CRYSTAL GROWTH & DESIGN
Effects of Forced Solution Flow on Protein-Crystal Quality and Growth Process Kadowaki,†
Yoshizaki,‡
Akio Izumi Satoshi Osamu Odawara,† and Shinichi Yoda*,†,‡
Adachi,‡
Hiroshi
2006 VOL. 6, NO. 10 2398-2403
Komatsu,‡,§
Tokyo Institute of Technology, Materials Interdisciplinary Graduate School of Science and Engineering, 4259 Nagatsuta, Midori-Ku, Yokohama 226-8502, Japan, Japan Aerospace Exploration Agency (JAXA), ISS Science Project Office, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505, Japan, and Iwate Prefectural UniVersity, Takizawa-mura, Iwate 020-0193, Japan ReceiVed NoVember 5, 2005; ReVised Manuscript ReceiVed July 8, 2006
ABSTRACT: X-ray diffraction experiments were performed to study the effect of forced solution flow on protein-crystal quality. The tetragonal crystals of hen egg white lysozyme were grown under flow in the rate range of 0 (quiescent condition) to 1000 µm/s. The quality of the crystals grown under flow was superior to that of crystals grown under quiescent conditions. To investigate the mechanism of the quality improvement, we observed the surface morphology of the (110) face using atomic force microscopy (AFM). Under quiescent conditions, the two-dimensional (2D) nuclei were randomly formed over the surface. Under solution flow, a hillock consisting of 2D islands was formed around the facet center. The step density under flow was higher than that under quiescent conditions. The step growth rate decreased with increasing flow rate because of the step-density increase. We found that there was a strong correlation between the crystal quality and the step growth rate. The results suggested that the slow growth decreases the molecular misorientation at the kink site, resulting in an improvement in crystal quality. 1. Introduction The effect of solution flow on protein-crystal quality through the growth process was studied as a counter experiment to crystal-quality improvement under microgravity. Conducting preliminary X-ray diffraction experiments with synchrotron radiation, we found that tetragonal lysozyme crystals grown under flow had better quality than those grown under quiescent conditions.1 However, it is not clear how flow improves the crystal quality. The growth process determines the crystal quality. Thus, it is necessary to investigate the effect of solution flow on the growth process to clarify the mechanism. The effects of flow on the growth of tetragonal lysozyme crystals have been studied through the investigations of growth rate, surface morphology, and nucleation rate.2-12 When lowly purified lysozyme (∼95%) was used, the face growth rate of crystal subjected to forced flow, which was comparable to natural convection (30-50 µm/s), significantly decreased with time and eventually ceased.2,3 Growth cessation was also observed when the crystal was suspended in an up-flow provided by thermosyphon.4 However, the reduction of face growth rate was unconfirmed when crystals grew at a higher flow rate (150 µm/s).5,6 When highly purified lysozyme (99.99%) was used, the face growth rate initially increased (250 µm/s) with increasing flow rate.7-9 A maximum in the face growth rate was also observed in a membrane crystallization device working at 1100 µm/s (purity ∼95%).10 The effect on surface morphology, the formation of step bunching under solution flow (25 µm/s), was confirmed by AFM observation (purity ∼99%).11 When steps moved parallel to the solution flow, step bunching developed within about 10 h. The deceleration in nucleation rate by gentle stirring12 or the reduction in kinetics fluctuations under flow (2.5 µm) were formed under 1000 µm/s. The adsorbed aggregates would locally enhance 2D nucleation and give rise to the hillock formation. However, the possibility of quality degradation by the adsorption of the aggregates should be considered. Their adsorption and subsequent incorporation into the crystal would produce various kinds of defects.19-21 In this case, this assumption fails to explain the quality improvement by flow. However, Kuznetsov et al. reported that liquid aggregates immediately restructured as crystalline multiple layer stacks (hillock) when they adsorbed the existing crystal surface.21 The stacks were well aligned with the underlying lattice. They suggested that the order of the molecules in the aggregate was guided by the underlying lattice, which served as an epitaxial substrate. If such an aggregate contributed to the formation of a hillock observed here, the crystal quality was not affected by aggregate adsorp-
Figure 7. (a) AFM images of the (110)a surface of a crystal grown under quiescent conditions. (b) The cross-sectional profile along line 1 shown in (a). (c) The cross-sectional profile along line 2. (d) AFM images of the (110)a surface of a crystal grown under solution flow (1000 µm/s). The black arrow indicates the direction of solution flow. (e) The cross-sectional profile along line 3 shown in (d). (f) The cross-sectional profile along line 4. (g) The cross-sectional profile along line 5. Table 2. Step Densities d110a and Estimated Local Slopes p110a of Quiescent and Flow Crystals (using step height h ) 5 nm) flow rate (µm/s) d110a (1/µm) p110a
0 0.80 4.00 × 10-3
100 1.00 5.00 × 10-3
500 1.20 6.00 × 10-3
700 1.33 6.65 × 10-3
1000 1.37 6.83 × 10-3
2402 Crystal Growth & Design, Vol. 6, No. 10, 2006
Figure 8. Flow-rate dependence of average face growth rate R110a (2) and average step growth rate υ110a (9).
tion. The details of the relations of aggregate adsorption to hillock formation and the quality will be described elsewhere. After formation of the growth hillock, the step density increases as follows. The solution flow increases interfacial concentration through the decrease in the thickness of the concentration boundary layer over the crystal.22,23 A higher interfacial concentration increases the 2D nucleation rate. The rapid 2D nucleation on the top of the underlying 2D island decreases the terrace width. The interfacial concentration increases with flow rate, which results in an increase in the step density. Step propagation is likely to be faster than step generation, so that the steps from the hillock gradually engulf the entire surface. Furthermore, we also attempted to find planar defects, stacking faults, and dislocations that will also affect the crystal quality. It is possible to detect these defects by AFM observation.19-21 However, no major defects were observed under solution flow or under quiescent conditions. This result coincides with that obtained from the previous study in which the lysozyme purity is equivalent to ours.24 3.3. Step Growth Rate. With respect to the correlation between the crystal quality and step growth rate, it has been proposed that a slower growth yields a higher-quality protein crystal.25 Therefore, the effect of solution flow on the step growth rate on the (110)a face was investigated. To estimate the average step growth rate υ110a, we measured the average face growth rates R110a and the average local slope p110a under quiescent and flow conditions. Figure 8 shows flow-rate dependence of the average R110a value. R110a increases with increasing flow rate, reaches a maximum value at 700 µm/s, and then slightly decreases. The initial increase in R110a can be attributed to the enhancement of solute transport toward the interface by flow. The decease in R110a under a higher flow rate agrees with that reported in previous papers.7-9,10 The decrease in R has been attributed to the flow-enhanced supply of impurities to the crystal interface. The impurities presumably affect crystal growth even in highly purified lysozyme solution. The average p110a value also increased with increasing flow rate, which was mentioned in section 3.2, because the step density increased (see Table 2.) The average υ110a was calculated from the relationship υ110a ) R110a/p110a. Consequently, we found that the average υ110a decreased with increasing flow rate (Figure 8). Despite the increase in R110a with increasing flow rate, the decrease in υ110a indicates that the flow effects were just reflected in the increase in the step density. 3.4. How Flow Improves Crystal Quality. We considered an improvement mechanism in terms of the relation between the crystal quality and the step growth rate. The correlation coefficients between each quality index and the step growth rate were examined. The correlation coefficients were found to
Kadowaki et al.
be 0.89 for maximum resolution limit, 0.93 for 〈I〉/〈σ(I)〉, 0.91 for Rmerge, and 0.42 for the B factor. This result indicated that there is a strong correlation between the crystal quality and the step growth rate (except for the B factor). When we discuss the contribution of the decrease in the step growth rate, a possible improvement mechanism is proposed as follows. The lower step growth rate was induced by the high step density, as mentioned above. Once the step density increases because of solution flow, surface diffusion field overlap is induced. The diffusion field overlap decreases the flux of protein molecules to a kink. Therefore, each molecule can have sufficient time to settle in its optimum orientation at the kink site before being interfered by the subsequent molecules, which leads to a more ordered crystal. In contrast, the flux of protein molecules to a kink site increases in faster step growth. In this condition, the molecules being trapped in nonstable configurations at the kink site are inconsistent with the regular periodic lattice. This is possible because protein molecules are anisotropic both in the shape and bonding site. Also, amino acid side chains of protein molecules are not rigid in structure, so they may arrange their residues to fit in the kink site even if they are disoriented. Recently, such rotational disorder of protein molecules was confirmed from surface observation using electron microscopy.26 According to this model, the key to quality improvement is the high step density induced by flow. 4. Conclusion The effect of forced solution flow on the quality of lysozyme crystal was investigated. The X-ray diffraction results clearly demonstrated that the quality of flow crystals was higher than that of quiescent crystals. The flow changed the surface morphology drastically. Because of the increase in step density at the hillock slope, the step growth rate for the flow crystal was lower than that for the quiescent crystal. We found a strong correlation between the crystal quality and step growth rate. A lower step growth rate yielded a higher quality. The results suggest that the slow growth improved quality through the decrease in misorientation upon molecular incorporation. Acknowledgment. We thank Mr. Iimura (A. E. S.) for sample purification and Dr. Igarashi and Dr. Suzuki (Photon Factory) for the beam-time allocated for this study and Dr. Sazaki (Tohoku University) for helpful comments and advice. We also thank Mr. Fukuyama (A. E. S.) for AFM analytical help and Dr. Rong and Mr. Sano (JAXA) for experimental help. Appreciation is also extended to Dr. Ishikawa (JAXA) for help in making a growth cell and to Dr. Paradis (JAXA) for his review of the manuscript. We greatly thank the reviewers for helpful comments and advice. References (1) Kadowaki, A.; Yoshizaki, I.; Rong, L.; Komatsu, H.; Odawara, O.; Yoda, S. J. Synchrotron Radiat. 2004, 11, 38-40. (2) Pusey, M.; Witherow, W.; Naumann, R. J. Cryst. Growth 1988, 90, 105-111. (3) Grant, M. L.; Saville, D. A. J. Cryst. Growth 1995, 153, 42-54. (4) Nyce, T. A.; Rosenberger, F. J. Cryst. Growth 1991, 110, 52-59. (5) Durbin,.S. D.; Feher, G. J. Cryst. Growth 1986, 76, 583-592. (6) Durbin,.S. D.; Feher, G. J. Mol. Biol. 1990, 212, 763-774. (7) Vekilov, P. G.; Rosenberger, F. J. Cryst. Growth 1998, 76, 251-261. (8) Vekilov, P. G.; Thomas, B. R.; Rosenberger, F. J. Phys. Chem. 1998, 102, 5208-5216. (9) Vekilov, P. G.; Rosenberger, F.; Lin, H.; Thomas, B. R. J. Cryst. Growth 1999, 196, 261-275. (10) Curcio, E.; Simone, S.; Di Profio, G.; Drioli, E.; Cassetta, A.; Lamba, D. J. Membr. Sci. 2005, 257, 134-143. (11) Rong, L.; Yoshizaki, I.; Komatsu, H.; Kadowaki, A.; Fukuyama, S.; Iimura, Y.; Yoda, S. J. Jpn. Soc. MicrograVity Appl. 2003, 20, 128131.
Effects of Forced Solution Flow on Protein-Crystal Quality (12) Adachi, H.; Takano, K.; Yoshimura, M.; Mori, Y.; Sasaki, T. Jpn. J. Appl. Phys. 2002, 41, L1025-L1027. (13) Thomas, B. R.; Vekilov, P. G.; Rosenberger, F. Acta Crystallogr., Sect. D 1996, 52, 766-784. (14) Sazaki, G.; Kurihara, K.; Nakada, T.; Miyashita, S.; Komatsu, H. J. Cryst. Growth 1996, 169, 355-360. (15) Collaborative Computational Project. Acta Crystallogr., Sect. D 1994, 50, 760-763. (16) Rossmann, M. G.; van Beek, C. G. Acta Crystallogr., Sect. D 1999, 55, 1631-1640. (17) Chernov, A. A. Modern Crystallography, Vol. III: Crystal Growth; Springer-Verlag: Berlin, 1984. (18) Vekilov, P. G.; Monaco, L. A.; Rosenberger, F. J. Cryst. Growth 1995, 156, 267-278. (19) McPherson, A. Crystallization of Biological Macromolecules; Cold Spring Harbor Laboratory Press: New York, 1999.
Crystal Growth & Design, Vol. 6, No. 10, 2006 2403 (20) Malkin, A. J.; Kuznetsov, Yu G.; McPherson, A. J. Struct. Biol. 1996, 117, 124-137. (21) Kuznetsov, Yu. G.; Malkin, A. J.; McPherson, A. Phys. ReV. B 1998, 58, 6097-6103. (22) Hasegawa, K. Ph.D. Thesis, Tohoku University, Sendai, Japan, 1997. (23) Rashkovich, L. N.; Shekunov, B. Yu. J. Cryst. Growth 1990, 100, 133-144. (24) Yoshizaki, I.; Sato, T.; Igarashi, N.; Natsuisaka, M.; Tanaka, N.; Komatsu, H.; Yoda, S. Acta Crystallogr., Sect. D 2001, 57, 16211629. (25) Yoshizaki, I.; Nakamura, H.; Sato, T.; Igarashi, N.; Komatsu, H.; Yoda, S. J. Cryst. Growth 2002, 237-239, 295-299. (26) Braun, N.; Tack, J.; Fischer, M.; Bacher, A.; Bachmann, L.; Weinkauf, S. J. Cryst. Growth 2000, 212, 270-282.
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