Effect of Protein Molecular Anisotropy on Crystal Growth† Hironori Hondoh*,# and Toshitaka Nakada‡ Research Faculty of Agriculture, Hokkaido UniVersity, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan, and Faculty of Science and Engineering, Ritsumeikan UniVersity, Noji-Higashi 1-1-1, Kusatsu, Shiga 525-8577, Japan
CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 12 4262–4267
ReceiVed June 30, 2008; ReVised Manuscript ReceiVed September 30, 2008
ABSTRACT: We have measured the difference in the growth rates of the (010) and (01j0) polar faces of hen egg-white lysozyme monoclinic crystals using optical microscopy, and observed the micro-topography of these faces with atomic force microscopy. Because of the lack of a rotational axis perpendicular to the b-axis, these two opposite faces are not symmetry related. The growth rate of the (010) face was faster than the (01j0) face at supersaturation levels of 0.84-3.5. The surface free energy of the step on the (010) face is calculated to be 8.9 ( 1.3 × 10-4 J/m2. Using atomic force microscopy to examine the crystal faces, the steps on the (010) face were smooth, straight, and parallel to the a- and c-axes, whereas the steps on the (01j0) face were rough and a complex shape even with highly purified lysozyme. The complex shape of the steps on the (01j0) face indicates the presence of adsorbed molecules on the terraces of the steps. The relatively slow growth of the (01j0) face may be explained by the adsorption of wrongly oriented lysozyme molecules with this face. Introduction Anisotropy is a key feature of protein structure. Because of the complexity of enzymes and receptors, these biomacromolecules only bind with their cognate substrates and ligands when they are in the correct orientation. The anisotropic shape and orientation of protein molecules also affect the growth of protein crystals. Protein molecules with optimal conformation and orientation can be incorporated into crystals. However, protein crystallization is hampered due to the complex shape of proteins. Crystallographic symmetry and molecular packing in a unit cell determine the molecular arrangement of crystal surfaces. The morphology and the surface property of (hkl) and (-h-k-l) faces of a non-centrosymmetric crystal should be different due to the polarity of the crystal. The morphology and molecular packing of the hen egg-white lysozyme monoclinic crystal, which does not have centrosymmetry, is shown in Figure 1.1,2 Although the molecular packing at the (010) and (01j0) faces of the monoclinic crystal is identical, different parts of the lysozyme molecule are exposed to the surfaces of the crystal because no rotational symmetry perpendicular to the b-axis exists. The N- and C-terminal regions of lysozyme appear on the (010) face, while the active site of lysozyme is exposed to the surface at the (01j0) face. In a previous paper, we reported a difference in the growth rates of the (010) and (01j0) faces of the hen egg-white lysozyme monoclinic crystal from the growth sector boundary of a crystal.2 Asai et al. and Heijna et al. also reported on the polar growth of the lysozyme monoclinic form by measuring the displacement of the crystal faces with optical microscopy.3,4 Although the polar growth of the monoclinic crystal is obviously demonstrated in this research with optical microscopy, the growth mode of these polar faces at each supersaturations is not clear. In addition, the growth mechanism in molecular scale is still obscure because the observation of these polar faces is difficult due to the elongated shape of the † 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. * To whom correspondence should be addressed. E-mail: hondoh@ abs.agr.hokudai.ac.jp. # Hokkaido University. ‡ Ritsumeikan University.
Figure 1. The morphology of the monoclinic lysozyme crystal (a) and molecular packing in the unit cell (b). Four molecules exist in the unit cell. Two molecules in the asymmetric unit are labeled as A and B, whereas the symmetry equivalent molecules are labeled as A′ and B′. The crystallographic orientation of (a) and (b) is identical. Reprinted with permission from ref 2. Copyright 2001 American Chemical Society.
crystal. Recently, the anisotropic surface topography of the (001) face of triclinic lysozyme crystals was observed with atomic
10.1021/cg800694j CCC: $40.75 2008 American Chemical Society Published on Web 10/30/2008
Protein Molecular Anisotropy on Crystal Growth
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force microscopy (AFM).5 This measurement clearly showed that the orientation of the lysozyme molecules on the surface of a crystal affects the surface topography. Unfortunately, the (hkl) and (-h-k-l) faces are not distinguished in their report because the absolute configuration of the triclinic crystal is not known. Therefore, the relation between molecular orientation and growth mechanisms remains unknown. In this paper, we report the supersaturation dependence of the growth rates of the polar (010) and (01j0) faces of the lysozyme monoclinic crystal and calculate the surface free energy of the steps on the (010) face. The surface topography on these two faces is also shown by using AFM to reveal the effect of protein anisotropy on the growth and surface topography of the crystals. Materials and Methods Crystallization of the Hen Egg-White Lysozyme Monoclinic Form. Six-times recrystallized hen egg-white lysozyme (Seikagaku Kogyo Corporation Ltd.) was used for growth rate and solubility measurements without further purification. Commercial lysozyme from Seikagaku Kogyo and highly purified lysozyme prepared by Maruwa Foods Industry Inc. from Seikagaku products were used for AFM observations. All other chemicals used were reagent grade. Monoclinic form crystals were prepared according to previously described methods.2,6 The buffer solution was 100 mM sodium acetate pH 4.5, and the precipitant was 40 mg/mL NaNO3 dissolved in the same buffer. Equal volumes of 20 mg/mL lysozyme in the buffer solution and the precipitant solution were mixed and incubated at 20 °C. Large rectangular shape crystals were obtained within one week. These crystals were used for further experiments. Growth Rate Measurements. The growth rate of a crystal was calculated from the displacement of the edge of a crystal within a fixed time period. One seed crystal was placed in the observation cell (0.5 mm thickness), and grown until the crystal was fixed to the cell. The b-axis of the crystal was oriented parallel to a horizontal axis. The temperature of the cell was kept at 20 ( 0.1 °C using Peltier devices. Prior to each measurement, the solution in the cell was replaced with a freshly prepared lysozyme solution (supersaturation of the solutions: 0.84-3.5). Two crystals were used for the measurements. To determine the supersaturation of a monoclinic lysozyme crystal under the measurement conditions, an equilibrium concentration of crystals was calculated from the solubility curve measured using two-beam interferometry.7 The supersaturation, σ, was defined by ln C/Ce, where C (mg/mL) is the lysozyme concentration and Ce (mg/mL) is an equilibrium concentration. AFM Observations. The crystals for in situ AFM observations were prepared as follows. Seed crystals were broken into several pieces with a Micro Tool Kit II (Hampton Research). The fragment was placed on a coverslip to orient the (010) or (01j0) faces down. The crystal was grown for several hours to allow the crystal to adhere to the coverslip. The morphology of the crystal was identified according to previous research.2 In-situ AFM observation of the surface topography on the (010) and (01j0) faces were performed with a NanoScope E (Digital Instruments) AFM operating in the contact mode at room temperature (25 °C). The supersaturation of the measurement solution was controlled by adjusting the concentration of lysozyme. Si3N4 cantilevers with a spring constant of 0.06 N/m were used. The AFM observations were performed with commercial lysozyme and highly purified lysozyme.
Results and Discussion Growth Rate of the {010} Faces. The solubility curve of the monoclinic crystal in the buffer solution of 100 mM sodium acetate, pH 4.5, containing 20 mg/mL NaNO3 was measured. This curve is identical to the previous results measured in a 50 mM sodium acetate buffer.2 The solubility of the monoclinic crystal at 20 °C was determined to be 0.94 mg/mL from the solubility curve. The supersaturation was calculated using this value. Figure 2 shows the normal growth rates, R of the (010) and (01j0) faces with σ. The horizontal error bars were estimated
Figure 2. Supersaturation dependence of the growth rates of the (010) and (01j0) faces of a monoclinic lysozyme crystal. The filled circles are for the (010) face, and the filled triangles are for the (01j0) face.
Figure 3. Morphological change of the (010) face grown at σ ) 3.0. Time points are (a) 0 and (b) 60 min. The scale bar in the figure represents 100 µm.
using the errors obtained from the solubility measurements. As shown in Figure 2, the growth rate of the (010) face is much higher than the (01j0) face in all ranges of σ we measured. The growth rates of these two faces did not decrease even at higher supersaturation levels. This is different from the growth rates of the crystal faces of the tetragonal lysozyme polymorph, which decrease if the σ level was over 2.5 because of cluster formation8 or the incorporation of impurities.9 The growth rates of tetragonal crystal faces had maxima at σ ) 2.5. Figure 3 shows that when σ was increased in the monoclinic form, a concave (010) face was observed. This morphological instability appeared when the σ exceeded ∼2.4. AFM Observations. The surface topography on the (010) face of the monoclinic crystals grown with commercial lysozyme (σ ) 0.7) is shown in Figure 4a. Numerous straight steps parallel to the c-axis were observed. The height of these steps was 5.8 ( 0.4 nm, which consists of a two-molecular height. Further-
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Figure 4. Deflection images of the {010} face of the monoclinic lysozyme crystal. The (010) face with commercial lysozyme at σ ) 0.7 (a) and σ ) 1.4 (b). The 2D islands, on which the covering steps are observed, are indicated by white arrows. The (010) face (c) and the (01j0) face (d) with highly purified lysozyme at σ ) 0.05. (e) Processed images of panel c. The single- and double-layered steps are indicated by red and black, respectively. The notations U and L show upper and lower steps.
more, many plane defects were found on the (010) face. These faults also have a definite crystallographic orientation that is parallel to the c-axis. These defects arise from the origin of the seed crystal which was crushed and oriented by artificial means.
As the direction of these defects tends to be along the c-axis, monoclinic lysozyme crystals may have the ability to cleave along the (100) plane. Figure 4b shows the results of the AFM topograph of commercial lysozyme at σ ) 1.4. The figure shows
Protein Molecular Anisotropy on Crystal Growth
Figure 5. The normal growth rate of the (010) face, R versus supersaturation.
the two-dimensional (2D) nucleation on the (010) face. This is the first direct observation of 2D nucleation of a lysozyme monoclinic crystal. The shapes of the islands are round and isotropic and the height corresponds to the monomolecular height with 2 nm. The 2D islands that formed on the narrow terrace were incorporated by coming steps moving from left to right. The steps on the 2D islands contacting with a doublelayered (DL) step were shown in Figure 4b. When the 2D island contacted with the coming step, the upper layer rapidly covered the surface of the island. This indicates that the growth rate of the upper layer is faster than the lower layer. To measure the detailed morphology and velocity of steps on the {010} faces, we carried out the AFM observations using highly purified lysozyme because it is a concern that the commercial lysozyme contains a small amount of impure proteins and it affects the micro-topography on the surface of crystals.10-12 The obtained AFM images on {010} faces of monoclinic crystals grown with highly purified lysozyme are shown in Figure 4c,d (σ ) 0.05). Figure 4c shows spiral steps observed on the (010) face. The straight steps along the c- or a-axes were observed on the (010) face. This micro-topography does not agree with the macromorphology of the monoclinic crystal. The developed faces of the monoclinic form are {101j}, {101} and {010}, whereas the steps on the (010) face are parallel to [100] or [001]. The macrobond analysis showed that the ledge energy of the steps on the (010) face parallel with the a- and c-axes is not significantly different from that parallel to the {101j} and {101}. The narrow (001) face is actually observed in some cases. Further, a zigzag pattern of splitting steps was formed diagonally from the bottom right to the top left of Figure 4c. This pattern is the result of the symmetry-induced interlacing13,14 which was facilitated by the 2-fold screw axis perpendicular to the (010) face. There are two different step heights on the (010) face. The height of the larger step is 4.9 ( 0.3 nm, which corresponds to a two-molecule height. The smaller step is 2.8 ( 0.2 nm, which is close to the height of one molecule. From the interlaced pattern in Figure 4c, we can obtain the relative velocity of steps in the different directions on the (010) face. Figure 4e is the processed image of Figure 4c, in which the single- (SL) and double-layered (DL) steps are distinguished by the color. At point A in Figure 4e, DL
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steps were separated into two SL steps. The step velocity of the lower layers of the splitting steps advancing to the [1j00] direction was clearly higher than the DL steps, and from the interlaced pattern, the upper layer will move faster than the lower layer. These results mean that the velocity of DL steps to the [100] direction is lower that any SL step moves to the [100] direction. Since protein crystal growth is limited by the incorporation of molecules,15 and the growth rate under this condition is quite low, the progress of DL steps to the [100] direction will be inhibited by reducing the incorporation rate of the lysozyme molecule into the step by forming DL steps. In contrast, the velocity of DL steps advancing to the [001] direction is higher than the lower SLs of the component as shown at point B in Figure 4e. The distance between the corner of the DL steps and the intersection of the SL and DL steps becomes longer when the steps advanced from the spiral center. This difference on the step velocity may arise from the Gibbs-Thomson effect on the curved SL steps because the faceted DL steps indicate that the DL steps are more stable than the SL steps. From point C to the D in Figure 4e, the one straight line with SL height was observed. The right side of this step is lower than the left side of the step. This means that the progress of the step from left to right was inhibited there even if the terrace around point D looks smooth after meeting with the coming step. Thus, a plane defect as shown in Figure 4a should be there even if the defect is not shown clearly. On the (01j0) face, the step and terrace topology is observed (Figure 4d). This topology is different from that observed for the (010) face. In contrast to the straight shape of steps on the (010) face, relatively rough-shaped steps were observed on the (01j0) face of the monoclinic crystal. In the tetragonal form of lysozyme, similar rough steps were observed if the commercial lysozyme was used.12,16 However, even with the highly purified lysozyme, the (01j0) face of the monoclinic crystal showed rough and complex steps. Two different step heights were also found on the (01j0) face. The heights of these steps were 4.0 ( 0.3 nm and 1.9 ( 0.3 nm. These values are similar to those observed for the (010) face. The step direction on the (01j0) face may be isotropic because we could not find any anisotropic topography on the (01j0) face. These results indicate that the impurities present on the sample are attached to the terraces or the ledges of the steps even with highly purified lysozyme. As the molecular packing in the steps on the (010) and (01j0) faces are identical, the adsorbed impurities should be located on the terraces of the steps. The step velocities in the direction of [100] were calculated using continuous AFM images including Figure 4c,d. The velocity of the step in the [100] direction at σ ) 0.05 on the (010) face was 9.2 × 103 nm/h, and on the (01j0) face was 4.0 × 102 nm/h. Even if we used highly purified lysozyme, the step velocity on the (010) face was evidently higher than that measured on the (01j0) face. Growth Mode and Surface Free Energy of the Steps of the {010} Faces. To determine the growth mode, the data was fitted by the empirical equation:17,18
R ∼ σn
(1)
where n is a constant. For n e 2, the growth is known to be governed by the spiral growth mode. For n > 2, the 2D nucleation on the surface limits the growth rate. Since the 2D nucleation was observed as shown in Figure 4b at σ ) 1.4 and to enable fitting using eq 1, we divided the data in two at σ ) 1.75. From this fitting, we obtained n ) 1.7 and n ) 5.4 for the low and high supersaturation sets of data, respectively. The results of AFM observation and this fitting indicate that the spiral
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growth is dominant in the growth of the (010) face until σ ) 1.75. The growth mode will change from the spiral growth mode to the 2D nucleation mode at σ ) ∼1.4-1.75, and the instability of the (010) face becomes pronounced if the σ reaches approximately 2.4. This instability confirms that the growth mode of the (010) face at higher supersaturation levels changes from incorporation-limited to diffusion-limited. The growth rate of the (01j0) face was very slow in comparison with the (010) face. By fitting all the data on the (01j0) face, n was determined to be 2.6. However, the identification of the growth mode of the (01j0) face was prohibited because impurities are likely to be involved in the step propagation on the (01j0) face and the error of the data was significantly high. To obtain the surface free energy of the step on the (010) face, we tried to fit the R versus σ data with the 2D nucleation growth model of the birth-and-spread type. The R is expressed as19-21
( 2σ3 )[exp(σ) - 1]
R ) k1 exp
2⁄3 1⁄6
σ
( )
exp -
k2 σ
(2)
where k1 and k2 are constants. The constant k2 is expressed as
πγ2 3kB2T2
(3)
γ ) R√hΩ
(4)
k2 ) Here we define γ as
where kB, T, R, h, and Ω are Boltzmann’s constant, the temperature, the surface free energy of a step, the step height and the molecular volume in a crystal, respectively. Using eq 2, we obtain:
{
ln
}
k2 R 2σ ) ln(k1) 2⁄3 3 σ σ [exp(σ) - 1] 1⁄6
(5)
Figure 5 shows the relation between ln{Rσ-1/6[exp(σ) - 1]-2/3} versus σ-1. From this figure, we can find the two linear characteristics in the supersaturation ranges of 0.29 e σ-1 e 0.57 (1.75 e σ e 3.5) and 0.57 e σ-1 e 1.2 (0.84 e σ e 1.75). The value σ ) 1.75, at which the growth mode was changed in this analysis, is identical with that obtained value from empirical analysis as shown above. The slope of the straight line in Figure 5 gives R ) 8.9 ( 1.3 × 10-4 J/m2 (γ(010) ) 2.0 ( 0.3 kBT) when h ) 31.25 × 10-10 [m] and Ω ) 2.664 × 10-26 [m3]. Asai et al. have reported the average surface energy of the steps on (010) and (01j0) faces as 4.5 ( 0.5 × 10-4 J/m2, which is half of our value.3 Because the reported value is the average of the two different faces (010) and (01j0), and the supersaturation range in their calculation includes the region of spiral-growth mode, the step free energy of the reported value should be different from our analysis. The obtained value in this analysis is 5-10 times smaller than the surface energies of the steps on the (101) face of the lysozyme monoclinic form.22 This small surface free energy of the step on the (010) face may be the origin of the relatively faster growth of the (010) face. Anisotropic Growth Rate. Although the molecular structure on the step ledge is identical, as shown above, the growth rates of the (010) and (01j0) faces are completely different. One of the possible reasons for this difference is the small surface free energy of the step on the (010) face. The adsorption of impure protein material on the (01j0) face may be the origin of the anisotropic growth. The rough step shape on the (01j 0) face implies the presence of such impurities. However, the
difference of the step velocities on the {010} faces with highly purified lysozyme indicates that quite small amounts of impure protein in the crystallization solution may efficiently inhibit the growth of the (01j0) face. An alternative, more likely reason is the self-poisoning with lysozyme molecules. Heijna et al. reported that the anisotropic growth of lysozyme monoclinic crystals might be the result of blocking by the wrongly oriented protein molecules covering the surface of the crystal.4 From the crystal packing of the lysozyme monoclinic crystal (PDBID: 5LYM), it is clear that the active site of lysozyme is aligned on the surface of the (01j0) face, whereas the active site is hidden on the (010) site. The active site of lysozyme is composed of hydrophobic residues including Trp62, Trp63, Leu75, Ile98, Ala107, and Trp108, which are important for substrate binding. These hydrophobic residues will attract not only substrates but also lysozyme molecules because of the nonspecific attractive forces between hydrophobic residues. As a result, only the growth of the (01j0) face may be inhibited by the adsorption of wrongly oriented lysozyme molecules. Conclusion The polar growth of the (010) and (01j0) faces of hen eggwhite lysozyme monoclinic crystals were investigated. The solubility of lysozyme monoclinic crystals was determined against 20 mg/mL NaNO3 in sodium acetate buffer, pH 4.5. The growth rate of the (010) face is faster than the (01j0) face in the ranges of supersaturation measured. Two-dimensional nucleation on the (010) face was investigated at σ ) 1.4, and the morphological instability of the (010) face was observed when supersaturation reached 2.4. This instability was due to the diffusion-limited growth mode. The surface free energy of the steps of the (010) face was determined to be 8.9 ( 1.3 × 10-4 J/m2, which are 5-10 times smaller than the step free energy of the (101) face. The small value of the step free energy of the (010) face may be the origin of the relatively fast growth of the (010) face. Straight and smooth steps parallel to the aand c-axes were found on the (010) face by AFM measurements, whereas rough and complex steps were observed on the (01j0) face. The complex steps on the (01j0) face indicate the presence of adsorbed molecules on the terraces of the steps on the (01j0) face. The adsorption of molecules may be the result of protein self-poisoning that is induced by the hydrophobic environment of the active site of lysozyme. This effect may be the cause of the observed growth inhibition of the (01j0) face. Acknowledgment. H.H. thanks Professor M. Kitamura, Kyoto University for valuable discussions.
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CG800694J
