Photon Factory Activity Report 2019 #37 (2020)

BL-20B/2018G547

Improvement of Hen Egg White Lysozyme Crystal Quality
by Control of Dehydration Process
Haruhiko Koizumi1*, Satoshi Uda2, Katsuo Tsukamoto3,4, Kenji Hanada5, Ryo Suzuki6,
Masaru Tachibana6, Kenichi Kojima7
1
Strategic Planning Office for Regional Revitalization, Mie University, 1577 Kurimamachiya-cho,
Tsu city 514-8507, Japan
2
Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577,
Japan
3
Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
4
Graduate School of Science, Tohoku University, 6-3 Aramaki, Aoba-ku, Sendai 980-8578, Japan
5
Aichi Synchrotron Radiation Center, 250-3 Minamiyamaguchi-cho, Seto 489-0965, Japan
6Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku,Yokohama 236-0027, Japan
7
Department of Education, Yokohama Soei University, 1 Miho-cho, Midori-ku, Yokohama 226-0015,
Japan
Equal-thickness fringes that are attributed to the Pendellösung effect were clearly observed in the region of a taperedtetragonal hen egg white (HEW) lysozyme crystal with a wedgelike 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 growthfrom solution, which plays an important role. Both the local quality and whole homogeneity of the crystals wereimproved by regulating the dehydration process. Oscillatory contrast was observed in X-ray topographs from regions of
the same thickness in the crystals. However, the area of such a contrast was quite small, which indicates the presence ofdefects, 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°.
1 Introduction
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 [1].
The dehydration process determines the crystal growthrate [1, 2], which indicates the importance of thehydration 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 difficult to directly observe
the hydration structure.
We have recently revealed that the crystal growth rateis increased and the crystal quality is improved when theconcentration of an added salt, such as NaCl, is higher inprotein solutions [3], which is attributed to the faster
dynamics of water around protein molecules [4, 5]. This
indicates that the dehydration process could have aneffect not only on the crystal growth rate but also on thecrystal quality. More recently, we have also clarified that
perfect crystals can be grown even for protein crystals, byusing glucose isomerase crystals [6, 7]. However, it has
not been understood why the grown glucose isomerase
crystals become perfect, although research concerning thequality of macromolecular crystals grown from solution
has been actively performed using proteins [8]. In ourprevious works [6, 7], glucose isomerase crystals weregrown 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 tothe growth of crystals with perfect quality.
In this report, we demonstrate that equal-thickness
fringes, which are attributed to the Pendellösung effect,can be observed in the region of a tapered tetragonal hen
egg white (HEW) lysozyme crystal with a wedgelikeedge 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 thewhole homogeneity of the grown crystals is improved byregulating the dynamics of water around protein
molecules. This could be a universal phenomenon for thecrystal growth of macromolecules from solution, which ischaracterized by the dehydration process.
2 Experiment
The HEW lysozyme used in this work was purchasedfrom Wako Pure Chemical Industries, Ltd.. Dialysis wasemployed to remove NaCl from the HEW lysozyme
solutions prior to crystal growth. The growth of tetragonal
HEW lysozyme crystals was conducted using a crosslinked seed crystal, which has been described in detailelsewhere [9]. Tetragonal HEW lysozyme crystals werealso grown under NaCl concentrations of 0.34, 0.68, 0.86,
and 1.20 mol/L. The crystals were grown using a growthcell (25 × 25 × 2 mm) with a growth time of 1 week.

Photon Factory Activity Report 2019 #37 (2020)

Table 1 Misorientation between Subgrains for Tetragonal
HEW Lysozyme Crystals Grown with Various
Concentrations of NaCl
NaCl concentration
supersaturation
misorientation
mol/L (w/v %)σdeg0.34 M (2.0 w/v%)
2.34
0.00143
0.68 M (4.0 w/v%)
2.390.00103
0.86 M (5.0 w/v%)
2.350.00069
1.20 M (7.0 w/v%)
2.320.00041

Figure 1: Synchrotron monochromatic-beam X-ray
topograph of a HEW lysozyme crystal obtainedusing the 110 reflection for a crystal grown with aNaCl concentration of 1.20 mol/L.
Monochromatic-beam
X-ray
topography
wasconducted at room temperature using the BL8S2
beamline at the Aichi Synchrotron Radiation Center(AichiSR) of Japan. A two-crystal monochromatorconsisting of a Si(111) crystal was located 16 m from thesource and was used to select an X-ray wavelength of λ =1.0 Å. Topographs were obtained using the 110 reflection
and were recorded on nuclear plates (Ilford) with an
exposure time of approximately 3 min.
XRD rocking-curve measurements were performed atroom temperature using the BL20B beamline at thePhoton Factory, part of the High Energy Accelerator
Research Organization (KEK) of Japan. The detailed
setup for the XRD rocking-curve measurements is
described elsewhere [9].
3 Results and Discussion
Figure 1 shows a synchrotron monochromatic-beam Xray topograph of a HEW lysozyme crystal obtained using
the 110 reflection. The crystal was grown with a NaCl
concentration of 1.20 mol/L, by which the dynamics ofwater around protein molecules are considered to becomemuch faster [4, 5]. The contrast in the center part in thetopograph 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 wedgelike edge. Thus, the observed fringes are not
Moiré interference but equal-thickness fringes, which areattributed to the Pendellösung effect. This indicates thatthe quality of the grown crystal is almost perfect: i.e., themisorientation between subgrains is only about 10−4°.
Table 1 shows the misorientation between subgrains in
crystals grown with various concentrations of NaCl. As
shown in Table 1, higher NaCl concentrations result insmaller misorientations. It was recently reported that anincrease in the salt concentration of an aqueous solution
results in faster dynamics of water around protein

molecules [4, 5]. Thus, the quality of a crystal composedof macromolecules is closely related to the dynamics ofwater around the macromolecules: i.e., the dehydration
process for crystal growth from solution.
We have recently observed clear oscillatory profiles inrocking curves for glucose isomerase crystals with
subgrain misorientations of about 10−4° [7]. However,rocking curves for tetragonal HEW lysozyme crystals
with subgrain misorientations of the order of 10−4° do nothave oscillatory profiles. This suggests that the quality of
the HEW lysozyme crystal is not perfect.
Let us discuss why the area of the oscillatory contrast
is small for the tetragonal HEW lysozyme crystal grownwith 1.20 mol/L NaCl. Strain is clearly observed in thecrystal using the 001 family of reflections, although nostrain is detected using the 110 family of reflections. Thissuggests that stress is applied in the direction of the c axis.
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 water-filled channels along the [001] direction in
tetragonal HEW lysozyme crystals. The diffusion
coefficient of this water is less than that for bulk watermolecules, which suggests that the water molecules forma structure in the channels. It has also been reported thatthe mobility of water molecules decreases when the NaCl
concentration is higher [10]. Therefore, the stress along
the c axis direction for tetragonal HEW lysozyme crystals
grown with 1.20 mol/L NaCl could be attributed to thestructure of the hydrogen bond network of watermolecules in the channels.
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(2018).
[4] K. Aoki et al., Appl. Phys. Lett. 103, 173704 (2013).
[5] K. Aoki et al., Phys. Chem. Chem. Phys. 18, 15060(2016).
[6] H. Koizumi et al., Cryst. Growth Des. 16, 4905
(2016).
[7] R. Suzuki et al., Proc. Natl. Acad. Sci. U. S. A. 115,3634 (2018).
[8] J. Helliwell, J. Cryst. Growth 90, 259 (1988).
[9] H. Koizumi et al., Cryst. Growth Des. 17, 6712
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[10] V. Morozov et al., Eur. Biophys. J. 24, 93 (1995).
* h_koizumi@sporr.mie-u.ac.jp