Photon Factory Activity Report 2019 #37 (2020)

BL-17A/2018G613

Electrostatic Potentials around the Proteins Crystallized byAmmonium Sulfate Preferably
Tyuji Hoshino1,* Yan Guo1 Liang Qu1 and Michiyoshi Nukaga2
Graduate School of Pharmaceutical Sciences, Chiba University,
1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
2
Faculty of Pharmaceutical Sciences, Josai International University,
Gumyo 1, Togane-shi Chiba 283-8555, Japan
11 Introduction
X-ray crystallography is the primary experimental
method for determining protein structure. The crystal
growth of protein molecules is, however, a critical step forthis method. In general, a variety of parameters has
influence on the molecular interaction of proteins and isrelated to the protein crystal growth. Hence, theoptimization of the crystallization condition is a timeconsuming process. In particular, the selection ofprecipitant agent is a key parameter for obtaining a goodquality of protein crystals. Ammonium sulfate (AS) andpolyethylene glycol (PEG) are the most popularprecipitants to induce protein crystallization. Some
proteins are easily crystallized by AS, while others are not.
Instead, some proteins are crystallized PEGs, preferably.
The reason why the preference of precipitant is different
among proteins is not still clear in spite of many previous
studies on the function of precipitants.
The protein solubility is affected by the ionic strength of
the solution and the net charge of the protein. The surface
tension at the water-protein interface and the activity ofwaters in the hydration layer are related to the solubility.
According to the well-accepted mechanism, Hofmeisterseries of ions for protein hydration, the strongly hydrated
anions such as sulfate and phosphate compete for watermolecules in the second hydration layer, which leads to thedecrease of the water activity in the first layer for solvating
the protein. The strongly hydrated anions further increase
the surface tension of the solvent, making the protein tominimize its solvent accessible surface area. The decreaseof water activity and the increase of surface tension
eventually result in the reduction of the protein solubilityby the salting-out effect. Another effect of salts on proteinsis the protein-ion interaction. The electric charge of protein
is an essential factor related to the solubility. The strongly
hydrated ions form strong protein-ion interaction, whichhave significant influence on the hydration of protein. AS
is one of the strong salting-out agents and widely used incrystallization due to its high solubility, low heat ofsolution, and low denaturation of protein structure. Hence,the protein crystal growth by AS is attributed to hydrationand dehydration effects involving competition for waters.
Although the salt-out mechanism is broadly accepted, theunderstanding of the reason for the preference ofprecipitants is still limited.

In our previous study [1], a kind of protein wascrystallized with three different precipitants, including ASand PEG. Computational analysis showed that AS ionswere anisotropically distributed around the protein, which
was reflected in the molecular packing of the crystals.
Because the molecular packing of protein crystal is linked
to space groups, the relationship between space groups andprecipitants was investigated for several kinds of proteins.
Furthermore, we performed X-ray structure analysis bygrowing single crystals for four kinds of proteins [2]. Twoof them are known to be easily crystallized by AS, while
the other two are not. Molecular dynamics simulations of
the four proteins with AS ions indicated that thedistribution of AS ions in the proteins easily crystallized
by AS was not random but highly anisotropic around theprotein with the ions localized in two areas. The localized
distribution was caused by the electrostatic potential
around the protein, and AS ions gathered at the regions
where the absolute value of the electrostatic potential washigh. The findings suggested that the shape of the isosurface of the electrostatic potential was responsible for theeasiness of the crystal growth with AS.
In this work, we picked up 100 crystal structures that had
been crystallized by high concentrations of AS. The
electrostatic potentials were drawn for the spacesurrounding the proteins. The shapes of electrostatic
potentials were contrasted to those of 20 proteins that had
been crystallized by PEGs. In addition, the electrostatic
potentials were examined for the proteins that had been
crystallized by low concentrations of AS and otherprecipitant agents. The difference in the shape ofelectrostatic potential was quantitatively characterized byan index representing the polarity. This study aims toclarify the reason for the preference for precipitants inprotein crystal growth.
2 Experiment
The crystal structures analyzed in this work were
downloaded from protein data bank (PDB) under thefollowing criteria. (1) Crystal was grown in the apo-form
without ligand or inhibitors. (2) As for 100 structures,proteins were crystallized by AS at high concentrations
over 2.0 M. (3) As for 10 structures, proteins werecrystallized by AS at low concentrations below 2.0 M. For
comparison, 20 crystal structures obtained by utilizing
PEGs were picked up. Additionally, 10 structures of thePhoton Factory Activity Report 2019 #37 (2020)

proteins crystallized by other kinds of salts or agents wereincluded. Totally, 140 crystal structures were surveyed inthis work.
The electrostatic potential around every protein wasdepicted for all the downloaded crystal structures in thefollowing manner. In preparation, the missing residues in
the crystal structure were added by the homology modelingwith Modeller 9.2. The pKa calculation was performed by
Propka 3.1, in which the pKa values of the titratable
residues were predicted by electrostatic continuum theory.
The protonation state of each titratable residue wasselected so that the net charge of the whole-body of theprotein became zero at a certain pH, i.e., iso-electric point.
The hydrogen atoms were generated by leap module ofAmberTools16. The electrostatic potential was obtained bysolving the Poisson-Boltzmann equation with theelectrostatic continuum method by Delphi 5.1, and thepotential map was visualized using Chimera 1.12 or
PyMOL 1.7.
Single crystal of chitosanase was grown by utilizing AS.
An Escherichia coli strain, Rosetta (DE3) pLysS, wastransformed with pET50b vector (Novagen) containing thegene coding N-terminal side-truncated chitosanase.
Chitosanase was expressed as a 6×His-fused Nus-tagconjugated form due to the use of pET50b vector. After thepre-culture in LB medium at 37°C, the protein wasexpressed at 28°C overnight by the induction with 0.2 mM
isopropyl-β-thiogalactopyranoside (IPTG) at an OD600
value of 0.6. The conjugated protein was purified a Co
metal-affinity chromatography, followed by the cleavageof 6×His-fused Nus-tag by human rhinovirus 3C (HRV3C) protease. The cleaved Nus-tag, HRV-3C protease, anduncleaved conjugated protein were removed by Ninitrilotriacetic acid (NTA) agarose resin. The protein was
further purified by gel filtration with a running buffer of 10mM Tris-HCl at pH 8.0 and 150 mM NaCl. Finally, theprotein was concentrated to 5.4 mg/mL.
The single crystal of chitosanase was grown by thesitting drop vapor diffusion method at 18°C. For setting up
a droplet for crystallization, 1.0 µL of protein solution wasmixed with 1.0 µL of the precipitant solution consisting of
100 mM sodium citrate at pH 5.0 and 3.4 M AS. The
mixture was placed on the well plate with the reservoir
filled with 300 µL of precipitant solution. The crystals
were grown within two weeks without any seedingtechnique. The crystals were cryo-protected by brief
immersion in a solution containing 25% (v/v) glycerol,
followed by freezing in liquid nitrogen.
X-ray diffraction data were collected at 100 K on BL17A beamline of photon-factory (PF, Tsukuba, Japan). The
diffraction data were indexed, scaled, and merged withXDS. Intensities were converted into structure factors, and5% of the reflections were flagged for Rfree calculations.
The protein structure was determined by the molecularreplacement with MolRep in CCP4 software package using
a structure obtained by the PEG-grown crystal (PDB code:1v5c) as a search model. Structure refinement and modelbuilding were carried out using Refmac and Phenix.
Molecular structure and electron density map werevisualized by COOT.

3 Results and Discussion
The electrostatic potentials were depicted for all the 140
proteins with determining the protonation state of every
titratable residue at a pH value in which the net charge ofthe protein was zero. Figure 1 shows the electrostatic
potentials at the surrounding space of the typical 20
proteins that are preferably crystallized by highconcentrations of AS. All the 20 proteins display acommon shape in electrostatic potential. The positive andnegative areas are almost equally separated, and each area
has a spherical lobe form. The protein molecule ispositioned at the boundary region of the two areas, that is,each spherical lobe stems from the protein and expandedin the opposite direction to each other. It should be notedthat the positive and negative areas distinctly split even attheir boundary, and the contact region is limited to theprotein. The electrostatic potentials around the 10 proteinsthat are preferably crystallized by PEGs are shown forcomparison. The separation between the positive andnegative areas is unclear and the electrostatic potential at
the boundary region is not neat for the proteins crystallizedby PEGs. The positive and negative areas are interlacedwith each other.

Figure 1: Positive and negative iso-surfaces ofelectrostatic potential around the selected 30 proteins. Outof 30, 20 proteins were crystallized by high concentrationsof AS, and 10 were by PEGs. The AS-crystallized 20
proteins are illustrated in the blue frame and the PEGcrystallized 10 are in the red one. The positive andnegative iso-surfaces are shown in the blue and red meshrepresentation, respectively. The contouring values of isosurfaces are +0.5 and -0.5 in the unit of kT/e. Protein
molecules are represented by green cartoons.
The electrostatic potentials for all the 140 proteins areshown in Figure 2. The initial 100 proteins have been
crystallized by AS at the concentrations above 2.0 M. As
described for the 20 typical proteins in Figure 1, thepositive and negative areas are distinctly separated, andeach of the areas has a spherical lobe form. In other words,

Photon Factory Activity Report 2019 #37 (2020)

the positive and negative areas of the electrostatic potential
clearly split, in which both areas are round in shape andalmost equal to each other in volume. The next 10 proteins
have been crystallized by AS at the concentrations below
2.0 M. The positive and negative areas are clearly
separated for some proteins such as 1skz and 1toh. At thesame time, the separation is not clear for a few otherproteins as 5go6 and 5wko. For the 20 proteins that havebeen crystallized by PEGs, the boundary between thepositive and negative areas is not clear, with some
exceptions like 1v5c. In 1ao6 and 1y62, the positive andnegative areas are separated, but the boundary is notdistinguishable. The additional 6, 2, and 2 proteins havebeen crystallized by sodium formate, glycerol, and others,respectively. The proteins crystallized by sodium formate
show the distinct separation of the positive and negative
areas as seen in the proteins crystallized by AS. Therefore,
it can be concluded that the positive and negative areas
neatly split in the electrostatic potentials for the proteinspreferably crystallized by the strongly hydrated ioncontaining precipitants such as AS and sodium formate.

Figure 2: Electrostatic potentials for the 140 proteins. The
contouring values of the positive and negative iso-surfaces
are +0.5 and -0.5 in the unit of kT/e, respectively. The
positive and negative iso-surfaces are shown in the blue
and red mesh representation, and the protein molecule isrepresented by the green cartoon. The AS-crystallized 110
proteins are displayed in the blue frame, and the PEGcrystallized 20 are in the red one. Six proteins in the green
frame were crystallized by sodium formate, and two
proteins in yellow were by glycerol. The rest two proteinsin pink were crystallized by ethylene glycol and sodium
chloride.
Among 20 crystal structures crystallized by PEGs, theelectrostatic potential of 1v5c exceptionally displayed aclear separation of negative and positive areas. Since theelectrostatic potential had the shape commonly observed

for the AS-crystallized proteins, the crystal growth of theprotein will be possible by AS, too. The PDB entry: 1v5c
is the crystal structure of the inactive form of chitosanase,which is an enzyme to hydrolyze the partly acetylated
chitosan. The inactive form of chitosanase was crystallized
by the precipitant of 20% (w/v) PEG4000, including 0.4M
AS at pH3.7 in a previous report. The space group of 1v5c
was I222, and the max resolution was 2.00 Å. In the presentwork, we tried the crystallization of chitosanase by AS. A
single crystal of chitosanase was grown under thecondition of 3.4 M AS at pH5.0. No PEGs or organic
ingredient was included in the precipitant solution. Thespace group of the AS-grown crystal was P212121, and themax resolution was 1.75 Å. Two chitosanase molecules
were included in the asymmetric unit, unlike 1v5c. The
structures of the two chitosanase molecules were almost
equal to each other, as shown in the drawing of theirsuperimposition (Figure 3a). The root-mean-squaredeviation (RMSD) between two molecules was 0.131 Å.
The structure of the AS-grown chitosanase is almost thesame as the PEG-grown one. The crystal structure of one
chitosanase molecule in the asymmetric unit wassuperimposed on the 1v5c structure (Figure 3b). The
RMSD between them was 0.136 Å.
One chitosanase molecule in the AS-grown crystal madecontact with six surrounding molecules in the crystal
packing. The electrostatic potential of the AS-grownchitosanase was drawn with the surrounding molecules(Figure 3c). The positive and negative spherical lobes ofthe electrostatic potential were located at the spaceunoccupied by other molecules in the crystal packing.
Other molecules were likely to be positioned at theintermediate between two spherical lobes. In other words,
the molecules in the crystal made contacts among them in
the areas where the absolute value of the electrostatic
potential was low. According to a simulation study of AS
ions in the protein crystal growth, the distribution of AS
ions around a protein was anisotropic if the protein waseasily crystallized by AS solution as a precipitant. Instead,
AS ions are likely to gather at the local areas of the protein
surface. The distribution of AS ions around the protein
coincided with the shape of the electrostatic potential.
Therefore, AS ions were considered to be attracted to theareas where the absolute value of the electrostatic potential
was high, that is, the space unoccupied by chitosanasemolecules in crystal packing. The electrostatic potential of
the PEG-grown chitosanase was also drawn with thesurrounding molecules for comparison (Figure 3d). The
locations of the surrounding molecules seem to correlatewith the electrostatic potential. The surrounding molecules,
with one exception, tended to make contact with the centermolecule at the intermediate areas between two sphericallobes where the absolute value of the electrostatic potential
was low. Furthermore, no molecule was observed in thepositive area of the electrostatic potential. Since the PEGgrown chitosanase was reported to be crystallized withincluding a low concentration of AS, the mixed AS ionsmight assist the protein growth with gathering at thepositive area even for the PEG-grown chitosanase.

Photon Factory Activity Report 2019 #37 (2020)

Figure 3: (a) Structures of the two chitosanase molecules
in an asymmetric unit of the AS-grown crystal. One
molecule colored green is superimposed on the othercolored cyan. (b) Superimposition of the AS-crystallized
chitosanase onto the PEG-crystallized one. Protein
molecules are represented by the cartoon, changing colors
from blue to red as the residue goes from N- to C-terminalsides for the AS-crystallized one. The PEG-crystallized
molecule is colored grey. Water molecules are not shownfor clarity. (c) Electrostatic potentials for the AScrystallized chitosanase with the surrounding molecules. A
molecule of interest is colored cyan, and the surrounding
ones are green. The positive and negative iso-surfaces are
drawn in blue and red, with their contouring values at +0.5
and -0.5 in the unit of kT/e, respectively. (d) Electrostatic
potentials for the PEG-crystallized chitosanase with thesurrounding molecules. The coloring and drawings are thesame as (c).
Acknowledgement
Calculations were performed at the Research Center for
Computational Science, Okazaki, Japan and at theInformation Technology Center of the University of Tokyo.
X-ray diffractions were acquired at the Photon Factory,
Tsukuba, Japan (proposal no. 2018G613). A part of thiswork was supported by a grant for Scientific Research C
from the Japan Society for the Promotion of Science.
References[1] Fudo, S., Qi, F., Nukaga, M., Hoshino Cryst. GrowthDes. 17, 534-542 (2017).
[2] Kitahara, M., Fudo, S., Yoneda, T., Nukaga, M.,
Hoshino, T. Cryst. Growth Des. 19, 11, 6004-6010(2019).
* hoshino@chiba-u.jp