An Extensive Study of Protein Phase Diagram Modification: Increasing

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CRYSTAL GROWTH & DESIGN

An Extensive Study of Protein Phase Diagram Modification: Increasing Macromolecular Crystallizability by Temperature Screening†

2008 VOL. 8, NO. 12 4277–4283

Yi-Bin Lin,#,‡ Dao-Wei Zhu,#,§ Tao Wang,‡ Jian Song,‡ Yong-Shui Zou,‡ Yong-Lian Zhang,⊥ and Sheng-Xiang Lin*,‡,§ Laboratory of Structural Biology with Visiting scientists, Institute of Biochemistry and Cell Biology (IBCB), Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Shanghai 200031 China, and Shanghai Key Laboratory of Molecular Andrology & State Key Laboratory of Molecular Biology, IBCB, SIBS, CAS, Shanghai 200031, China, and Laboratory of Molecular Endocrinology and Oncology, Centre Hospitalier UniVersite´ LaVal (CHUL) Research Center (CHUQ) and LaVal UniVersity, Que´bec, G1V 4G2, Canada ReceiVed July 1, 2008; ReVised Manuscript ReceiVed October 18, 2008

ABSTRACT: A new parameter “relative crystallizability” for protein crystallization has been proposed, and its relationship with protein solubility and crystallization success has been studied (Zhu et al. J. Struct. Biol. 2006, 154, 297). Here we further construct the phase diagrams of a larger number of proteins, study the phase modification as a function of temperature, and establish the relationship between the nucleation zone area (SN) and crystallization success. The phase diagrams of 10 proteins were constructed and their SN were compared, demonstrating that temperature modifies the protein nucleation zone. Such modification can significantly enlarge the SN and increase protein crystallizability. For example, the SN of ribonuclease S and trypsin increases by 2.4- and 1.6-fold when the temperature moves to 277 K from 295 K, while at the same time the crystallization hits increase from 20.8% to 42.9% and 12.5% to 25%, respectively. SN of chymotrypsinogen A and concanavalin A increases by 1.6- and 1.7-fold (277 to 295 K), while the hits increase from 37.5% to 54.2% and 43.3% to 73.4%, respectively. Such an excellent agreement strongly supports the validity of protein “relative crystallizability”, and crystallization screening at several temperatures can significantly increase the success for most proteins. A new protein epididymal-specific lipocalin was crystallized by varying temperature, yielding quickly the first crystals, and complete data sets have been collected at 1.97 Å. Introduction With the recent development of structural genomics and biological sciences, unprecedented numbers of macromolecule structures need to be determined. 1 X-ray crystallography is the prevalent method of structural biology and will continue to play an increasing role in the understanding of molecular interactions. Despite numerous advances in the field, crystallography remains an integrated process including several steps for successful structure determination. Often identified as a bottleneck along this pathway is crystallization of the target molecule.2-4 Thus far, a sophisticated method for rational protein crystal growth (PCG) is not yet available. Rational crystallization has been investigated for decades.5-9 As a result of continuous work being carried out during recent years, Zhu et al. have proposed a new measurable parameter “relative crystallizability” for proteins and have evaluated its relationship to crystallization success in the widely used Sparse Matrix Screen, represented as “hits”.10,11 The nucleation zone where spontaneous nucleation occurs has been used as a reference for crystallizability, or “relative crystallizability”, and is defined as the proportion of this zone area over the total phase area within the experimental protein and precipitating agent concentration range in the diagram. Here we attempt to further † Part of the special issue (Vol 8, issue 12) on the 12th International Conference on the Crystallization of Biological Macromolecules, Cancun, Mexico, May 6-9, 2008. * Corresponding author and visiting scientist at SIBS. E-mail: structuralbiology@ sibs.ac.cn. # Co-first authors. ‡ Laboratory of Structural Biology with visiting scientists, IBCB, SIBS, CAS. ⊥ Shanghai Key Laboratory of Molecular Andrology, State Key Laboratory of Molecular Biology, IBCB, SIBS, CAS. § Laboratory of Molecular Endocrinology and Oncology, CHUL Research Center (CHUQ) and Laval University.

establish and compare a large number of phase diagrams for various proteins, study the relation between nucleation zone and crystallizability, and facilitate the obtainment of first crystals making use of crystallizability. The phase diagrams of lysozyme, ribonuclease A, ribonuclease S, trypsin, concanavalin A, chymotrypsinogen A, papain, catalase, and proteinase K are constructed and their nucleation zone areas have been measured. These phase diagrams demonstrate that temperature change modifies the precipitation behavior of proteins and can significantly enlarge nucleation zones with modified temperature, thus increasing protein crystallizability. Such an excellent correspondence strongly supports the validity of protein “relative crystallizability”, to facilitate the finding of the first crystals. Epididymal-specific lipocalin (ELP16) is a newly found protein, belonging to the lipocalin (LCN) family, cloned from Rattus norVegicus (Norway rat). It may play very important roles in sperm maturation and egg fertilization. 12 In the crystallization of ELP16, the crystallizability was studied to facilitate the growth of first crystals while complete phase diagrams have been obtained. The results suggest that such a study helps to orient the crystallization condition, thus facilitating the obtainment of first crystals. Results Effect of Temperature on PCG Success. Protein crystallizability study was used to design and implement customized crystallization strategies for 10 proteins. To begin the crystallization of these proteins, they were screened against 24 conditions (Sparse Matrix Screening conditions with PEG, here after abbreviated as SMS-PEG)11 at different temperatures (e.g., 277 K, 285 and 295 K). The success rate of each protein at

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Table 1. Success Rate of Protein Crystallization by Experimental “Hits” in Sparse Matrix Screeninga success rate at different temperatures (%) temperature (K) lysozyme (chicken egg white) catalase (bovine liver) ribonuclease A (type III bovine pancreas) ribonuclease S (bovine pancreas) trypsin (bovine pancreas) concanavalin A (cancanvalin ensiformis) papain (papaya latex) proteinase K (tritirachium album) chymotrypsinogen A (bovine) ELP16 (Rattus norVegicus) a

277

285

62.5

54.2

288 58.3

20.8 42.9 25 4.2 4.2 4.2 37.5

16.7 33.3 16.7 4.2 12.5 8.3 45.8 12.5

295 29.2 54.2 8.3 20.8 12.5 8.2 16.7 12.5 54.2 16.7

300 50

20.8

“hits” dependence on temperature decremental decremental decremental decremental decremental incremental incremental incremental incremental incremental

Crystals were obtained at different temperatures, using the 24 conditions with PEG as precipitating agent in Sparse Matrix Screen.

Figure 1. Effects of temperature on model protein crystallization. (a) Comparison of hits at different temperatures. (b) Best choice temperature contributes to hits improvements. The best means that crystals were obtained under the optimum temperature. The worst means that crystals were obtained under disadvantageous conditions. The experimental results were statistically analyzed using One-Way ANOVA and One Sample T Test.*The mean difference is significant at the 0.05 level (LSD test).**The mean difference is significant at the 0.01 level (LSD test).

different temperatures was evaluated by the number of “hits” obtained in such a screening. The precipitating agents used in the crystallization experiments are polyethylene glycol (PEG) polymers with different chain lengths. The results are listed in the Table 1. The temperature had different effects for various proteins. Some proteins can be crystallized easily at lower temperatures. Others, however, yield crystals more readily at higher temperatures. For example, crystals of lysozyme, ribonuclease A, and ribonuclease S have been grown with 62.5% (15/24), 20.8% (4/24), and 33.3% (8/24) “hits” at 277 K compared to 29.2% (7/24), 8.3% (2/24), and 25% (6/24) at 295 K, respectively, in the screening. For these proteins, more crystallization conditions have been found at lower temperatures, with novel conditions discovered. For example, the crystals of ribonuclease S were not found at the condition 0.2 M ammonium acetate, 0.1 M sodium citrate pH 5.6, 25% PEG 4000 at 295 K, but the crystals were obtained under the same conditions at lower temperatures. The same phenomenon was found in lysozyme, catalase, and tryspin. In contrast, concanavalin A, chymotrypsinogen A and proteinase K yielded crystals with more conditions at higher temperatures, where novel conditions have been found in addition to those reported in the literature. Crystals of chymotrypsinogen A were obtained at 0.15 M ammonium acetate, 0.1 M sodium citrate pH 5.6, 20% PEG 4000, 295 K, and 0.2 M sodium acetate, 0.1 M tris-HCl pH 8.5, 30% PEG 4000, 295 K, but not at lower temperatures. Similar results were found for concavalin A. New crystals were obtained in two conditions: 0.2 M zinc acete at 0.1 M sodium cacodylate pH 6.5, 12% PEG 8000, 295 K, as well as 0.1 M sodium acetate pH 4.5, 20% PEG 4000, 295 K, but not at lower temperatures (Figures 2b and 4a, respectively). Table 1 shows the results of the crystallization trials (also see Materials and Methods) using SMS-

PEGs, with samples set up at different temperatures (e.g., 277, 285, and 295 K). One-Way ANOVA statistics analysis of the extensive experiments results in a significance < 0.001, demonstrating significantly higher hits at the most suitable temperature than other temperatures for different proteins, while the hits’ difference is very significant between the most suitable and the least suitable temperatures used for screening (Figure 1a). In fact, this happens in more than 90% of proteins in our experiments. One Sample T Test shows that even though the overall hits for all crystallizations at the best temperature are also significantly higher than other temperatures, the average ratio of hits’ increase reaches 65.1% with 95% confidence (Figure 1b). Two-Dimensional Phase Diagrams and the Nucleation Zone Area Measurement. In the two-dimensional-phase diagrams (protein concentration - the precipitating agent concentration), the solubility, nucleation, and precipitation curves delineate the different states of the protein (Figure 2).4 Phase diagrams plotting protein concentration versus precipitating agent concentration at various temperatures have been established experimentally for sample proteins (Figure 2). From the phase diagrams we can see that the precipitation curves of some proteins, for example, concanavalin A, papain, proteinase K, and chymotrypsinogen A, move to higher protein concentrations and precipitating agent concentrations, when the temperature shifts from 277 K (lower) to 295 K (higher). But their nucleation curves move less than those in the other group leading to an increase of the nucleation zone. The precipitation curves of lysozyme, ribonucleases A, and trypsin move to higher protein concentrations and precipitating agent concentrations, when the temperature shifts from 277 K (lower) to 295 K (higher). But their nucleation curves move even more significantly in the same direction, leading to the reduction of the

Study of Protein Phase Diagram Modification

Figure 2

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Figure 2. Two-dimensional-phase diagrams of test proteins: lysozyme (a), concanavalin A (b), ribonuclease A (c), ribonuclease S (d), chymotrypsinogen A (e), trypsin (f), papain (g), proteinase K (h) and catalase (i). Solubility curve (in red) was determined from the residual concentration in equilibrium with crystals 50-days after the initiation of crystallization at varying temperatures. Nucleation and precipitation curves are plotted in black and green, respectively. In this diagram the precipitation zone is denoted by the region above the precipitation curve, while the nucleation zone, where spontaneous nucleation occurs, lies between the nucleation and precipitation curves. The metastable zone is situated between the nucleation and solubility curves where crystals grow, while the area below the solubility curve is the undersaturated zone. Lines are drawn using Origin 6.0.

nucleation zone. The extent of the moving depends on the particular protein. The nucleation curve of lysozyme moves to much lower protein and precipitating agent concentrations from 295 to 277 K and crystals can be observed in the crystallization trials with 2 mg/mL lysozyme and 15% PEG (w/v) at 277 K. Though it was at 20 mg/mL and 15% PEG that initial crystals

were found in the screening under 295 K (Figure 2a).13 The augmentation of the nucleation zone facilitates PCG success, while similar results were found for other proteins. The phase diagram modification with a significant move for the precipitation curve, or simultaneous variations for the precipitation and nucleation curves, for proteins with retrograde or direct tem-

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Figure 3. Dependence of protein relative crystallizability and PCG success on temperature, and their interrelationships: (a) percentage of “hits” with the Spare Matrix Screen, (b) relative crystallizability, expressed as a ratio of the nucleation zone and the total phase area, (c) solubility curve of selected proteins under different temperatures. Concanavalin and ELP solubility dependence curves are amplified in the insert of C. The solubility of ribonuclease S, trypsin, papain, ribonuclease A, chymotrypsinogen A, concanavalin A, lysozyme, proteinase K, and catalase (mg/mL) were determined at 21% PEG4000, 15% PEG4000, 15% PEG8000, 21% PEG4000, 21% PEG4000, 6%PEG4000, 15% PEG4000, 22%PEG8000, 4% PEG8000. Solubility of the new ELP 16 was determined at 6% PEG 4000 and 10% iso-propanol.

perature dependence, respectively, have been evidenced by extensive experimentation in this work. Figure 3a,b shows the results of nucleation zone area comparisons with hits. Nucleation zone area (SN) of ribonuclease S, lysozyme, ribnuclease A, and trypsin increases by 2.4-, 1.3-, 3.1-, and 1.6-fold, when the temperature shifts from 295 to 277 K, while at the same time the crystallization hits increase from 20.8% to 42.9%, 29.2% to 62.5%, 8.3% to 16.7% and 12.5% to 25%, respectively. The nucleation zone of catalase increases by 2.3-fold when the temperature shifts from 285 to 303 K, while the hits increase from 50% to 58.3%. SN of chymotrypsinogen A, papain, proteinase K, and concanavalin A increases by 1.6-, 1.3-, 3.8-, and 1.7-fold, when the temperature changes from 277 to 295 K, while the hits increase from 37.5% to 54.2%, 4.2% to 16.7%, 4.2% to 12.5% and 43.3% to 73.4%, respectively. The ELP16 nucleation zone increases by 1.92-fold when shifting from 288 to 300 K, compared to the hits increase from 12.5% to 20.8% with the same temperature change. The modification of hits is thus in good agreement with the SN change. The phase diagrams and crystal pictures of concavalin A and new purified ELP16 protein are shown in Figure 4. In this crystallization condition at pH 4.6, concanavalin A crystals were found at 295 and 285 K, but no crystal was obtained at 277 K (Figure 4a). From the phase diagrams we can see that the nucleation zone became smaller and smaller from higher to

lower temperature. When the temperature changes to 277 K, no crystal is obtained. Similar results can be seen from the phase diagrams of ELP16 in which SN changes from 14.1% to 27.1% from 288 to 300 K with solubility decrease (Figure 4b). Relative crystals can be seen from this figure, and it demonstrates that screening at different temperatures helps to improve the PCG success. Protein Solubility. Figure 3c shows the solubility of selected proteins under different temperatures. Under our experimental conditions the concanavalin A, chymotrypsinogen A, papain, ELP16 and proteinase K have retrograde solubility dependence on temperature. Whereas the second group, represented by lysozyme, trypsin, catalase, ribonuclease A, and ribonuclease S, has incremental solubility (called direct effect) depending on temperature.4 Thus, the dependence of relative crystallizability on temperature is demonstrated to be closely related to solubility modification10 (Figure 2a,c) and PCG screen against different temperatures can lead to a higher success rate. The Crystallization of a New Protein Epididymal-Specific Lipocalin (ELP16). The initial crystallization screening was performed with 24 conditions of SMS-PEG at 285, 296, and 300 K. After several days, 83% conditions (20/24) resulted in precipitation at 285 K, while only 50% (12/24) conditions at 300 K in the drops. This suggests that this protein has the characteristics of proteins with retrograde temperature dependence. Crystals were then found in 3 and 4 conditions at 285

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Figure 4. Phase diagrams and crystal photos of ELP16 and concanvalin A at difference temperatures. (a) Phase diagrams of concanvalin A were obtained at different temperatures: 277, 285, and 295 K. Solubility curve, nucleation curve, and precipitation curve are plotted in red, black, and green, respectively. These phase diagrams were determined by crystallization at pH 4.6 (NaAc-HCl), in the presence of PEG 4000 from 6 to 18% (w/v), while the protein concentration varied from 1 to 60 mg/mL. (b) Phase diagrams of ELP were determined at 0.1 M Hepes-Na. pH 7.7, 10% isopropanol, PEG 4000 (from 0.5 to 30%, w/v) and 0.5-9 mg/mL protein (protein with 0.5 mM detergent of C12E8). The multiwells were placed in incubators at different temperatures (288, 295, and 300 K). The crystal pictures were obtained with a digital camera. The abscissa is Hepes precipitant concentration (%, w/v), and the ordinate is the protein concentration (mg/mL).

and 296 K, while in 5 conditions at 300 K. The crystals appear more readily at high temperatures, while the precipitations were easily found at lower temperature. We further optimized the crystals with the variation of salt, buffer pH, and PEG concentration at 300 K based on the initial crystallization conditions. The biggest crystal (about 0.3 mm × 0.15 mm × 0.1 mm) was obtained in the mother solution containing pH 7.7 Hepes-Na, 5% PEG 4000, 10% iso-propanol. This result strongly supports that screening at different temperatures can facilitate the choice of the most suitable condition where crystallization success is higher than at other temperatures, contributing to the elimination of this rate-limiting step in PCG. The fact that most proteins have temperature-dependent solubility further supports the validity of the method.10,14 The data of the ELP16 crystals have now been collected to 1.97 Å in 19-BM Beam-line at APS (Advanced Photon Source, Chicago, USA). Discussion Relative Crystallizability Correlates with Nucleation Zone. To verify the validity of using the nucleation zone area for the measurement of relative crystallizability, we carried out systematic experiments for different proteins using SMS-PEG.11 To avoid a possible architectural pH effect due to temperature modification, we closely monitored this pH dependence under the conditions of the SMS-PEG. The solubility change versus temperature test showed virtually insignificant reverse temperature dependence for the proteins in our experiments in the presence of the different salts used in SMS-PEG. No significant pH change was noticed under the temperature used.10 Thus, it

was pertinent to perform a statistical analysis on the set of 24 SMS-PEG conditions in this screening. The numbers of conditions producing protein crystals in the above screening, the “hits”, are used to evaluate the success rate. Under our experimental conditions the concanavalin A, proteinase K, ELP16, papain and chymotrypinogen A have incremental ”hits” with temperature. Lysozyme, trypsin, catalase, ribonuclease A and ribonuclease S, however, have decremental “hits” dependence on temperature. From Figure 3a,b we can see that the changes of “hits” are in excellent agreement with the study of nucleation zone area, strongly demonstrating the correlation between such an area and the crystallization success. The experimental results support the validity of our initial idea. More importantly, the application of relative crystallizability can act as a guide to significantly broaden the crystallization conditions in PCG experiments. Screening at Different Temperature Helps to Improve the PCG Success. Much excellent work has shown that 86% of the tested proteins demonstrated solubility dependence on temperature.14,15 In this study, we have focused on the modification of the nucleation zone at different temperatures, and how to use the relative crystallizability to obtain the first crystals more rapidly, thus implementing a more rational approach to PCG. In our experiments, finding the most suitable temperature contributes to the hits increase by 65.1% (Figure 1b). Some new crystallization conditions were found when screening at different temperatures. Similar results have also been found for other selected proteins. Eight, three, five, and two new crystallization conditions were found for lysozyme, trypsin, ribonuclease S, and catalase at lower temperatures compared to the

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reported conditions. Crystals of concanavalin A and chymotrypsinogen A were obtained from one and four new conditions, respectively. These results demonstrate that screening at different temperatures will help to find new conditions, facilitating the rational crystallization of new proteins. Materials and Methods Most proteins were purchased from Sigma/Aldrich; their names and sources are listed in Table 1. All crystallizations were carried out by vapor diffusion in hanging drops, initiated by mixing an equal volume of protein and reservoir solution. Crystal-solution phase diagrams were designed to plot the initial protein concentration versus the precipitating agent concentration under each crystallization condition. The multiwells were placed in incubators at different temperatures (277, 285, and 295 K). Crystals were observed with an optical microscope (LEICA MZ APO). In the phase diagram plotting protein concentration versus precipitating agent concentration, nucleation and precipitation curves were determined by visual examination at 50-fold magnification. After an equilibration period of 50 days, the residual protein concentration in equilibrium with crystals was titrated using a dye-binding assay to determine the solubility curve.16,17 Under such conditions equilibrium can be reached at 95% level after approximately 10 days. The precipitation zone is denoted by the region above the precipitation curve while the nucleation zone, where spontaneous nucleation occurs, lies between the precipitation and nucleation curves. The metastable zone is situated between the nucleation and solubility curves where crystals grow, while the area below the solubility curve is the undersaturated zone (e.g., Figure 2). Lysozyme phase diagrams were determined after crystallization at pH 7.5 in Hepes-Na buffer, in the presence of 6-27% (w/v) PEG 4000, and 1-60 mg/mL protein. Ribonucleases A and S phase diagrams were determined by crystallization at pH 4.5 (NaAc-HCl), in the presence of 0.2 M MgCl2 and PEG 4000 from 12 to 33%, while the protein concentration varied from 1 to 60 mg/mL. The phase diagrams for trypsin were determined after crystallization at pH 4.6 in NaAc buffer, in the presence of 0.2 M ammonium sulfate and 9-27% (w/v) PEG 4000, and 1-60 mg/mL protein. Chymotrypsinogen A phase diagrams were determined by crystallization at pH 4.6 (NaAc-HCl), in the presence of 0.2 M ammonium sulfate and PEG 4000 from 15 to 33%, while the protein concentration varied from 1 to 60 mg/mL. The phase diagrams for concanavalin A were determined after crystallization at pH 6.5 in sodium cacodylate buffer, in the presence of 0.2 M Zn(Ac)2 and 9-21% (w/v) PEG 4000, and 1-30 mg/mL protein (Figure 2b) or 0.1 M sodium acetate pH 4.5, 20% PEG 4000 and 1-60 mg/mL protein (Figure 4a). The phase diagrams for papain were determined after crystallization at pH 6.5 in sodium cacodylate buffer, in the presence of 0.2 M ammonium sulfate and 5-25% (w/v) PEG 8000, and 1-60 mg/mL protein. Catalase phase diagrams were determined after crystallization at 0.1 M bicine pH 8.5, PEG 8000 (from 2 to 15%, w/v), and 1-30 mg/ml protein. Proteinase K phase diagrams were determined at 0.05 M cacodylate, pH 6.5, 0.08 M Mg(Ac)2, PEG 8000 (from 20 to 33%, w/v), and 5-20 mg/mL). ELP16 phase diagrams were determined at 0.1 M Hepes-Na, pH 7.7, 10% iso-propanol, PEG 4000 (from 0.5 to 30%, w/V) and 0.5-9 mg/mL protein in the presence of 0.5 mM C12E8. Calculated Area of Phase Diagram (Sp) and Nucleation Zone (SN). The nucleation zone area (SN) has been calculated using the following expression:

Sn ) b(x1 - x2) +

∫xa y1(x) dx - ∫xa y2(x) dx 1

2

(1)

where a is the highest concentration of precipitant for a set of experiment, and b is the highest protein concentration for set of experiment. The x1 is a precipitant concentration at b for the precipitant curve. The x2 is a precipitant concentration at b for the nucleation curve. The y1(x) is the protein concentration as a function of precipitant for the precipitant curve, and y2(x) is the protein concentration as a function

of precipitant for the nucleation curve. Both y1(x) and y2(x) are power functions of second order. The calculated total area of phase diagram (Sp) has been used with the following expression:

Sp ) a × b

(2)

where a is the highest concentration of precipitant and b is the lowest protein concentration for the set of experiment. ELP16 was expressed as insoluble inclusion bodies in Escherichia coli. A succeed protocol was found to renature the protein. After gel filtration chromatography, homogeneous proteins were obtained.11

Acknowledgment. We acknowledge our colleagues Mr. Jiong Chen and Mr. Zeyong Chen for helpful assistance. This work was supported by a collaborative agreement between SIBS and CHUL Research Center and CAS Creation grant KSCX1-YWR-54.

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