Effect of Additives on the Crystallization of Lysozyme and Chymotrypsinogen A Jie
Lu,*,†
Xiu-Juan
Wang,‡
and Chi-Bun
Ching‡
Institute of Chemical and Engineering Sciences, and Chemical and Process Engineering Center, National University of Singapore, Block 28, Ayer Rajha Crescent, Unit No. 02-08, Singapore 139939
CRYSTAL GROWTH & DESIGN 2003 VOL. 3, NO. 1 83-87
Received September 4, 2002
ABSTRACT: We report the effect of some electrolyte and nonelectrolyte additives on the crystallization of lysozyme and chymotrypsinogen A. In particular, the influence of glycerol and dimethyl sulfoxide on protein’s solubility and the critical supersaturation for explosive nucleation has been studied. The two additives are found to increase the solubility, decrease crystallization enthalpy, and promote nucleation. Introduction The knowledge of three-dimensional (3D) molecular structure gives us useful information about the mechanism of action of biomolecules at the atomic level. Such information forms a very important part of biology, as well as medicine, biotechnology, and any field dependent upon new applications of biomacromolecules.1 X-ray diffraction is the most reliable method to determine the structure of such large molecules, but it can only be applied provided that suitable crystals are obtained. Obtaining good quality macromolecular crystals is thus a critical step within the process of X-ray structural analysis.2 Although many techniques for protein crystallization have been developed, successful crystallization is still largely empirical and operator-dependent, and finding precise optimal conditions for maximal yield of high quality crystals is still a challenge.3-6 A large number of works have indicated that the control of protein crystallization by additives is potentially very useful for crystal structure determination, as well as for industrial applications.7 Additives are normally applied to improve the order of poorly diffracting crystals or to reduce the branching of crystals and the growth of crystal clusters through suppressing random aggregation of protein molecules.8,9 To date, the use of nonionic and zwitterionic detergents in the crystallization of membrane proteins and some soluble proteins has been well-established and has become routine practice.10 Nondetergent solubilizers/stabilizers are also applied in protein crystallization popularly. However, the precise roles of various additives to the protein crystallization process are still not well-understood, and the choice of additives is largely empirical.8,11 To gain some insights into the underlying mechanisms of the effect of additives on protein crystallization, we investigate the influence of additives on crystal habit, solubility, and critical supersaturation for explosive nucleation of lysozyme and chymotrypsinogen A. The results show that additives such as glycerol and dimethyl sulfoxide (DMSO) can increase protein’s solu* To whom correspondence should be addressed. Fax: 65-6873 4805. E-mail:
[email protected]. † Institute of Chemical and Engineering Sciences. ‡ Chemical and Process Engineering Center.
bility, reduce the surface energy of the crystal, and promote nucleation in protein crystallization. Experimental Section Proteins and Additives. Chymotrypsinogen A (C-4879, 6× crystallization) and lysozyme (62971, crystalline powder) were purchased from Sigma-Aldrich. Six times crystallized lysozyme for study on solution phase behavior was purchased from Seikagaku. All proteins were used without further purification. The additives tested in crystallization experiments include electrolytes (CsCl, MgCl2, and CaCl2) and nonelectrolytes such as diols (ethyl glycol, hexanediol, 2-methyl-2,4-pentanediol (MPD), and pentanediol), glycerol, poly(ethylene glycol)s (PEGs), DMSO, polysaccharides (sorbitan, dextran, and lipopolysaccharide), alditols (xylitol, lactitol, and mannitol), and detergents (MEGA-8 and zwittergent 3-18). Experimental Procedures. Sodium acetate buffer (0.1 M) at pH 4.8 and 0.01 M citrate buffer at pH 5.25 were prepared with ultrafiltered, deionized water. Sodium azide, at a concentration of 0.05% (w/v), was added to all buffer solutions as an antimicrobial agent. Because chymotrypsinogen A tends to deteriorate at pH values above 5.0, 0.2 mM phenylmethylsulfonyl fluoride (PMSF) was added into citrate buffer to suppress its self-cleavage and self-digestion.12 Protein stock solutions were prepared by dissolving protein powder in buffers and then filtered through 0.22 µm filters (Millex-VV) for further experiments. The concentration of protein solution was determined by measuring the absorbance at 280 nm with UV spectroscopy (Shimadzu UV-2550). Precipitant solutions were prepared by dissolving the required amount of ammonium sulfate or sodium chloride together with additives in buffers. The pH of solutions was measured by a digital pH meter (Mettler Toledo 320) and adjusted by the addition of small volumes of NaOH or HAc solution. The batch method was chosen to perform crystallization experiments. The starting material was made up of equal volumes of protein solution and precipitant solution. The total solution volume in one batch crystallization was about 0.61.6 mL. Crystallizations were not seeded, and trials were incubated in a circulator (PolyScience) over 2 weeks. Final samples were observed with a microscope with an attached CCD video camera (Olympus BX51), and images were analyzed by software (analySIS). The solubility of lysozyme at various temperatures and precipitant/additive concentrations was measured at pH 4.8 in 0.1 M acetate buffer, while solubility of chymotrypsinogen A was measured at pH 5.25 in 0.01 M citrate buffer. Solidliquid equilibrium was approached through both crystallization and dissolution. Dissolving lasted 3 days, while the period of crystallization was over 2 weeks. The supernatant in equilibrium with a macroscopically observable solid was then filtered
10.1021/cg0200412 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/07/2002
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Figure 1. Micrographs of lysozyme crystals obtained with/without additives in 0.10 M NaAc/HAc buffer at pH 4.8 and 22.0 °C: (a) no additive; (b) 1% (w/v) PEG 4000; (c) 3.75% (w/v) PEG 1500; (d) 12.5% (v/v) ethyl glycol; (e) 12.5% (v/v) DMSO; (f) 25% (v/v) glycerol; (g) 12.5% (v/v) 1,3-pentanediol; (h) 0.15% (w/v) lipopolysaccharide; and (i) 0.6% (w/v) MEGA-8. through 0.1 µm filters (Millex-VV). The concentration of diluted supernatant was determined spectroscopically and verified by refractive meter (Kru¨ss) until the refractive index remained unchanged at the equilibrium state. The solubility of each sample was measured in duplicate. Nucleation from solution is strongly dependent on supersaturation. There exists a critical supersaturation range Sc that separates the region where no measurable nucleation occurs from that where abundant new phase forms instantaneously (referred to as “explosive nucleation” in this paper), and this critical supersaturation Sc is generally affected by many parameters such as buffer, pH, precipitant/additive, stirring, seed, cooling/heating rate, crystallizer, external fields, etc.13-18 In this study, explosive nucleation was approached through decreasing/increasing the temperature of saturated protein solution at a desired precipitant/additive concentration at a rate of 6 °C/h. The chosen cooling/heating rate ensured that protein solution could not undergo liquid-liquid phase separation or gelation.19,20 A laser generator (Interlink TS-N) generating a laser beam of 660 nm was applied to detect turbidity. The stirring bar ensured proper mixing and rapid temperature equilibration of the protein solution in a crystallizer. The temperature of the crystallizer was controlled by a circulator (Julabo). After the temperature reached a certain value, the solution became opaque or translucent from clear, which indicated the presence of explosive nucleation. The turbidity temperature was denoted as Tturbid, and the supersaturation ratio of protein solution at this critical temperature Tturbid was referred to as the critical supersaturation for explosive nucleation, Sc. Microscopic observation showed that the final solid was crystalline precipitate.
Results and Discussion Batch Crystallization. When the starting concentrations after the mixing of chymotrypsinogen A and
ammonium sulfate (AS) were 14.1 mg/mL and 20% (w/ v), respectively, the addition of 0.8 (w/v) or 0.24% PEG 4000 or 4.0% PEGS 1500 resulted in gelation at both 4.0 and 22.0 °C. When 0.2% (w/v) CsCl, 0.2% (w/v) MgCl2, 1% (w/v) CaCl2, 10% (v/v) glycerol, or 2.5% (v/v) DMSO were added, respectively, into crystallizing solution at the starting concentrations of AS and chymotrypsinogen A of 18% and 20.0 mg/mL, no detectable crystals formed at 4.0 °C, whereas rodlike crystals appeared without additives or in the presence of 2.5% glycerol or 1.25% DMSO. In a recipe using sodium chloride as the precipitant (the starting concentrations after mixing of NaCl and lysozyme were 7.5% (w/v) and 20 mg/mL, respectively), lysozyme crystals appeared needlelike without the addition of additives (Figure 1a). When 1% PEG 4000 was added, rodlike crystals formed (Figure 1b). When 3.75% PEG1500 was added, prism crystals formed but were apt to aggregate (Figure 1c). However, in the presence of 12.5% (v/v) ethyl glycol, the crystals became smaller needlelike (Figure 1d). Such additives as DMSO (Figure 1e), glycerol (Figure 1f), hexanediol, MPD, pentanediol (Figure 1g), sorbitan, dextran, lipopolysaccharide (Figure 1h), xylitol, lactitol, mannitol, MEGA-8 (Figure 1i), and zwittergent 3-18 could also result in prism crystals. As for electrolyte additives, 1% (w/v) MgCl2 showed little effect on the crystal habit of lysozyme, while using 0.5% (w/v) CsCl or 1% CaCl2 (w/v) as additives made prism crystals occurr. The additives tested in this work showed no marked effect on the crystal habit of chymotrypsinogen A;
Effect of Additives on Lysozyme and Chymotrypsinogen A
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Figure 3. Lysozyme solubility at different temperatures under different solvent conditions. The solubility data at NaCl concentration 2.0 and 3.0% are from the work of Forsythe, Judge, and Pusey.24
Figure 2. Micrographs of chymotrypsinogen A crystals obtained gradually under temperature up from 4.0 to 22.0 °C in 0.01 M citrate buffer at pH 5.25. The starting concentrations of protein and ammonium sulfate are 10 mg/mL and 20% (w/ v), respectively: (a) no additive; (b) thicker crystals formed in the presence of 10% (v/v) glycerol; and (c) clusters appeared.
crystals appeared long and rodlike. For example, when using 10% (v/v) glycerol as the additive, crystals became a bit thicker, but white crystal clusters also formed (Figure 2). After comparing the results of crystallization experiments in the presence of additives with those without additives, and not considering the probable effect of impurities, we can suggest that additives have different influences on the crystallization region and crystal habit of different proteins, and the effect of additives on protein crystallization is dependent on their concentrations. It is worth noting that in this study, ethyl glycol (EG) can cause lysozyme crystals to be smaller and needlelike. Sauter et al.21 have also observed more and smaller crystals forming in 0.10 M sodium acetate buffer, pH 4.6, with 1.0 M NaCl in the presence of ethylene glycol. But Farnum and Zukoski11 have reported that ethylene glycol can weaken attractions and increase the solubility of lysozyme in 0.05 M sodium acetate buffer, pH 4.6, with 0.35 M NaCl. One method to explain the discrepancy is that the effect of EG on the attractive forces between lysozyme molecules may be dependent on the ionic strength, and/or EG can promote nucleation. PEGs may induce a depletion attraction between particle pairs and can hydrate as well as alter the dielectric constant of the solvent,22 so that the addition of PEGs into the chymotrypsinogen A crystallization batch can result in gelation with precipitant ammonium
Figure 4. Chymotrypsinogen A solubility at different temperatures under different solvent conditions.
sulfate at high concentrations. On the other hand, our experimental results from lysozyme crystallization suggest that the addition of PEGs with low molecular weights at low concentrations is favorable for lysozyme crystallization at high ionic strength, and the influence varies with their concentrations and molecular weights. Therefore, there may exist an optimal choice among ionic strength, PEGs concentration, and molecular weight when using PEG as additive.23 Solubility. The solubility of lysozyme and chymotrypsinogen A under different solvent conditions in the presence of DMSO or glycerol is shown in Figures 3 and 4 (the lines are included to guide the eye). The measured solubility of lysozyme at 2.5% NaCl concentration is consistent with the solubility data for tetragonal crystals of chicken egg white lysozyme by Forsythe, Judge, and Pusey.24 Some works have indicated that the effect of additives on solubility strongly correlates to the strength of molecule interactions.11,25 As shown in Figures 3 and 4, protein solubility increases in the presence of 10% (v/v) glycerol or 1% (v/v) DMSO, indicating that repulsions are induced (attractions are reduced) between protein molecules in the presence of additives. Figure 5 shows that the solubility of lysozyme in 0.1 M sodium acetate buffer, at pH 4.8, with 2.5% (w/v) NaCl increases with the concentration of DMSO or glycerol. Further-
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dinated effect of additives on the interactions between protein molecules, solubility (corresponded to supersaturation), and interfacial energy. As seen from Figure 1e,f, using glycerol and DMSO as additives, prism crystals of lysozyme form instead of needlelike crystals. It can be explained that although glycerol and DMSO promote nucleation, the addition of glycerol or DMSO can increase protein-protein repulsion and solubility, thus supersaturation is decreased, which can result in better quality crystals. Conclusions Figure 5. Lysozyme solubility at 22.0 °C as a function of the concentration of additives. Table 1. Effect of Additives on Crystallization Enthalpy ∆Hxtal and the Critical Supersaturation Sc of Lysozyme and Chymotrypsinogen A protein
solution conditions
∆Hxtal (kJ/mol)
Sc
lys lys lys chy chy chy
2.5% NaCl, no additive 2.5% NaCl, 1% DMSO 2.5% NaCl, 10% glycerol 20% AS, no additive 20% AS, 10% glycerol 20% AS, 1% DMSO
-73.7 -52.8 -44.8 79.8 63.1 27.2
4.38-4.88 4.15-4.36 3.45-3.60 4.83-5.08 2.17-2.38 1.78-2.00
more, the increment of solubility decreases as the concentration of additive increases. When the concentration of DMSO is higher than 2%, the solubility of lysozyme increases very slowly. Crystallization Enthalpy. Following Ewing, Forsythe, and Pusey,26 we fit the solubility using van’t Hoff’s equation and obtained crystallization enthalpy, ∆Hxtal. The values of ∆Hxtal are summarized and compared in Table 1. As expected, the change in enthalpy ∆Hxtal of lysozyme is negative, while that of chymotrypsinogen A is positive; that is, heat is released when a lysozyme crystal forms, while the crystallization of chymotrypsinogen A is an endothermic process. Differences in solution conditions and crystal forms may account for the disparate enthalpy values in Table 1. Crystallization enthalpy ∆Hxtal can be regarded as a reflection of the nature of intermolecular contacts and hydration. The larger ∆Hxtal corresponds to the larger surface energy of the crystal.27 In this study, ∆Hxtal decreases in the presence of glycerol or DMSO, which suggests that these two additives can reduce surface energy of the crystal and thus promote nucleation. Critical Supersaturation. The critical supersaturation Sc for explosive nucleation is also presented in Table 1. As for lysozyme, the critical supersaturation Sc in the presence of 1% DMSO or 10% glycerol is a bit lower than that without additives. While for chymotrypsinogen A, the addition of 10% glycerol or 1% DMSO results in much lower Sc, which indicates that nucleation is notably promoted by additives. It is found that the tendency of the effect of additives on Sc is quite consistent with that on solubility and ∆Hxtal, which can confirm that above two additives can increase solubility, decrease the attractive strength between protein molecules, reduce surface energy of the crystal, and promote nucleation. Apparently, the ultimate influence of additives on nucleation process is determined by the coor-
Different additives show various influences on the crystallization of lysozyme and chymotrypsinogen A. Some probable mechanisms of the effect of additives on protein crystallization may include that additives can affect the interactions between protein molecules, change protein’s solubility, shift thermodynamic equilibrium, alter surface energy of the crystal, and affect nucleation in protein crystallization. This investigation contributes to a better understanding of the effect of additives involved in macromolecular crystallization, which may be useful for optimizing crystallization with additives. Further work will focus on the correlation between the rate of nucleation or growth and the effect of additives on nucleation or growth. Solution and particle behaviors and specific interactions in the presence of additives will be addressed. Much more theoretical work also needs to be conducted. Acknowledgment. This research is supported by the Institute of Chemical and Engineering Sciences (ICES), Singapore, and the National Natural Science Foundation of China (No. 20106010). References (1) Stewart, L.; Clark, R.; Behnke, C. Drug Discovery Today 2002, 7, 187-196. (2) Chayen, N. E. J. Cryst. Growth 1999, 198/199, 649-655. (3) Wiencek, J. M. Annu. Rev. Biomed. Eng. 1999, 1, 505-534. (4) Hu, H.; Hale, T.; Yang, X.; Wilson, L. J. J. Cryst. Growth 2001, 232, 86-92. (5) McPherson, A. Eur. J. Biochem. 1990, 189, 1-23. (6) Schwartz, A. M.; Berglund, K. A. J. Cryst. Growth 2000, 210, 753-760. (7) Markman, O.; Roh, C.; Roberts, M. F.; Teeter, M. M. J. Cryst. Growth 1996, 160, 382-388. (8) Tanaka, S.; Ataka, M.; Kubota, T.; Soga, T.; Homma, K.; Lee, W. C.; Tanokura, M. J. Cryst. Growth 2002, 234, 247254. (9) Hamana, H.; Moriyama, H.; Shinozawa, T.; Tanaka, N. Acta Crystallogr. 1999, D55, 345-346. (10) Guan, R. J.; Wang, M.; Liu, X. Q.; Wang, D. C. J. Cryst. Growth 2001, 231, 273-279. (11) Farnum, M.; Zukoski, C. Biophys. J. 1999, 76, 2716-2726. (12) Velev, O. D.; Kaler, E. W.; Lenhoff, A. M. Biophys. J. 1998, 75, 2682-2697. (13) So¨hnel, O.; Garside, J. Precipitation; Butterworth-Heinemann Ltd.: Oxford, 1992; Chapter 3, pp 52-53. (14) Dixit, N. M.; Zukoski, C. F. J. Colloid Interface Sci. 2000, 228, 359-371. (15) Galkin, O.; Vekilov, P. G. J. Cryst. Growth 2001, 232, 6376. (16) Bhamidi, V.; Skrzypczak-Jankun, E.; Schall, C. A. J. Cryst. Growth 2001, 232, 77-85. (17) Izmailov, A. F.; Myerson, A. S. J. Chem. Phys. 2000, 112, 4357-4364. (18) Paxton, T. E.; Sambanis, A.; Rousseau, R. W. Langmuir 2001, 17, 3076-3079.
Effect of Additives on Lysozyme and Chymotrypsinogen A (19) Broid, M. L.; Tominc, T. M.; Saxowsky, M. D. Phys. Rev. E 1996, 53, 6325-6335. (20) Grigsby, J. J.; Blanch, H. W.; Prausnitz, J. M. Biophys. Chem. 2001, 91, 231-243. (21) Sauter, C.; Ng, J. D.; Lorber, B.; Keith, G.; Prion, P.; Hosseini, M. W.; Lehn, J. M.; Giege, R. J. Cryst. Growth 1999, 196, 365-376. (22) Tardieu, A.; Finet, S.; Bonnete, F. J. Cryst. Growth 2001, 232, 1-9. (23) Jaramillo-Flores, M. E.; Soriano-Garcia, M.; Moreno, A. J. Mol. Struct. 1998, 444, 155-164.
Crystal Growth & Design, Vol. 3, No. 1, 2003 87 (24) Forsythe, E. L.; Judge, R. A.; Pusey, M. L. J. Chem. Eng. Data 1999, 44, 637-640. (25) Kulkarni, A. M.; Zukoski, C. F. Langmuir 2002, 18, 30903099. (26) Ewing, F.; Forsythe, E.; Pusey, M. Acta Crystallogr. 1994, D50, 424-428. (27) Ninomiya, K.; Yamamoto, T.; Oheda, T.; Sato, K.; Sazaki, G.; Matsuura, Y. J. Cryst. Growth 2001, 222, 311-316.
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