CRYSTAL

GROWTH
& DESIGN

The Effect of Ionic Liquids on Protein Crystallization and X-ray

Diffraction Resolution

2009
VOL. 9, NO. 8
34633469

Russell A. Judge,* Sumiko Takahashi, Kenton L. Longenecker, Elizabeth H. Fry,

Cele Abad-Zapatero, and Mark L. Chiu
Department of Structural Biology, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, Illinois 60064
ReceiVed February 6, 2009; ReVised Manuscript ReceiVed April 6, 2009

ABSTRACT: Ionic liquids exhibit a variety of properties that make them attractive solvents for biomaterials. Given the potential
for productive interaction between ionic liquids and biological macromolecules, we investigated the use of ionic liquids as precipitating
agents and additives for protein crystallization for six model proteins (lysozyme, catalase, myoglobin, trypsin, glucose isomerase,
and xylanase). The ionic liquids produced changes in crystal morphology and mediated significant increases in crystal size in some
cases. Crystals grown using ionic liquids as precipitating agents or as additives provided X-ray diffraction resolution similar to or
better than that obtained without ionic liquids. Based upon the experiments performed with model proteins, the ionic liquids were
used as additives for the crystallization of the poorly diffracting monoclonal antibody 106.3 Fab in complex with the B-type natriuretic
peptide (5-13). The ionic liquids improved the crystallization behavior and provided improved diffraction resulting in the determination
of the structure. Ionic liquids should be considered as useful additives for the crystallization of other proteins.
Introduction
Ionic liquids, also known as molten salts, are liquids
composed entirely of ions with melting points under 100 C.1-4
They are generally organic salts where one or both of the
counterions can be customized for specific applications. In
general, they are thermally stable, nonflammable, and exhibit
very low vapor pressure. Ionic liquids can dissolve a wide range
of organic and inorganic compounds and can be water miscible.
Because of these properties, ionic liquids have been used as
solvents for conducting a wide range of chemical reactions, in
some cases replacing more toxic solvents, earning them the
reputation of being environmentally friendly solvents.5
Ionic liquids are also finding application with biomaterials.
As solvents, they have been used to solubilize protein and
DNA,6,7 cellulose,8 and reconstitute silk fibroin.9 Of particular
interest, ionic liquids are employed in biocatalysis10-13 while
preserving enzyme stability.14-16 Ionic liquids have also been
reported to increase enzymatic activity, selectivity, and yield
in comparison to other solution systems.17-21 Imidazolium-based
ionic liquids have also been used to enhance protein folding
and suppress aggregation.22
Given these applications with biomaterials and their ability
to participate in ionic, hydrophobic, and hydrogen bond interactions, ionic liquids are potential agents for use in protein
crystallization experiments. In this respect, ethyl ammonium
nitrate has been used as a precipitating agent for the crystallization of lysozyme, providing crystals with good diffraction.23
Pusey et al. (2007) further developed this idea by exploring the
use of three ionic liquids on the crystallization behavior of four
model proteins.24 In some instances, the ionic liquids acted as
precipitating agents and in other cases as additives. While some
morphology improvements were observed, the diffraction quality
of the crystals was not assessed.
In this study, we have undertaken a wider scope, utilizing
16 ionic liquids that comprise different cation (imidazolium,
phosphonium, ammonium) and anion (borate, halide, sulfate,
* Corresponding author. Phone 847-935-1343; fax 847-938-1083; e-mail:
Russell.judge@abbott.com.

acetate, and sulfonate) structures, for crystallization experiments

with six model proteins (chicken egg white lysozyme, bovine
liver catalase, horse myoglobin, bovine pancreas trypsin, glucose
isomerase, and xylanase). These water-miscible ionic liquids
were screened as protein crystallization precipitating agents and
additives. The ionic liquids were selected based on their
solubility in standard protein crystallization precipitant solutions.
Some of the crystals grown in these ionic liquid solutions were
tested for X-ray diffraction quality. On the basis of the results
of these experiments, the ionic liquids were used to improve
the crystallization behavior and diffraction resolution for the
monoclonal antibody (mAb) 106.3 fragment antigen binding
region (Fab) in complex with its cognate epitope, the truncated
B-type natriuretic peptide (BNP (5-13)).25
Methods
Chemicals and Model Proteins. Chicken egg white lysozyme
(Sigma Chemicals, St. Louis, MO, L-6876) was dissolved in
0.1 M sodium acetate buffer pH 4.7 to give a 50 mg/mL
solution, based on prior experimental experience.26 Bovine liver
catalase (Sigma Chemicals, C9322) was dissolved in 25 mM
HEPES buffer pH 7.0 to give a 30 mg/mL solution. Horse
skeletal myoglobin (Sigma Chemicals, M0630) was dissolved
in distilled water to give a 20 mg/mL solution.27 Bovine
pancreas trypsin (Sigma Chemicals, T1426) was dissolved in
25 mM HEPES pH 7.0, 10 mM calcium chloride, and 10 mg/
mL benzamidine hydrochloride (to inhibit autoproteolytic activity) to give a 60 mg/mL solution. Glucose isomerase from
Streptomyces rubiginosus (Hampton Research, Aliso Viejo, CA)
was dialyzed against 10 mM HEPES pH 7.0 to a concentration
of 18 mg/mL (Hampton Research). Xylanase from Trichoderma
longibrachiatum (Hampton Research, Aliso Viejo, CA) was
diluted with distilled water to give a solution of 18 mg/mL
(Hampton Research). Sodium acetate and HEPES were titrated
to the specified pH using 1 M hydrochloric acid and 1 M sodium
hydroxide, respectively (Hampton Research, Aliso Viejo, CA).
All buffer and precipitant solutions for protein crystallization
were obtained from Hampton Research (Aliso Viejo, CA). The
benzamidine hydrochloride was obtained from Sigma Chemicals

10.1021/cg900140b CCC: $40.75 2009 American Chemical Society

Published on Web 05/04/2009

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Crystal Growth & Design, Vol. 9, No. 8, 2009

Judge et al.

Table 1. Structures and Molecular Weight of Ionic Liquids Used in This Study

(B6506). The ionic liquids (Table 1) were purchased from Merck

(KGaA, Darmstadt, Germany). The majority of the ionic liquids
were supplied as liquids. Seven (tetraethylammonium bromide,
n-butylpyridinium chloride, tetrabutylphosphonium bromide,
benzyltriethylammonium chloride, 1-ethyl-3-methylimidazolium
chloride, 1-butyl-2,3-dimethylimidazolium tetrafluoroborate and
1,3-dimethylimidazolium methyl sulfate) however were supplied
as solids. For these ionic liquids, stock aqueous solutions were
made at 50% (w/v).
Model Protein Crystallization Trials Using Ionic Liquids
As Precipitants. The crystallization trials of the six model
proteins were tested in 96-well format screening plates

(Intelliplate, Art Robbins Instruments, Sunnyvale, CA) using

the sitting drop vapor diffusion technique. Twelve different
reservoir concentrations of each ionic liquid from 3 to 36%
(v/v or w/v depending on the supplied state of the ionic
liquid) were used with increments of 3%, setup at three buffer
conditions using 0.1 M sodium acetate pH 5.0, 0.1 M HEPES
pH 7.0 and 0.1 M Tris pH 8.5. The upper value of the ionic
liquid was based on saturation of the ionic liquids in aqueous
solution. Given the molecular weight of the ionic liquids,
this upper boundary was approximately 1.0-2.5 M. The
crystallization drops were made by adding 0.5 L of protein
solution to 0.5 L of reservoir solution. The crystallization

Effect of Ionic Liquids on Protein Crystallization

Crystal Growth & Design, Vol. 9, No. 8, 2009 3465

Table 2. Lysozyme Crystallization Results with Ionic Liquidsa

ionic liquids

0.1 M sodium acetate pH 5.0

1-ethyl-3-methylimidazolium tetrafluoroborate
1-butyl-3-methylimidazolium chloride
1-hexyl-3-methylimidazolium chloride
1-ethyl-3-methylimidazolium trifluoromethanesulfonate
triisobutyl (methyl) phosphonium p-toluenesulfonate
tetraethylammonium bromide
n-butylpyridinium chloride
tetrabutylphosphonium bromide
benzyltriethylammonium chloride
1-ethyl-3-methylimidazolium chloride
1-butyl-2,3-dimethylimidazolium tetrafluoroborate
1,3-dimethylimidazolium methyl sulfate
1-butyl-3-methylimidazolium trifluoroacetate
a

0.1 M HEPES pH 7.0

0.1 M Tris pH 8.5

30% (v/v)

3 and 6% (v/v)
9-18% (v/v)

3-12% (v/v)
9-27% (w/v)

9% (v/v)
3-12% (v/v)
6-30% (w/v)

6 and 9% (v/v)
3-12% (v/v)
6-27% (w/v)

3-33% (w/v)

9% (w/v)

The ionic liquid concentration range where crystals were obtained is displayed for each buffer.

drops equilibrated against a 100 L reservoir in crystallization

plates that were incubated at 16 C.
Model Protein Crystallization Trials Using Ionic Liquids
As Crystallization Additives. The six model proteins were
crystallized using known crystallization conditions, with the
inclusion of 0, 2, and 5% of each ionic liquid in the reservoir.
This corresponds to approximately 0.05-0.15 M depending on
molecular weight, which is typical of concentrations used for
many crystallization additives. The reservoir crystallization
solutions were for lysozyme - 5% (w/v) NaCl, 0.1 M sodium
acetate pH 4.7; for myoglobin - 3.2 M ammonium sulfate; for
trypsin - 2.0 M ammonium sulfate, 0.1 M Tris-HCl pH 8.5;
for catalase - 8% (w/v) PEG 8000, 0.1 M Tris-HCl pH 8.5;
for glucose isomerase - 1.6 M ammonium sulfate, 0.1 M TrisHCl pH 8.0; for xylanase - 1.4 M sodium potassium phosphate
pH 7.5. Crystallization trials were conducted by vapor diffusion
using the hanging drop method in 24-well Linbro plates which
were incubated at 16 C. Drops were set using 2 L of protein
added to 2 L reservoir solution and equilibrated against 1 mL
of reservoir solution.
Preparation and Crystallization of the mAb 106.3 Fab
in Complex with the BNP (5-13) Peptide. Production and
purification of mAb 106.3 (IgG1, ) were performed as
previously described.25,28 Briefly, mAb 106.3 was produced in
a murine hybridoma cell line (Scios, Inc.) grown in serum-free
media. Fab fragments were prepared by digestion of IgG 103.6
using immobilized papain (Pierce Biotechnology, Inc., Rockford,
IL) at 1:100 (w/w) papain/antibody ratio in 0.05 M phosphate
buffer, pH 7.2, containing 0.15 M NaCl, 1 mM D,L-dithiothreitol
(DTT) and 1 mM ethylene diamine tetraacetic acid (EDTA)
incubated at room temperature overnight. Digestion was stopped
by separation of the papain gel in a centrifuge. Undigested IgG
and Fc fragments were removed by chromatographic separation
using a Protein A Poros A50 column (Applied Biosystems,
Foster City, CA). Higher than 95% purity of the isolated Fab
fragments was achieved by using a G2000SWxl HPLC column
(Tosoh Corporation, Tokyo, Japan) for final purification. Purified
protein was transferred into 20 mM Tris buffer, pH 8.0,
containing 50 mM NaCl.
The Fab-peptide complex was prepared by addition of
N-acetyl-Val-Gln-Gly-Ser-Gly-Ala-Phe-Gly-Arg peptide in a 1:1
molar mixture with purified Fab106.3 yielding a final concentration of 17 mg/mL in a 20 mM Tris buffer containing 50 mM
NaCl, pH 7.4. Crystals were grown at room temperature
(23 C) in a vapor diffusion setup of 1 L complex mixed with
1 L of reservoir solution over a 1 mL reservoir of 15-20%
(w/v) polyethylene glycol monomethyl ether 5000, 1.0 M
sodium formate, 0.1 M sodium citrate pH 5.6, with and without
5% (v/v) ionic liquids as additives. X-ray diffraction data were

collected at 100 K using 1 wavelength radiation on the 17BM beamline of the Advanced Photon Source at Argonne
National Laboratory, Chicago, IL, USA.
Results
Ionic Liquids As Precipitants for the Six Model
Proteins. Of the six model proteins tested, only trypsin and
lysozyme yielded crystals. Trypsin crystals were only obtained
with 1-butyl-3-methylimidazolium trifluoroacetate (24-33%
v/v) at pH 7.0, while lysozyme crystallized in a number of ionic
liquids listed in Table 2. Lysozyme crystals appeared within
minutes in triisobutyl (methyl) phosphonium p-toluenesulfonate.
Tetraethylammonium bromide was able to induce lysozyme
crystallization in the widest range of concentration (from 9-27%
(w/v)). Both triisobutyl (methyl) phosphonium p-toluenesulfonate and tetraethylammonium bromide produced crystals
at all three pH values. Of the six cases where ionic liquids
produced lysozyme crystals at pH 7.0, in three instances the
cation was 1-ethyl-3-methylimidazolium. Overall pH 7.0 was
the most effective in generating protein crystals. For myoglobin,
catalase, glucose isomerase, and xylanase, there was no general
observed pattern for protein precipitation or solubilization with
pH or selected ionic liquids. Only in the case of two ionic
liquids, 1-ethyl-3-methylimidazolium tetrafluoroborate and 1-butyl-3-methylimidazolium chloride, was a pH trend observed in
that the number of proteins precipitated by these ionic liquids
decreased with increasing pH (4 and 3 respectively at pH 5.0,
2 each at pH 7.0 and none for either at pH 8.5).
To test protein diffraction, lysozyme crystals grown in 30%
(w/v) of 1-ethyl-3-methylimidazolium chloride with 0.1 M
HEPES pH 7.0 were cryo-cooled with 25% (v/v) glycerol in
a stream of 100 K gaseous nitrogen, using an Oxford Cryostream (Oxford Cryosystems, Oxford, UK). The ionic liquids
used in this study did not display cryo-protectant properties
and therefore for cryo-cooling a cryo-protectant such as
glycerol was employed. Diffraction data were collected to
1.9 using a MAR-CCD detector system (Rayonix, Evanston, IL, USA) with a Rigaku RU-2000 rotating anode X-ray
generator (Rigaku, The Woodlands, TX, USA) operating at
100 mA and 50 kV. The diffraction pattern is illustrated in
Figure 1. The space group was P43212 with unit cell a ) b
) 78.6 , c ) 37.0 , which is the same as that commonly
reported for tetragonal lysozyme grown in sodium chloride
solutions (5% (w/v) NaCl, 0.1 M sodium acetate buffer pH
5.2), and the diffraction resolution is comparable or marginally better than that reported for lysozyme diffraction using
in-house X-ray sources.26 Note in terms of molarity, 5%
(w/v) NaCl is 0.86 M while 30% (w/v) 1-ethyl-3-methylimidazolium chloride is 2.4 M.

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Crystal Growth & Design, Vol. 9, No. 8, 2009

Figure 1. Diffraction pattern for lysozyme grown in 30% (w/v) 1-ethyl3-methylimidazolium chloride, 0.1 M HEPES pH 7.0. The diffraction
resolution was 1.9 .

Figure 2. Lysozyme crystals in 0.1 M Tris pH 8.5 were grown in (a)

6% (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate and
(b) 6% (w/v) tetraethylammonium bromide.

Judge et al.

phosphonium p-toluenesulfonate had the opposite effect with

crystal numbers increasing with increasing ionic liquid concentration.
Ionic Liquids As Crystallization Additives for the Six
Model Proteins. As crystallization additives, the selected ionic
liquids provided larger crystals or different crystal morphologies
in reference to the control experiments and in some cases
amorphous precipitation or no observable effect (Table 3).
Changes in crystal morphology and size for glucose isomerase,
lysozyme, trypsin, and xylanase were observed (Figure 4). In
the case of glucose isomerase, triisobutyl (methyl) phosphonium
p-toluenesulfonate changed the crystal morphology from rods
to hexagonal plates. For trypsin, the same ionic liquid produced
clusters of small needles instead of short prisms. For xylanase,
1-butyl-3-methylimidazolium chloride changed the crystal morphology from overlapping bunches of many thin plates to thicker
and larger crystals (similar to that reported for xylanase by Pusey
et al.24). A number of ionic liquids generated larger trypsin
crystals, with 1-ethyl-3-methylimidazolium chloride inducing
larger crystals for both lysozyme and trypsin. No effect was
observed for catalase, and myoglobin was precipitated in every
instance.
Crystallization Results for the mAb 106.3 Fab Complex
with BNP (5-13) Peptide. Crystals were initially obtained by
vapor diffusion with a reservoir of 15-20% (w/v) polyethylene
glycol monomethyl ether 5000, 1.0 M sodium formate, 0.1 M
sodium citrate pH 5.6 (Figure 5a). Individual crystals were
separated, cryo-cooled, and tested for X-ray diffraction at the
Advanced Photon Source beamline 17BM. The best of these
initial crystals diffracted to 2.8 ; however, better data was
desired. Typically additives can be successfully used to optimize
crystals, and so the Hampton Additive screen (Hampton
Research, Aliso Viejo, CA) was applied. One additive, 0.1 M
calcium chloride, provided crystals (Figure 5b) that were
separated and tested for X-ray diffraction at the Advanced
Photon Source. There was however no improvement in diffraction data.
Given that in our prior experiments with model proteins, ionic
liquids as additives had shown the capability to change crystal
morphology, ionic liquids were added as 5% (v/v) to the initial
crystallization condition. Both triisobutyl-(methyl)-phosphonium-p-toluenesulfonate and 1-butyl-3-methylimidazolium tetrafluoroborate produced improved crystals. Crystals with 1-butyl3-methylimidazolium tetrafluoroborate (Figure 5c) were more
favorable. The crystals were thicker and grew with individual
crystal blades being more separated from each other. Individual
crystals were separated and data were collected to 2.1 at the
Advanced Photon Source, and the structure was determined.25
Discussion

The crystal habit of lysozyme varied with different ionic

liquids. For example, in 0.1 M Tris pH 8.5 with 6% (v/v)
1-ethyl-3-methylimidazolium trifluoromethanesulfonate, lysozyme
crystals were long thin prisms, while 6% (w/v) of tetraethylammonium bromide produced lysozyme crystals with a shorter
aspect ratio (Figure 2). It was also observed that crystal number,
morphology, and size changed with ionic liquid concentration.
For example, Figure 3, illustrates lysozyme crystallization in
the presence of increasing concentrations of 1-ethyl-3-methylimidazolium chloride with 0.1 M HEPES pH 7.0. As the ionic
liquid concentration increases the number of crystals per drop
decreases and better formed crystals are obtained. This trend
was also observed for 1-butyl-3-methylimidazolium chloride and
tetraethylammonium bromide at pH 7.0. Triisobutyl (methyl)

Following the concepts that ionic liquids can be used to

enhance protein stability and folding, we examined the effect
of ionic liquids on protein crystallization. When ionic liquids
were used as the sole precipitation agent, only two (lysozyme
and trypsin) out of the six model proteins tested provided
crystals. The ionic liquids that were used in this study are
observed to be weak precipitation agents for these proteins in
comparison to salts typically used in protein crystallization.29
Of the two proteins that did crystallize, lysozyme crystallized
in multiple ionic liquids (6 out of the 13 tested) and at multiple
pH values. Ionic liquids have the potential to serve as bases or
acids based on the presence of different cations and anions.
Although halide, sulfate and sulfonate ions are fully dissociated,
and hence not likely to affect pH, the ammonium, phosphonium,

Effect of Ionic Liquids on Protein Crystallization

Crystal Growth & Design, Vol. 9, No. 8, 2009 3467

Figure 3. Lysozyme crystals grown with increasing concentrations of 1-ethyl-3-methylimidazolium chloride in 0.1 M HEPES pH 7.0, (a) 6%, (b)
9%, (c) 12%, (d) 15%, (e) 18%, and (f) 21% (w/v).
Table 3. The Effect of Ionic Liquids As Crystallization Additivesa
ionic liquids

catalase

glucose isomerase

lysozyme

myoglobin

trypsin

xylanase

1-ethyl-3-methylimidazolium tetrafluoroborate
1-butyl-3-methylimidazolium chloride
1-hexyl-3-methylimidazolium chloride
1-ethyl-3-methylimidazolium trifluoromethanesulfonate
triisobutyl (methyl) phosphonium p-toluenesulfonate
tetraethylammonium bromide
n-butylpyridinium chloride
tetrabutylphosphonium bromide
benzyltriethylammonium chloride
1-ethyl-3-methylimidazolium chloride
1-butyl-2,3-dimethylimidazolium tetrafluoroborate
1,3-dimethylimidazolium methyl sulfate
1-butyl-3-methylimidazolium methyl sulfate
1-butyl-3-methylimidazolium trifluoroacetate
1-butyl-3-methylimidazolium tetrafluoroborate
1-butyl-3-methylimidazolium octyl sulfate

N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
P

P
N
P
P
M
N
N
P
P
N
P
N
N
P
P
P

N
N
N
N
N
N
N
N
N
Si
N
N
N
N
N
P

P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P

Si
P
P
Si
M
Si
Si
P
P
Si
P
Si
P
Si
P
Si

N
M
N
N
N
N
N
P
N
N
N
N
N
N
N
N

Key: Si, size increase, M, morphology change, P, precipitation, N, no observable effect.

pyridinium, borate, and acetate ions caused significant pH

changes at higher concentrations. Nonetheless, lysozyme still
yielded crystals at very low pH values in the example of
triisobutyl (methyl) phosphonium p-toluenesulfonate.
As a cautionary note Baker and Heller,30 report denaturation
for cytochrome c and denaturation with aggregation for human
serum albumin when the ionic liquid 1-butyl-3-methylimidazolium chloride was present at 50% (v/v). At concentrations
below 25% (v/v) both proteins retained their native structures.
For this same ionic liquid, in this study, lysozyme crystallized
at concentrations of 30% (v/v) or less (Table 2). Concentrations
significantly greater than 30% (v/v) were not investigated. While
ionic liquid interactions with individual proteins will likely be
variable, there is the possibility that high concentrations of ionic
liquids could lead to protein denaturation.
The application of ionic liquids as protein crystallization
additives was more successful. For the six model proteins tested,
four displayed significant changes in morphology or crystal size.
In the case of the mAb 106.3 Fab BNP (5-13) peptide complex,
the subtle change in morphology was critical to the eventual
structure determination.25 Here the ionic liquids had provided
improvement in both crystal form and diffraction, where the
use of a commercial additive screen had not. For the ionic liquids
used in this study, some influenced the crystallization behavior
of multiple proteins. The cation 1-butyl-3-methylimidazolium

was successful in reducing the number of crystal blades and

provided improved separation of the blades for both xylanase
and the BNP-Fab complex. The anion was different in each
case. Triisobutyl (methyl) phosphonium p-toluenesulfonate was
useful in modifying the crystal form of both glucose isomerase
and the BNP-Fab. Except for trypsin however, only a small
number of the 16 ionic liquids tested affected protein crystallization, and it would be preliminary at this point to extrapolate
specific effects of individual ionic liquids to a broader range of
proteins. It would therefore be prudent to utilize a wide range
of ionic liquids in an additive screen.
Lange et al.22 in working with imidazolium-based ionic
liquids suggest that additives that are excluded from the protein
surface tend to stabilize proteins and promote salting out, while
additives that preferentially bind favor protein denaturation and
solubility. These authors further classify these ionic liquids as
preferentially bound and slightly to moderately chaotropic
agents. In the case of lysozyme crystallized with 1-ethyl-3methylimidazolium chloride (Figure 3), the ionic liquid does
not appear to be salting out the protein as crystal numbers
decrease with increasing ionic liquid concentration, which would
indicate (according to Lange et al.22) an ionic liquid that is
binding to the protein and potentially acting through increasing
protein solubility. To determine if the ionic liquids used in this

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Crystal Growth & Design, Vol. 9, No. 8, 2009

Judge et al.

Figure 4. The effect of ionic liquids as crystallization additives for glucose isomerase in (a) control conditions and (b) with 2% (v/v) triisobutylmethyl-phosphonium-p-toluenesulfonate; lysozyme in (c) control conditions and (d) with 5% (w/v) 1-ethyl-3-methylimidazolium chloride; trypsin
in (e) control conditions and (f) with 2%v/v triisobutyl-methyl-phosphonium p-toluenesulfonate and (g) with 5% (w/v) 1-ethyl-3-methylimidazolium
chloride; xylanase in (h) control conditions and (i) with 5% (v/v) 1-butyl-3-methylimidazolium chloride.

Figure 5. Crystals of mAb 106.3 Fab complexed with BNP (5-13) peptide, (a) initial crystals, (b) with 0.1 M calcium chloride as additive, and (c)
with 5%(v/v) 1-butyl-3-methylimidazolium tetrafluoroborate as additive.

study are binding to the protein, X-ray analyses of lysozyme

and trypsin crystals grown using ionic liquids as additives was
undertaken.
Lysozyme crystals grown in 5% (w/v) NaCl, 0.1 M sodium
acetate pH 4.7 with 5% (w/v) 1-ethyl-3-methylimidazolium
chloride as an additive showed an increase in size over control
experiments without the ionic liquid. These crystals were
mounted in the X-ray beam with 25% (v/v) glycerol as
cryoprotectant. Diffraction data were collected to 1.9 using
the X-ray equipment described previously. The space group was
P43212, with unit cell parameters, a ) b ) 78.6 and c )
36.9 . The structure was solved by molecular replacement
using 4LYM (Protein Data Bank).31 No clear evidence for ionic
liquid binding to lysozyme was observed in the structure.
Of the six proteins tested, trypsin showed the highest
sensitivity to the use of ionic liquids as additives. The trypsin
crystals exhibited changes in morphology and significant
increases in crystal size in the presence of nine ionic liquids
(Table 3). To determine if ionic liquids bound to trypsin, a

trypsin crystal grown in 2.0 M ammonium sulfate, 0.1 M TrisHCl pH 8.5 with 5% (w/v) 1-ethyl-3-methylimidazolium
chloride was selected for X-ray diffraction analysis. Cryoprotectant of 25% (v/v) glycerol was used and diffraction data
collected to 1.8 using the X-ray equipment previously
described. The same space group was found as that reported
for trypsin grown in these conditions. No ionic liquid ions were
observed in the crystal structure. Also, no ionic liquid ions were
observed in the structure of the mAb 106.3 Fab BNP (5-13)
peptide complex. The possible effect of these ionic liquids on
crystal morphology and crystal size was therefore likely due to
subtle changes in solution conditions (pH, ionic strength)
providing a change in protein solubility or crystal growth
kinetics, rather than as a result of protein binding.
In terms of crystal diffraction, crystals of lysozyme grown
in the presence of ionic liquids as the sole precipitating agent
exhibited high resolution diffraction similar to that observed
for crystals grown in the presence of more traditionally used
precipitants such as sodium chloride. The same was observed

Effect of Ionic Liquids on Protein Crystallization

for lysoyzme and trypsin when the ionic liquids were used as
additives. Model proteins such as lysozyme and trypsin however
crystallize and diffract robustly, and so it is unlikely to expect
that the ionic liquids would significantly improve the diffraction
resolution of these proteins. For the limited samples tested in
this study however, no negative impact on X-ray diffraction
resolution was observed. A better test case is a poorly diffracting
protein such as the mAb 106.3 Fab BNP (5-13) peptide complex.
In this case where the ionic liquids were used as additives, the
diffraction resolution was indeed significantly improved.
Given the nature of ionic liquids, the potential for customizing
cations and anions provides the opportunity to create tailored
combinations of ionic liquids. In this study, we have examined
a limited set and range of ionic liquids. While the results of
this study show potential, a further step will be to conduct
broader studies to determine if ionic liquids can be customized
to optimize protein crystallization.
Conclusions
The ionic liquids used in this study performed as weak
precipitating agents and were more successful when used as
additives. The X-ray diffraction resolution of crystals grown
with ionic liquids was comparable or better than that obtained
without their use. In the case of the mAb 106.3 Fab BNP (513) peptide complex, improved crystal form and diffraction were
obtained when ionic liquids were used, in contrast to the use of
additives from a commercially available additive screen, which
failed to provide improvement. The mode of action of ionic
liquids as additives seems to be through changes in solution
properties rather than through binding to the protein. It is
expected that a screen of ionic liquid additives will be useful
for the crystallization of other proteins.
Acknowledgment. We thank Sergey Tetin of Abbott Laboratories for the purified sample of the mAb 106.3 Fab BNP (513) peptide complex. X-ray diffraction data were collected in
the facilities of the Industrial Macromolecular Crystallography
Association Collaborative Access Team (IMCA-CAT) at the
Advanced Photon Source. These facilities are supported by the
companies of the Industrial Macromolecular Crystallography
Association.
Note Added after ASAP Publication. After this paper was
published ASAP May 4, 2009, changes were made; the corrected
version was reposted May 11, 2009.

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