mM), crystallization events were reproducibly observed for the PILs EAF, EAP, EAPv, and EAA; however, visual crystal quality was variable. At the higher concentration studied (200 mM), crystallization events were reproducibly observed for only two PILs, EAP and EAA. The visual crystal quality for trypsin grown with EAA as an additive was highly reproducible with seven out of eight repeats scoring 6. Visual crystal quality for experiments containing the EAP additive was variable, scoring between 4 and 6. Interestingly, EAN, which has previously been employed in protein crystallization

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Figure 7. Summary of the results of crystallization trial 2; trypsin/ JCSGþ/PIL additive after two months. Number indicates score of crystallization events (subwell 1/subwell 2) c-microcrystalline, 4-crystals*, 5-crystals**, and 6-crystals***; if not listed crystallization did not occur in either subwell of any experiment. Wells are colored according to the legend to highlight the influence of the PIL on crystallization and the reproducibility observed in the experiment.

both as an additive and as a precipitant, was not successful in promoting the crystallization of trypsin in this study. Indeed, none of the nitrate containing PILs were found to be effective additives in the buffered crystallization solution used. While it is interesting that all 10 PILs were found to perturb the crystallization of trypsin, it is not common to modify an optimized system. A more realistic protocol was sought which incorporates PIL additives into a typical screening methodology used to identify crystallization conditions for difficult to crystallize proteins. Commonly used approaches at the Collaborative Crystallization Center (C3) employ additive screens and sparse matrices in high throughput crystallization trials. It was decided to replicate the scenario where the optimal crystallization conditions are not known for a protein; a common approach in this case is to employ a sparse matrix screen which contains a wide range of buffers, crowding agents, and precipitants. The investigation undertaken used the PIL additives in conjunction with the sparse matrix currently employed at C3, the JCSGþ crystallization screen. This approach was trialed for the crystallization of trypsin, lysozyme, and glucose isomerase, Trials 2, 3, and 4, respectively. Every combination of PIL and JCSGþ condition was investigated for each of the proteins. The control experiments for each of the proteins, using water as an additive, all resulted in at least one condition scoring 6 across the 96 conditions in the JCSGþ screen. However, trypsin did not crystallize well with several of the PILs: EAMs, EAN, EOAN, DEOAN, and TEOAN, Trial 2/Table 2. This was also observed in Trial 1, where crystallization was prevented by these PILs at both 20 mM and 200 mM concentrations. This inhibition of the crystallization of trypsin by several of the PILs cannot 1783



Figure 8. Depiction of all 11 crystal structures for lysozyme in well F12 of crystallization Trial 3 overlaid. Areas of commonality in the structures of lysozyme are colored uniformly royal blue. The HEPES buffer cation (all structures), chloride (all structures), nitrite (3-10) and ethylammonium (3-2 and 3-4) identified are colored to highlight their location (white = carbon, blue = nitrogen, yellow = sulfur, red = oxygen, and green = chloride).

be attributed to the pH of the experiments. The PIL additives inhibiting the crystallization of trypsin included both PILs whose aqueous solutions are both highly acidic (EAMs 2 M solution, pH 1.83) and mildly acidic (EAN 2 M solution, pH 5.54), Table 1. In addition, the crystallization events for the water control experiments occurred over a variety of pH conditions (4.59.5) indicating that the crystallization of trypsin is not sensitive to pH. While more analogues would need to be investigated for a definitive conclusion, these PILs may prevent precipitation of the trypsin through increased solubilization. For lysozyme (Trial 3) all of the PIL additives resulted in at least one crystallization event scoring 6, Tables 3 and 4, respectively. For lysozyme, the average nucleation time was increased in the presence of the PILs with respect to the water control experiment, again indicating that increased solubility caused by the PILs may be a contributing factor. Lysozyme has previously been shown by Garlitz et al. to have increased stability in the PIL EAN.9 In is therefore unsurprising that when used as a crystallization additive, EAN and the similar PILs; EOAN, TEOAN and DEOAN improve the number of successful experiments from 10 in the water control to >13, Table 3, with most of these containing crystals that scored better on average than the control, Table S4 in the Supporting Information. Glucose isomerase in Trial 4, similar to lysozyme, had at least one crystallization experiment which scored 6 in the presence of each of the PIL additives. However, in the presence of the PIL additives the average nucleation time of glucose isomerase decreased with respect to the control plate, and significantly better reproducibility was observed compared to the control experiment, Table S4, Supporting Information. In this case, it can not be said that the PIL is increasing the solubility of the protein;

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conversely, it is acting as a precipitant, causing faster crystallization of the glucose isomerase. In order to investigate the effect of the PIL additives on the crystal quality, whether the ionic liquids were being incorporated into the protein and whether they significantly changed the structure of the protein, crystal structures for lysozyme were determined for crystals grown under identical conditions with the exception of the PIL additive. In each of the 11 crystallization experiments conducted in crystallization Trial 3 for lysozyme, crystals scoring 4, 5, or 6 were observed in condition F12 which contained Jeffamine M-600 and HEPES buffer in addition to the PIL additive. These 11 crystal structures for lysozyme were resolved individually to better than 1.46 Å resolution. On the whole, the models were identical and the PILs did not seem to make any significant difference to the crystal quality or model structure, as is illustrated in Figure 6 which depicts the 11 crystal structures overlaid with areas of commonality colored royal blue to highlight incorporated molecules. Apart from slight variation in the orientation of some side-chain amino acids, there is very little difference in the structures, and the individually placed HEPES buffer molecule and the chloride ion were identified in each of the resolved structures. In two cases, density that could be interpreted as fragments of the PILs was identified within the crystal structure. One nitrite group was identified within the crystal structure of lysozyme grown from 3 to 11 F12 with DEOAN as the PIL additive, and presumably the nitrite is present as a decomposition product of the nitrate group in the ionic liquid. Additionally, an ethylammonium cation was found to have incorporated in two of the lysozyme crystals. First in the lysozyme crystal grown from 3 to 2 F12, which had EAF as the PIL additive ethylammonium formate, and for the lysozyme crystal grown from 3 to 4 F12, which had EAMs as the PIL additive, where the ethylammonium moiety was identified in the same pocket as the HEPES buffer molecule. Despite these examples of minor PIL incorporation, there appears to be no significant change in the lysozyme crystal structure was caused by the growth of the crystals in media containing PIL additives.

’ CONCLUSION Like other studies into the use of ionic liquids as additives for protein crystallization,9,10,12 this series of 10 PILs have been found to influence the crystallization of trypsin, lysozyme, and glucose isomerase. We have found that PILs may be usefully used as additives in crystallization experiments, and that concentrations of 100200 mM may be very appropriate. In many cases, the PIL additives were effective at increasing the size and quality of protein crystals and also improved the reproducibility of protein crystallization events. However, the ionic liquids employed did not significantly change the structure of the lysozyme. The influence of the PIL additives on crystallization was different for each protein investigated, and therefore it can be concluded that the effect of the additive on protein crystallization is protein specific. The protein specificity is due to changes in pH, density, and viscosity of the solutions caused by the additives and also the interaction of ionic liquid fragments with the protein. This interaction has been observed in two of the refined lysozyme crystal structures with ionic liquid cations located in the core of the lysozyme structure. Using the PILs in conjunction with sparse matrix screens, such as the JCSGþ screen used in this study, increases the likelihood of identifying crystallization conditions which can be further optimized. This is a methodology 1784


Crystal Growth & Design which we are actively applying to the crystallization of proteins which are difficult to crystallize; these PILs have already been shown to be useful in our lab in generating crystals of diffraction quality for a difficult project.39

’ ASSOCIATED CONTENT

bS

Supporting Information. Tabulated data from each of the crystallization trials and the contents of JCSGþ crystallization screen. Physicochemical characterization of 2 M solutions of each of the PILs. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT C.J.D. is the recipient of an Australian Research Council (ARC) Federation Fellowship. DFK is the recipient of a CSIRO OCE postdoctoral fellowship. This work was also partly supported by an ARC Discovery Project grant, DP0666961. This research was undertaken in part on the MX beamline at the Australian Synchrotron, Victoria, Australia. We thank the scientists at MX-1, the Australian Synchrotron, for their help in obtaining the X-ray data for this work and the staff of the Collaborative Crystallisation Centre, as well as Pat Pilling for useful comments on the manuscript. ’ REFERENCES (1) Wasserscheid, P.; Welton, T., Ionic Liquids in Synthesis; Wiley-VCH: Weinheim, 2003. (2) MacFarlane, D. R.; Seddon, K. R. Aust. J. Chem. 2007, 60 (1), 3–5. (3) Jain, N.; Kumar, A.; Chauhan, S.; Chauhan, S. M. S. Tetrahedron 2005, 61 (5), 1015–1060. (4) Cantone, S.; Hanefeld, U.; Basso, A. Green Chem. 2007, 9 (9), 954–971. (5) Brautigam, S.; Bringer-Meyer, S.; Weuster-Botz, D. Tetrahedron: Asymmetry 2007, 18 (16), 1883–1887. (6) Pfruender, H.; Amidjojo, M.; Kragl, U.; Weuster-Botz, D. Angew. Chem., Int. Ed. 2004, 43 (34), 4529–4531. (7) Pfruender, H.; Jones, R.; Weuster-Botz, D. J. Biotechnol. 2006, 124 (1), 182–190. (8) Weuster-Botz, D. Chem. Rec. 2007, 7 (6), 334–340. (9) Garlitz, J. A.; Summers, C. A.; Flowers, R. A.; Borgstahl, G. E. O. Acta Crystallogr., Sect D: Biol. Crystallogr. 1999, 55, 2037–2038. (10) Hekmat, D.; Hebel, D.; Joswig, S.; Schmidt, M.; Weuster-Botz, D. Biotechnol. Lett. 2007, 29 (11), 1703–1711. (11) Li, X. X.; Xu, X. D.; Dan, Y. Y.; Feng, J.; Ge, L.; Zhang, M. L. Cryst. Res. Technol. 2008, 43 (10), 1062–1068. (12) Pusey, M. L.; Paley, M. S.; Turner, M. B.; Rogers, R. D. Cryst. Growth Des. 2007, 7 (4), 787–793. (13) Judge, R. A.; Takahashi, S.; Longenecker, K. L.; Fry, E. H.; Abad-Zapatero, C.; Chiu, M. L. Cryst. Growth Des. 2009, 9 (8), 3463–3469. (14) Lau, R. M.; Sorgedrager, M. J.; Carrea, G.; van Rantwijk, F.; Secundo, F.; Sheldon, R. A. Green Chem. 2004, 6 (9), 483–487. (15) De Diego, T.; Lozano, P.; Gmouh, S.; Vaultier, M.; Iborra, J. L. Biotechnol. Bioeng. 2004, 88 (7), 916–924. (16) De Diego, T.; Lozano, P.; Gmouh, S.; Vaultier, M.; Iborra, J. L. Biomacromolecules 2005, 6 (3), 1457–1464.

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