Photon Factory Activity Report 2018 #36 (2019)
BL-5A, AR-NW12A/2018G613

Anisotropic Distribution of Ammonium Sulfate Ions in Protein Crystallization
Tyuji Hoshino1,* Mariko Kitahara1 Satoshi Fudo1 Tomoki Yoneda1 and Michiyoshi Nukaga2
1
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
1 Introduction
X-ray crystal analysis is one of the most popular
approaches to determine the protein structure. The growthof single crystal is a critical step in the X-ray
crystallography. Ammonium sulfate (AS) is one of themost popular precipitants to induce crystallization ofprotein molecules. AS is, however, not always effective forall kinds of proteins in crystal growth. Namely, some
proteins are readily crystallized by utilizing AS as aprecipitant, but other proteins are not. The reason for thisdifference has been discussed in literature, but isn’t still
well-understood. Like AS, polyethylene glycols (PEGs)
are frequently utilized in protein crystallization. Crystal
growth of some kinds of proteins is effectively induced byPEGs, instead of AS. The cause for the dependency ofprotein crystallization on the precipitants has been barely
studied in the atomistic level so far.
In our previous study, crystals of a single kind ofprotein were grown with three different precipitants,
including AS and PEG. Molecular dynamics (MD)simulations were carried out for the proteins in thepresence of precipitants. The simulations showed thatammonium and sulfate ions (AS ions) played roles not onlyof decreasing the protein solubility but also of restricting
the contact sites on the protein surface to other protein
molecules, which was reflected in the molecular packingof the crystal structure. Since molecular packing of protein
crystals is highly linked to space groups, the relationshipbetween space groups and precipitants was also
investigated for several kinds of proteins by surveying
more than a thousand of crystal structures.
In this work, we performed X-ray structure analysis by
growing single crystals for four kinds of proteins; humancarbonic anhydrase II (CAII), horse myoglobin (Mb), henegg white lysozyme (HEWL), and human serum albumin(HSA). The former two proteins, CAII and Mb, are known
to be easily crystallized by AS. On the other hand, the latter
ones, HEWL and HSA, are known to be not. No structure
has been deposited in the protein data bank (PDB) for
HEWL crystallized with AS due to its difficulty incrystallization. To our knowledge, only photo image andcrystallographic data of HEWL crystals by AS areavailable. Since the crystal growth of HSA by AS is alsodifficult, no PDB entry of HSA crystallized with AS isavailable. A description of the HSA crystal was seen in areport on the study about the cross-linking of the protein
crystals. It should be noted that a large number of crystal
structures are available in PDB both for HEWL and HSA,

but all of them were obtained by growing crystals with
precipitants other than AS. We also performed MD
simulations for the four proteins in the calculation models
with and without AS ions. The AS distribution around theprotein was deduced from the simulation trajectory. Inaddition, the electrostatic potentials were drawn for thespace surrounding the proteins. To clarify the reason forreadiness for and difficulty in crystal growth by AS, thesecomputational results were compared among the proteins.
2 ExperimentProtein single crystals were grown for CAII, Mb,HEWL, and HSA at the AS concentrations of 2.7, 3.2, 1.2,
and 1.2 M, respectively. The max resolutions for therespective proteins were 1.32, 1.32, 1.13, and 3.90 Å. The
major precipitant reported in the literature forcrystallization of CAII and Mb is AS, and the space groups
are P1211 for CAII and P6 or P1211 for Mb. Therefore, thecrystals obtained in this work reproduced the previousreports. The space group of the HEWL crystal grown byAS was P1, which was different from the most popularspace group of HEWL crystals, P43212. The max
resolution for HEWL in this study, 1.13 Å, was better than
the average max resolution of the crystal structures ofHEWL deposited in PDB, 1.73 Å. The space group of theHSA crystal by AS was C121, and the max resolution was2.92 Å. The major space group of HSA crystals depositedin PDB is C121, and the average max resolution is 2.60 Å.
In terms of the resolution, the quality of the HSA crystal
grown by AS was inferior to the crystals obtained by otherprecipitants.
Calculation models for CAII, Mb, HEWL, and HSA
were built from the crystal structures obtained above. MD
simulations were performed for 200 ns in the presence ofAS and also for 200 ns in the absence of AS. The
simulations with AS were executed in triplicate forreproducibility. The concentrations of AS ions in thecalculation models were set to be equal to the initial droplet
in the experimental setup for protein crystallization, andother minor ingredients were omitted from the models. For
reference, the simulations without protein were also
performed in the presence of the same concentrations ofAS as the respective models.
3 Results and Discussion

Photon Factory Activity Report 2018 #36 (2019)

Figure 1. Representative structures of the simulation
trajectory for (a) CAII, (b) Mb, (c) HEWL, and (d) HSA.
Protein molecules are represented by the cartoon, changing
colors from blue to red as the residue goes from N- to Cterminal sides. N, S, and O atoms of AS ions are coloredblue, yellow, and red, respectively. Sodium and chloride
ions and water molecules are not shown for clarity.
The average structure of the protein was obtained fromthe simulation trajectory for the last 20 ns. A snapshot
structure that was the closest to the average one wasextracted from the trajectory and defined as therepresentative structure (Figure 1). The representative

structures demonstrate a keen difference in the AS
distributions between the easily crystalized proteins by AS,
CAII and Mb, and the not easily crystallized ones, HEWL
and HSA. That is, the distribution of AS ions was markedly
anisotropic for CAII and Mb. On the other hand, thedistribution was isotropic for HEWL and HSA.
In CAII, AS ions were localized at two regions in thesolvent, and the regions made contacts with the proteinfrom both sides on an axis across the protein. Since thedistributions of ammonium ions and sulfate ones werealmost identical to each other, electrical polarity caused by
AS ions was zero everywhere around CAII. AS
distributions were compatible among the three simulations,and particularly the AS sparse areas around the protein arewell matched among the three. In Mb, AS ions werelocalized at two regions, in which Mb was sandwiched
between the two regions as in CAII. The AS dense areas
were common among the three simulations. Electricalpolarity due to AS ions was almost zero everywhere
because of the identical distributions of ammonium andsulfate ions. In HEWL, AS ions were not localized in all of
the three simulations, and instead, AS ions sparsely
distributed in the solvent. The gatherings of AS ions weretemporarily observed at local areas, while the positions
were different among the simulations. In HSA, AS ionswere not localized as well as HEWL. Small gatherings of
AS ions were observed at a few regions near the protein
surface. When no protein was included in the calculation
model, AS ions were randomly distributed in solvent. The
conformational changes of the tertiary structures werehardly observed for all the proteins, while noticeable
increases in atom fluctuation were occasionally seen at theloop domain for HEWL.
The motions of protein and precipitant molecules weremonitored all through the 200 ns simulation, and thesnapshot structures at 0, 50, 100 ns were extracted fromsimulation trajectory both for the models with and without
AS. In every simulation without AS, the protein molecule
substantially moved in the solvent, and the direction of theprincipal molecular axis was ceaselessly altered. Since themolecular weights of Mb and HEWL are small comparedto those of CAII and HSA, the displacements of the centerof mass for Mb and HEWL were much larger than those of
CAII and HSA. In the presence of AS ions, the motion of
the protein molecule was reduced especially for CAII andMb. In the case of CAII with AS, not only the center ofmass but also the direction of the molecular axis did noteasily alter. The initial setup of AS positions, in which ASions were randomly placed in the solvent, was reflected in
the AS distribution at 0 ns. At 50 ns, AS ions wererelocated not to cover all over the protein surface but tomake contacts with CAII partially. The AS distribution
was not uniform, and instead, the localization of AS ions
was observed. The localized distribution became
remarkable at 100 ns. In the simulation of Mb with AS,
while AS distribution at 0 ns was random, AS ions werearranged to form a localized distribution at 50 ns. AS denseareas appeared at two regions around the protein, in which
two AS dense areas and the protein were almost aligned ina straight line. The positions of AS dense areas werePhoton Factory Activity Report 2018 #36 (2019)scarcely changed at 100 ns, which suggested that thedistribution was stable. In HEWL with AS, AS ions wererandomly distributed during the simulation. The random
distribution of AS ions suggested that the surrounding
space of HEWL was unfavorable for the gathering of ions.
In HSA with AS, the AS dispersed distribution was kept
through the simulation for 100 ns.

Figure 2. Distributions of AS ions in the simulations for(a) CAII, (b) Mb, (c) HEWL, and (d) HSA. The
distributions of AS ions are depicted in mesh, according to
the rate of the presence of ions for the last 20 ns. The
population of ammonium ions is colored from light to deep
with the increase of ion density in blue and that of sulfate
ions is colored in red.

In order to explain the localized distribution of AS ions,the iso-surface of electrostatic potential around the protein
was depicted (Figure 3). In CAII and Mb, the positive andnegative areas of electrostatic potential were clearly
separated, in which both areas had spherical shapes andwere almost equal to each other in volume. The AS
gatherings observed in the simulations were compatible
with the iso-surface of electrostatic potential. AS ions werelikely to be located at the regions where the absolute valueof electrostatic potential was high. That is, thesurroundings of the protein were polarized, and the electricfield lines were crowded at these polar regions. In contrast,a marked difference was observed in volume between thepositive and negative areas in HEWL and HSA. In HEWL,
the whole protein was completely covered with thepositive potential surface. AS ions were not inclined togather at specific areas on the protein surface. In HSA, theelectrostatic potential was greatly biased to the negative.
Most of AS ions were not strongly attracted to specificlocal areas, while there was a small region where AS ionsgathered due to the positive electrostatic potential (Figure3d). Consequently, the electrostatic potential is responsible
for the localized distribution of AS ions, which leads to thestabilization of the mixture of AS ions and the protein incases of CAII and Mb. It is known that the protein crystals
contain a large amount of solvent. The protein atoms
occupy only about 43% of the unit cell in volume onaverage. The distributions of precipitant agents and thepacking of protein molecules were suggested to becomplementary to each other. Therefore, AS ions willstabilize the protein crystals by staying at the space notoccupied by the protein atoms.
It will be expected from the findings of this study that an
introduction of amino acid mutation to alter theelectrostatic potential around a protein can enhance
crystallization even if the experimental condition for thecrystal growth of the protein with AS is not wellestablished. The optimal zone of precipitant concentration
is sometimes narrow for crystal growth with AS. The
electrostatic potential will be utilized as a guide to how tomodify recombinant proteins suitable for the crystal
growth with AS. For example, when a protein crystal isobtained with a precipitant other than AS, but theresolution of X-ray diffraction is not satisfactorily high, abetter quality of protein crystals is occasionally required.
Modeling of the protein structure from the low-resolution
X-ray diffraction, modification of the model byintroducing amino acid mutation, and calculation of theelectrostatic potential for the modified model will give agood suggestion on the adequate modification of theprotein to promote the crystallization with AS. The
electrostatic potential significantly depends on the pH ofsolution. Multiple calculations of the electrostatic
potentials for the models changing the protonation statesof the titratable residues will provide the information on
the appropriate pH condition. Hence, the analysis of theelectrostatic potential enables us to take the rational
strategy in experiments.

Photon Factory Activity Report 2018 #36 (2019)

structures were determined using the molecularreplacement method. Based on the determined protein
structure, 200 ns MD simulations were performed intriplicate with the models including AS ions at the same
concentration as the experimental condition in the crystal
growth for every protein. A 200 ns simulation without ASions was also performed for comparison. The presence of
AS ions reduced the motion of the proteins during thesimulations, especially for CAII and Mb. The distributionof AS ions was not random but highly anisotropic aroundthe protein with the ions localized at two areas. The
localized distribution was caused by the electrostatic
potential around the protein, and AS ions gathered at theregions where the absolute value of the electrostatic
potential was high. In CAII and Mb, the positive andnegative areas of electrostatic potential were almost
equally separated, and both areas had a spherical shape. In
contrast, either one of the positive or negative areas wasdominant in HEWL and HSA. Therefore, the shape of theiso-surface of the electrostatic potential is highly
responsible for the readiness in the crystal growth with AS.
If electrostatic potential around a protein can be controlled
by adjusting pH or introducing amino acid mutation, thequality of crystallization by AS will be improved even forthe proteins which are not easily crystallized by AS.
Acknowledgement
Calculations were performed at Research Center for
Computational Science, Okazaki, Japan and at Information
Technology Center of the University of Tokyo.
* hoshino@chiba-u.jp

Figure 3. Positive and negative iso-surfaces ofelectrostatic potential around the proteins for (a) CAII, (b)Mb, (c) HEWL, and (d) HSA. The positive and negative
iso-surfaces are shown in the blue and red meshrepresentation, respectively. The contouring values of isosurfaces are (a) +0.43 and -0.14 for CAII, (b) +0.39 and 0.13 for Mb, (c) +1.75 and -0.004 for HEWL, and (d) +0.43
and -1.96 for HSA in unit of kT/e. Protein molecules arerepresented by green cartoons. Every electrostatic potential
was obtained from the simulation in the absence of AS.
In summary, protein crystals were grown for CAII andMb, which are easily crystallized by AS, and also for
HEWL and HSA, which are not. X-ray diffractions wereacquired from the respective protein crystals, and the