Improvement of Hen Egg White lysozyme Crystal Quality by

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Improvement of Hen Egg White lysozyme Crystal Quality by Control of Dehydration Process Haruhiko Koizumi, Satoshi Uda, Katsuo Tsukamoto, Kenji Hanada, Ryo Suzuki, Masaru Tachibana, and Kenichi Kojima Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.9b01084 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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Crystal Growth & Design

Improvement of Hen Egg White lysozyme Crystal Quality by Control of Dehydration Process ∗,†

Haruhiko Koizumi,

Suzuki,

†Strategic

Satoshi Uda,

⊥

‡

Katsuo Tsukamoto,

Masaru Tachibana,

⊥

¶,§

Kenji Hanada,

and Kenichi Kojima

∥

Ryo

#

Planning Oce for Regional Revitalization, Mie University, 1577 Kurimamachiya-cho, Tsu city 514-8507, Japan

‡Institute

for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan

¶Graduate

School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

§Graduate

School of Science, Tohoku University, 6-3 Aramaki, Aoba-ku, Sendai 980-8578, Japan

∥Aichi

Synchrotron Radiation Center, 250-3 Minamiyamaguchi-cho, Seto 489-0965, Japan

⊥Graduate

School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan

#Department

of Education, Yokohama Soei University, 1 Miho-cho, Midori-ku, Yokohama 226-0015, Japan E-mail: [email protected]

1

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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Equal-thickness fringes that are attributed to the Pendello¨sung eect were clearly observed in the region of a tapered tetragonal hen egg white (HEW) lysozyme crystal with a wedge-like edge, which indicates that the misorientation between subgrains was on the order of 10−4 ◦ . This is attributed to control of the dehydration process for crystal growth from solution, which plays an important role. Both the local quality and whole homogeneity of the crystals were improved by regulating the dehydration process. Oscillatory contrast was observed in X-ray topographs from regions of the same thickness in the crystals (see Movie S1). However, the area of such contrast was quite small, which indicates the presence of defects, even in crystals for which equal-thickness fringes could be observed. We also discuss why the area of oscillatory contrast was small for tetragonal HEW lysozyme crystals with misorientations between subgrains on the order of 10−4 ◦ .

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Introduction What dominates the formation of defects in crystals composed of macromolecules that are grown from solution? Defects in crystalline materials such as metals, Si, GaN, and Ba(NO3 )2 are predominantly dislocations. 14 Dislocations are also the dominant defects in organic crystals composed of small molecules. 57 However, in the case of protein crystals grown from solution, the increase in the full width at half maximum (FWHM) of X-ray diraction (XRD) rocking curves cannot be explained solely by the presence of dislocations. 8 This suggests that the broadening eect except for dislocations, i.e., imperfections exist in a crystal composed of macromolecules for crystal growth from solution. Many types of crystals composed of macromolecules can be grown from solution, such as proteins, colloids, and three-dimensional (3D) photonic crystals. 3D photonic crystals allow versatile manipulation of light 911 by suppressing electromagnetic propagation and modication of dispersion around a specic wavelength region due to a full band gap. However, the presence of defects and inhomogeneity adversely aects the performance of devices based on 3D photonic crystals. In the case of protein crystals, this can lead to a signicant decrease in precision when analyzing their 3D structure. Therefore, improvement of the local quality and whole homogeneity of crystals composed of macromolecules is an important subject in crystal growth from solution. The dehydration process plays a dominant role during crystal growth from solution when atoms or molecules are incorporated into the steps on the crystal surface. 12 The dehydration process determines the crystal growth rate, 12,13 which indicates the importance of the hydration structure for crystal growth from solution. However, the relationship between the dehydration process and the mechanism of crystal growth is not yet fully understood, because it is dicult to directly observe the hydration structure. We have recently revealed that the crystal growth rate is increased and the crystal quality is improved when the concentration of an added salt, such as NaCl, is higher in protein solutions, 14 which is attributed to the faster dynamics of water around protein molecules. 15,16 3



This indicates that the dehydration process could have an eect not only on the crystal growth rate but also on the crystal quality. More recently, we have also claried that perfect crystals can be grown even for protein crystals, by using glucose isomerase crystals. 17,18 However, it has not been understood why the grown glucose isomerase crystals become perfect, although research concerning the quality of macromolecular crystals grown from solution has been actively performed using proteins. 19,20 In our previous works, 17,18 glucose isomerase crystals were grown under the faster dynamics of water around protein molecules. Therefore, it is quite important to elucidate whether the faster dynamics of water is closely related to the growth of crystals with perfect quality. In this article, we demonstrate that equal-thickness fringes, which are attributed to the Pendello¨sung eect, can be observed in the region of a tapered tetragonal hen egg white (HEW) lysozyme crystal with a wedge-like edge when the dynamics of water around the protein molecules are controlled by the concentration of an added salt. This suggests that the misorientation between subgrains in the crystal is quite small, i.e., the crystal quality is almost perfect. The results also indicate that the whole homogeneity of the grown crystals is improved by regulating the dynamics of water around protein molecules. This could be a universal phenomenon for the crystal growth of macromolecules from solution, which is characterized by the dehydration process. Therefore, the growth technique to improve crystal quality with a focus on control of the dehydration process could have potential for the growth of macromolecular crystals with high quality and high homogeneity, which would lead to high diraction eciency (diractivity) for protein crystals and fabrication of highquality 3D photonic crystals.

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Experimental Crystal Growth

The HEW lysozyme used in this work was purchased from Wako Pure Chemical Industries, Ltd. Dialysis was employed to remove NaCl from the HEW lysozyme solutions prior to crystal growth. The growth of tetragonal HEW lysozmye crystals was conducted using a cross-linked seed crystal, which has been described in detail elsewhere. 21 Growth solutions containing 12 mg/mL HEW lysozyme together with 1.20 mol/L NaCl in a 100 mM sodium acetate buer at pH 4.5 were prepared. The supersaturation ratio for the HEW lysozyme solution, σ (= ln( CCeq ), where C is the concentration of the solution and Ceq is the solubility), was determined from data reported by Cacioppo and Pusey, 22 and was calculated to be 2.32. Tetragonal HEW lysozyme crystals were also grown under NaCl concentrations of 0.34, 0.68 and 0.86 mol/L. Details of the growth conditions have been described in a previous report. 21 The crystals were grown using a growth cell (25×25×2 mm) with a growth time of 1 week.

X-ray Topography and XRD Rocking-curve Measurements

Monochromatic-beam X-ray topography was conducted at room temperature using the BL8S2 beamline at the Aichi Synchrotron Radiation Center (AichiSR), Japan. A two-crystal monochromator consisting of a Si(111) crystal was located 16 m from the source and was used to select an X-ray wavelength of λ = 1.0 Å. Topographs were obtained using the 110 reection and were recorded on nuclear plates (Ilford) with an exposure time of approximately 3 min. XRD rocking-curve measurements were performed at room temperature using the BL20B beamline at the Photon Factory, part of the High Energy Accelerator Research Organization (KEK), Japan. The detailed setup for the XRD rocking-curve measurements is described elsewhere. 21 XRD rocking-curve proles were measured using the 110 family (110, 220, 330, 440, 770, 11,11,0 and 12,12,0) and 001 family (004, 008, 0016) of reections for two tetragonal 5



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HEW lysozyme crystals to estimate physical values. The beam spot size at the region being analyzed was 258 µm (40 pixels). Two-dimensional (2D) mapping of the misorientation between subgrains in a crystal was also performed using a beam spot size of 64.5 µm (10 pixels).

Results and discussion Figure 1 shows a synchrotron monochromatic-beam X-ray topograph of a HEW lysozyme crystal obtained using the 110 reection. The crystal was grown with a NaCl concentration of 1.20 mol/L, by which the dynamics of water around protein molecules are considered to become much faster. 15,16 The contrast in the center part in the topograph is attributed to strain in the seed crystal, although no contrast due to dislocations is observed. As shown in Figure 1, clear straight fringes are observed in the region of a tapered tetragonal HEW lysozyme crystal with a wedge-like edge. Thus, the observed fringes are not Moire´ interference but equal-thickness fringes, which are attributed to the Pendello¨sung eect. This indicates that the quality of the grown crystal is almost perfect, i.e., the misorientation between subgrains is only about 10−4 ◦ . Next, let us consider the subgrain misorientation in HEW lysozyme crystals grown with a NaCl concentration of 1.20 mol/L through analysis of the FWHM, βM , measured from the XRD rocking curves. βM is given by a convolution of the sample's ‘ true ’ mosaicity prole with the intrinsic FWHM, β0 , and the instrumental resolution function, βins , and thus β0 and βins have to be deconvoluted from βM . Details concerning the analysis of misorientations have been described in previous reports. 8,14 The misorientation between subgrains in a crystal can be estimated using the following equation: 1,2,14

sin2 2θB sin2 2θB 2 = β Kα + KL . adj λ2 λ2

(1)

2 2 2 2 Here, βadj is the FWHM adjusted to account for β0 and βins (βadj = βM − β02 − βins ), θB is the

6


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Bragg angle, and λ is the X-ray wavelength. Kα and KL are related to tilting and particle size, respectively, which are dened in previous reports. 8,14 Figure 2 shows the relationship between

sin2 2θB 2 βadj λ2

and

sin2 2θB λ2

The relationship between

for crystals grown with a NaCl concentration of 1.20 mol/L.


and

sin2 2θB λ2

is linear, and the slope corresponds to the

misorientation between subgrains in the crystal. From the linear t to the data in Figure 2, the misorientation between subgrains can be estimated to be 0.00041◦ for the crystal grown with 1.20 mol/L NaCl. This is also on the order of 10−4 ◦ , similar to that for glucose isomerase crystals. 17 Table 1 shows the misorientation between subgrains in crystals grown with various concentrations of NaCl. The values for NaCl concentrations of 0.34, 0.68 and 0.86 mol/L are taken from Ref. 14. Table 1 shows that higher NaCl concentrations result in smaller misorientations. It was recently reported that an increase in the salt concentration of an aqueous solution results in faster dynamics of water around protein molecules. 15,16 Thus, the quality of a crystal composed of macromolecules is closely related to the dynamics of water around the macromolecules, i.e., the dehydration process for crystal growth from solution. These results could be useful for improving the quality of grown protein crystals, which would lead to accurate 3D structures of protein molecules for X-ray structural analysis. We next consider the whole homogeneity of crystals grown with various concentrations of NaCl. The whole homogeneity of grown crystals are important for improving the performance of devices and for accurate analysis. In this article, the analysis of 2D mapping of the subgrain misorientation was performed for the rst time. Figure 3 shows 2D mapping of the misorientation between subgrains in tetragonal HEW lysozyme crystals grown with 0.34, 0.68, 0.86 and 1.20 mol/L NaCl solutions. Analyses were performed using regions of the crystals with the same thickness. As shown in Figure 3, the whole homogeneity of the crystallinity is improved with increasing NaCl concentration. In particular, it seems that the crystal quality of the {110} growth sectors is worse than that of the {101} ones with an decrease in NaCl concentration. This suggests that the dynamics of water around HEW 7



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lysozyme molecules has an eect on the whole homogeneity of grown crystals. Therefore, control of the dehydration process is important for improving not only the local quality but also the whole homogeneity of crystals composed of macromolecules. We have recently observed clear oscillatory proles in rocking curves for glucose isomerase crystals with subgrain misorientations of about 10−4 ◦ . 18 However, do rocking curves for tetragonal HEW lysozyme crystals with subgrain misorientations of the order of 10−4

◦

also

have oscillatory proles? Figure 1 shows clear oscillations in regions of tetragonal HEW lysozyme crystals with the same thickness grown with 1.20 mol/L NaCl. The thickness of the measured crystal was 293 µm. A series of X-ray topographic images is shown in Movie S1, obtained using the same crystal as that used for X-ray topography. Such oscillations have also been more clearly observed with glucose isomerase crystals (see Movie S1 of Ref. 20), and lead to the oscillatory prole of the rocking curves. 18 However, similar results were not observed for tetragonal HEW lysozyme crystals grown with 1.20 mol/L NaCl, because of the weak diraction intensity. 18 In particular, the area of oscillatory contrast was much smaller than that for glucose isomerase crystals. 18 This suggests that the quality of the HEW lysozyme crystal is not perfect. Figure 4(a) shows the prole of oscillatory rocking curves simulated for the 110 reection for a tetragonal HEW lysozyme crystal with a thickness of 293 µm. The prole was calculated using the following equation and assuming a linear absorption coecient, an electric susceptibility and a relative permittivity of 0.33 mm−1 , 0.001 and 23, respectively: 18,23

I=r

Iga I¯ga + (1 − r) , Io Io

(2)

where

Iga exp(−µH/cosθB ) = Io W2 + 1

{

(

sin2

) ( )} √ √ 2+1 πH W 2 + 1 χπH W + sinh2 , Λ Λ

8


(3)

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and

[ { ( )} { ( )}] I¯ga 1 −µH ϵ −µH ϵ = exp 1− √ + exp 1+ √ . (4) Io 4(W 2 + 1) cosθB cosθB W2 + 1 W2 + 1 Here, r is the ratio of Iga /Io to I¯ga /Io with 0≤r≤1. The ratio, 1 − r , for the average curve corresponds to the degree of smearing or background in the oscillatory curve, which originates from the resolution limit due to the angular divergence of the beam. 24,25 In this simulation,

r was 0.38. The horizontal axis is expressed using the W scale, which is the parameter that represents the deviation from the diraction condition, given by:

W =

2ΛsinθB (θB − θ), λ

(5)

π Vc cosθB . re λ |F |

(6)

where

Λ=

Here, Λ is the period of the Pendello¨sung fringes in the Laue (transmission) case, Vc is the volume of the unit cell, re is the classical electron radius (2.82×10−5 Å), and |F | is the amplitude of the structure factor. As shown in Figure 4(a), simulated oscillatory rocking curves can be clearly observed when the quality of a tetragonal HEW lysozyme crystal with a thickness of 293 µm is perfect. However, oscillatory rocking curves could not be observed from the grown tetragonal HEW lysozyme crystal (see Figure 4(b)), but the measured FWHM value for the rocking-curve prole was only 0.00120◦ (4.32 arcsec). For the symmetrical Laue case, the FWHM for the rocking curve for a perfect crystal, ωp , can be estimated using the following equation: 23,26

ωp =

λ 1 . sinθB Λ

(7)

The value of ωp for the 110 reection obtained for tetragonal HEW lysozyme crystals is estimated to be 0.000133◦ (0.479 arcsec), substituting λ = 1.0 Å, θB = 0.512◦ , Vc = 2.37×10−25 m3 and |F | = 1,096. The measured FWHM is ten times larger than that estimated, which 9



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indicates that the grown crystal includes defects. Therefore, indistinct oscillatory contrast in the X-ray topograph could be predominantly caused by defects in the tetragonal HEW lysozyme crystal, whereas when the quality of glucose isomerase crystals is close to perfect, as in the case of Si and Ge, clear oscillatory contrasts are evident in the X-ray topographs. Finally, let us discuss why the area of the oscillatory contrast is small for the tetragonal HEW lysozyme crystal grown with 1.20 mol/L NaCl. The strain in the crystal can be estimated using the following equation: 1,2,8,27 2 βadj = Kα + Kϵ tan2 θB ,

(8)

where Kϵ is related to the local strain in the crystal, which has been dened in a previous 2 report. 8 Figure 5(a) shows the relationship between βadj and tan2 θB for the 001 family of

reections obtained from crystals grown with 1.20 mol/L NaCl. Strain is clearly observed in the crystal using the 001 family of reections. In contrast, the values are almost constant in the case of the 110 family of reections, although the values for the low-order reections increase slightly, which is attributed to the broadening contribution due to subgrain size (see Figure 5(b)). This means that no strain is detected using the 110 family of reections. The misorientation between subgrains can be estimated to be 0.00031◦ from the linear t to the data in Figure 5(a). This value is almost the same as that obtained using the 110 family of reections (0.00041◦ ). However, the strain in the crystal was estimated to be 32.8 µϵ. This suggests that stress is applied in the direction of the c -axis, which would lead to the worse crystal quality of the {110} growth sectors (see Figure 3). Therefore, the indistinct oscillatory contrast could be mainly caused by stress in the c -axis direction. However, the origin of the strain is not yet fully understood. There are the water-lled channels along the [001] direction in tetragonal HEW lysozyme crystals. The diusion coecient of this water is less than that for bulk water molecules, 28 which suggests that the water molecules form a structure in the channels. It has also been reported that the mobility of water

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molecules decreases when the NaCl concentration is higher. 28 Therefore, the stress along the

c -axis direction for tetragonal HEW lysozyme crystals grown with 1.20 mol/L NaCl could be attributed to the structure of the hydrogen bond network of water molecules in the channels.

Conclusion We have demonstrated that both the local quality and the whole homogeneity of tetragonal HEW lysozyme crystals can be improved by controlling the dynamics of water around the macromolecules, i.e., the dehydration process for crystal growth from solution, which was veried by XRD rocking-curve measurements. In the case of tetragonal HEW lysozyme crystals grown with 1.20 mol/L NaCl, equal-thickness fringes attributed to the Pendello¨sung eect in the region of a tapered crystal with a wedge-like edge were clearly observed. This is attributed to subgrain misorientation of the order of 10−4 ◦ . Oscillatory contrast could also readily be observed in a region with the same thickness of a tetragonal HEW lysozyme crystal grown with 1.20 mol/L NaCl. Therefore, control of the dehydration process for crystal growth from solution is important for achieving high-quality and high-homogeneity macromolecular crystals.

Acknowledgement The authors thank Dr. H. Sugiyama and Dr. K. Hirano of KEK for their help with XRD rocking-curve measurements. XRD rocking-curve measurements were performed at the Photon Factory under the auspices of the Photon Factory Program Advisory Committee of KEK (Proposal No. 2018G547). This work was carried out under the Inter-university Cooperative Research Program of the Institute for Materials Research, Tohoku University (Proposal Nos. 18K0109 and 19K0029). This work was supported in part by a Kakenhi Grant-in-Aid for Scientic Research (No. 16K06708) from the Japan Society for the Promotion of Science (JSPS). 11



Supporting Information Available Movie S1: Series of X-ray topographic images

12


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Table and Figure captions Table 1: Misorientation between subgrains for tetragonal HEW lysozyme crystals grown with various concentrations of NaCl. Figure 1: Synchrotron monochromatic-beam X-ray topograph of HEW lysozyme crystal obtained using 110 reection for crystal grown with NaCl concentration of 1.20 mol/L. Figure 2: Relationship between


and

sin2 2θB λ2

for tetragonal HEW lysozyme

crystals grown with NaCl concentration of 1.20 mol/L. Analyses were performed using a beam spot size of 258 µm (40 pixels). Figure 3: 2D mapping for misorientation between subgrains in tetragonal HEW lysozyme crystals grown with NaCl concentrations of (a) 1.20 mol/L, (b) 0.86 mol/L, (c) 0.68 mol/L and (d) 0.34 mol/L. Analyses were performed using a beam spot size of 64.5

µm (10 pixels). Figure 4: (a) Simulated and (b) measured proles of oscillatory rocking curves for 110 reection obtained from tetragonal HEW lysozyme crystal with thickness of 293 µm. 2 Figure 5: Relationship between βadj and tan2 θB for tetragonal HEW lysozyme crystals

grown with NaCl concentration of 1.20 mol/L; (a) 001 family of reections and (b) 110 family of reections. Analyses were performed using a beam spot size of 258 µm (40 pixels).

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Table 1: Misorientation between subgrains for tetragonal HEW lysozyme crystals grown with various concentrations of NaCl. NaCl concentration 0.34 mol/L[a] (2.0 w/v%) 0.68 mol/L[a] (4.0 w/v%) 0.86 mol/L[a] (5.0 w/v%) 1.20 mol/L (7.0 w/v%) [a] Ref. 14

Supersaturation, σ 2.34

Misorientation 0.00143◦

2.39

0.00103◦

2.35

0.00069◦

2.32

0.00041◦

17



Figure 1: H. Koizumi et al .

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For Table of Contents Use Only

Title: " Improvement of Hen Egg White lysozyme Crystal Quality by Control of Dehydration Process" Author(s): Koizumi, Haruhiko; Uda, Satoshi; Tsukamoto, Katsuo; Hanada, Kenji; Suzuki, Ryo; Tachibana, Masaru; Kojima, Kenichi

Control of the dehydration process for crystal growth from solution is important for achieving high-quality and high-homogeneity macromolecular crystals. Equal-thickness fringes that are attributed to the Pendellösung effect were clearly observed in the region of a tapered tetragonal hen egg white (HEW) lysozyme crystal with a wedge-like edge by regulating the dehydration process, which indicates that the misorientation between subgrains was on the order of 10‒4˚. The finding could be a breakthrough of the growth of high-quality and high-homogeneity 3D photonic crystals made from macromolecules.


Improvement of Hen Egg White lysozyme Crystal Quality by

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