Article pubs.acs.org/crystal
Effects of a Forced Solution Flow on the Step Advancement on {110} Faces of Tetragonal Lysozyme Crystals: Direct Visualization of Individual Steps under a Forced Solution Flow Mihoko Maruyama,*,† Hisato Kawahara,† Gen Sazaki,†,§ Syou Maki,†,○ Yoshinori Takahashi,† Hiroshi Y. Yoshikawa,†,∥ Shigeru Sugiyama,‡ Hiroaki Adachi,†,⊥ Kazufumi Takano,⊥,# Hiroyoshi Matsumura,†,⊥ Tsuyoshi Inoue,†,⊥ Satoshi Murakami,⊥,∇ and Yusuke Mori†,⊥ †
Graduate School of Engineering, and ‡Graduate School of Science, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0087, Japan The Institute of Low Temperature Science, Hokkaido University, N19-W8, Kita-ku, Sapporo 060-0819, Japan ∥ Department of Chemistry, Saitama University, 255, Shimookubo, Sakura-ku, Saitama, Saitama 338-8570, Japan ⊥ SOSHO Inc., 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan # Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan ∇ Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan §
ABSTRACT: To grow protein crystals of better quality, there still exists an open question whether a solution flow should be suppressed or intentionally introduced. To obtain a comprehensive understanding of the effects of a solution flow, we directly measured the velocities of individual elementary steps (on {110} faces of tetragonal lysozyme crystals) under a forced solution flow, for the first time, by laser confocal microscopy combined with differential interference contrast microscopy. When we used crystals grown by a twodimensional (2D) nucleation growth mechanism in a solution of commercial lysozyme (98.5% purity, from Seikagaku Co.), while increasing the solution flow rate, the step velocity decreased monotonically. We confirmed that this decrease in the step velocity with flow rate was due to the enhancement of the mass transfer of impurity (mainly covalently bonded dimer), by the observation using a lysozyme further purified (dimer was removed). In contrast, when we used crystals grown by a spiral growth mechanism in a commercial lysozyme solution, with increasing the flow rate, the step velocity increased and had the maximum at the flow rate of 10 μm/s, and then decreased monotonically. Also, the step velocity was 2−4 times higher than in the case of the 2D nucleation growth. These results demonstrate that the growth of spiral steps is less affected by impurities because the density of spiral steps is much higher than that of 2D island ones.
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insight into the effects of solution flow on the growth of protein crystals. Vekilov et al. observed crystal surfaces grown by a spiral growth mechanism using a Michelson interferometer and calculated step velocities from the average motions of spiral growth hillocks.21,22 They reported that as the flow rate increased, the step velocities increased (20 μm/s, V⟨110⟩ then gradually decreased as the flow rate increased; V⟨001⟩ also showed a similar tendency, although the amount of the increase at 10 μm/s is much smaller. The appearance of the maximum growth rate reproduced the results reported by Vekilov et al.21,22 However, they measured the normal growth rate and step velocity from the changes in the average height profiles of spiral growth hillocks by Michelson interferometry. In addition, as compared to the results of the 2D nucleation growth mechanism using the commercial lysozyme (Figure 3c), we found that the velocities of the spiral steps in the ⟨110⟩ and 2860
dx.doi.org/10.1021/cg300025b | Cryst. Growth Des. 2012, 12, 2856−2863
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Article
dimer, resulting in the increase in the step velocity (Figure 6e). In contrast, at the solution flow of >10 μm/s, the increase in the mass transfer of the dimer dominated the advancement of elementary steps, even in the case of spiral growth. Recently, Sleutel et al. measured the velocities of spiral steps and 2D island ones, as a function of supersaturation, on tetragonal lysozyme crystals grown via purified and commercial solutions by LCM-DIM.47 They found that the morphology and step dynamics of spiral hillocks were less affected by the presence of impurities than those of 2D islands. Although Sleutel et al. measured the step velocities in stagnant solutions, their findings are essentially the same as those in this study. Therefore, their results also strongly support the conclusion obtained in this study. Our group, so far, has obtained 15 kinds of protein crystals of better quality by intentionally stirring protein solutions.9−19 The results may be partly due to the experimental conditions in which the mass transfer of the solute became more significant than that of the impurity via a solution flow. To prove this hypothesis, we need to further perform systematic studies by changing growth mechanisms, flow rates, and types and amounts of impurities. Finally, we emphasize that the effects of a forced flow and the difference between 2D nucleation growth and spiral growth can be applied not only to protein crystals, but also to various kinds of crystals. Because the growth of small-molecule crystals is generally more limited by the mass transfer of a solute than protein crystals, a solution flow will play a more significant role in the growth of smallmolecule crystals.
impurity depletion zone was affected more significantly than the solute depletion zone. Impurity models can be classified into two categories:39 (1) impurity molecules adsorbing at kink sites leading to kink blocking,40 and (2) impurity molecules adsorbing on terraces leading to step pinning.41 It is reported that dimer molecules mainly adsorb randomly on terraces of {101} and {110} faces of tetragonal lysozyme crystals.39,42 Hence, we have to take into account the latter case. In the case of spiral growth, exposure time of a terrace to dimers in a solution is significantly shorter than that of the 2D nucleation growth, because of the much higher step density at the same supersaturation. In the case of spiral growth, the terrace exposure time is as follows:43 τspiral = λ /vstep
(1)
where λ is step spacing, and vstep is step velocity. In our experiments, vstep of spiral growth ranged from 2 to 11 nm/s, and λ was about several micrometers. From these experimental data, τspiral is estimated to be from a few minutes to several tens minutes. On the other hand, the terrace exposure time of 2D nucleation growth is expressed as follows:43 τ2D = h/R =
h 2 h[πvstep J ]1/3
(2)
where h is step height, R is normal velocity, and J is the 2D nucleation rate on the surface. In our experiments, vstep of 2D nucleation growth ranged from 1 to 3.5 nm/s, and J was estimated, on the basis of our observation, to be 3.6 × 104 s−1 m−2. From these experimental data, τ2D is estimated to be several hundreds minutes. Comparing τspiral with τ2D, it is apparent that τspiral is significantly shorter than τ2D. Hence, the amount of density of adsorbed dimer molecules for one terrace between the adjacent spiral steps should be much smaller than the density between the 2D island steps (Figure 6d). As a consequence, the impurity effects of dimers on the spiral steps can be significantly less than those on the 2D islands. This difference can satisfactorily explain the faster velocity (2−4 times) of the spiral steps, as compared to the 2D island steps. Recently, Dai et al. revealed, by direct observation, using a fluorescent-labeled lysozyme, that lysozyme molecules need a certain amount of time for the adsorption.44 Consequently, we should expect that dimers also need a certain amount time for their adsorption. If this expectation is true, the adsorption of a dimer onto a terrace between spiral steps becomes more difficult than in the case of 2D islands, resulting in smaller impurity effects on the spiral steps. Tsukamoto et al. slightly dissolved {110} faces of tetragonal lysozyme crystals grown by spiral growth and 2D nucleation growth mechanisms and observed the crystal surfaces. They found that the densities of shallow etch pits, which indicate point defects caused by impurity molecules incorporated into a crystal surface, were significantly lower on the spiral growth surface than on the 2D nucleation growth surface.45,46 This strongly supports our findings. Under the solution flow, with an increasing flow rate, the mass transfer of both the solute lysozyme and the impurity dimer was enhanced. However, because of the significantly weaker impurity effects on the spiral steps relative to the 2D island ones, at the solution flow rate of ≤10 μm/s, the increase in the mass transfer of the solute probably surpassed that of
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CONCLUSIONS In this study, we directly measured the step velocities on the {110} faces of tetragonal lysozyme crystals under the solution flows of various rates, for the first time, utilizing LCM-DIM. During the experiments, we used the crystals grown by the 2D nucleation growth and spiral growth mechanisms in the solutions of commercial and purified lysozymes. The key results are as follows: (1) In the case of 2D nucleation growth in the commercial lysozyme solution, by increasing the solution flow rate, the step velocity decreased monotonically. This was the result of an enhancement of the mass transfer of the dimer to the crystal surfaces. (2) The explanation of (1) was supported by the step velocity measurement using the purified lysozyme. (3) In the case of spiral growth in the commercial lysozyme solution, by increasing the flow rate, the step velocity increased and exhibited the maximum level (10 μm/s); then, it decreased monotonically. The step velocities were 2−4 times higher than those of 2D nucleation growth. (4) The results of (3) were successfully explained by the much smaller exposure time of a terrace to dimer molecules in the solution because of the much higher step density relative to 2D nucleation growth.
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AUTHOR INFORMATION
Corresponding Author
*Tel.:+81668797705. Fax: +81668797708. E-mail: maruyama@ cryst.eei.eng.osaka-u.ac.jp. 2861
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Present Address
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Laboratory of Molecular Chemistry, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1, Nishikiori-kita,Tondabayashi, Osaka Pref. 584-8540, Japan.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partly supported by a Core Research for Evolution Science and Technology (CREST) grant to Y.M., and by the Science and Technology Incubation Program from the Japan Science and Technology Agency to H.M.
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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on May 11, 2012, with errors to the Reference Section. The corrected version was reposted on May 17, 2012. 2863
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