Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
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Two-dimensional crystalline array formations of proteins by use of the
self-assembled monolayer at the air/water interface
Noriyuki Ishii1,2
1
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba
Central-6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
2
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho,
Koganei, Tokyo 184-8588, Japan
Two-dimensional crystals have been successfully used to obtain protein structural information by electron microscopy in
combination with tomography computing and cryogenic methods although the resolution of protein structure obtained by
this approach in two-dimensional crystals is often within the medium range. The subsequent combination of the structures
derived by electron microscopy combined with X-ray or NMR structures of their components is required for detailed
reconstruction. In this chapter, we have assessed the two-dimensional crystallization method using two novel chemical
compounds, amphiphilic β-cyclodextrin (β-CD) derivative (C18CD) and lipophilic isologue of oxazine-dye Nile Blue
(Chromoionophore I). The lateral diffusion of the monolayer formed at the air/water interface facilitates two-dimensional
crystalline array formation with model proteins, ferritin, and catalase.
Keywords: monolayer; ferritin; catalase; cyclodextrin derivative; Chromoionophore I; surface pressure; transmission
electron microscopy
1. Introduction
Crystallization on a monolayer substrate is an elegant method because it is possible to work with very dilute protein
solution and still generate a locally high concentration of protein constrained in two dimensions [1]. Amphiphilic
monolayer can be spread over the whole air/water interface driven by surface tension to form a flat one-molecule thick
film [2, 3]. The monolayer film provides a substrate for protein binding, and the protein retains sufficient mobility
leading to a protein molecule in a monolayer of closely packed proteins at the interface [4]. The adsorption of protein to
an amphiphilic monolayer limits the protein molecules to a few orientations relative to the monolayer at the interface,
which appears to facilitate crystallization. Consequently, the organized two-dimensional crystals are suitable for
structure determination by electron crystallography [5, 6].
Cyclodextrins (CDs) are widely known as typical examples of organic host compounds that can include hydrophobic
guest molecules in their hydrophobic cavity [7]. The CD molecules are cyclic oligosaccharides containing six (α-CD),
seven (β-CD), eight (γ-CD) or more anhydroglucose units joined by α-1,4 glucosidic linkages. Amphiphilic (lipophilic)
CD derivatives are usually synthesized by introducing hydrophobic alkyl chains into the CD molecules [8]. In our
study, the alkyl chain was linked at the O-6 position, and the resultant S-octadecyl (C18CD) CD derivative was used to
assess the adsorption capability as a template for protein two-dimensional array formation at the air/water interface, e.g.
by changing various experimental conditions such as the compression rate and the relaxation time after spreading the
solution, and so forth.
Another candidate for self-assembled protein-adsorption monolayer is Chromoionophore I. Chromoionophore I
belongs to a class of proton selective ionophore compounds. The molecule was synthesized as a lipophilic isologue of
highly basic oxazine-dye, Nile Blue [9, 10]. The structure formula of Chromoionophore I shows the molecule consists
of a C17-alkyl chain, and a hydrophobic benzo[a]phenoxazine moiety. These hydrophobic domains appear to act as a
flotage on the water surface, and the diethylamino moiety is expected to act as a potential binding site for substrates.
Proteins with net negative surface charge are promising candidates for binding substrates.
2. Materials and methods
An amphiphilic heptakis(6-alkylthio-6-deoxy)-β-cyclodextrin (CD) derivative, octadecylated CD derivative (C18CD)
was synthesized [8] and studied for its monolayer behavior at the air/water interface on the basis of surface pressuremolecular area (π-A) isotherms [11]. In the synthesis of C18CD, the degree of substitution with octadecyl chains was
6.1 on average against 7 candidates of the hydroxyl moieties in the primary face of β-CD. The C18CD was dissolved in
chloroform solution to give a concentration of 1.0 × 10-4 mol/l. Chloroform (spectroscopy grade) was purchased from
the Kishida Chemical Co., Ltd. (Osaka, Japan). Chromoionophore I (ETH 5294: [9-(diethylamino)-5octadecanoilimino-5H-benzo[a]phenoxazine], molecular wt. 583.85) was purchased from Fulka (Buchs, Switzerland).
Chromoionophore I was dissolved in chloroform solution at a concentration of 1.7 × 10-3 mol/l.
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Fig. 1
(a) The structure of an
amphiphilic β-CD derivative, C18CD. (b)
π-A isotherms of C18CD derivative
spread on a pure water phase. Following
evaporation of the solvent (5 min), the
monolayer was compressed at a rate of
2.1 × 10-3 nm2/molecule s. (c) Surface
pressure change for the C18CD
monolayers. C18CD solution was spread
onto the subphase containing ferritin
(plain line), and onto the same subphase
without ferritin (broken line).
The π-A isotherms were recorded
using a KSV-Minitrough (KSV
Instrument Ltd.) or a laboratory
handmade Langmuir-Blodgett (LB)
trough (surface area, 870 cm2) both
with a Wilhelmy type microbalance
using a platinum plate. The CD
derivative, C18CD was dissolved in
chloroform
solution
with
a
concentration of 1.0 × 10-4 mol/l, and
was spread on the water subphase
(Milli Q water) of KSV-Minitrough,
the temperature of which was
automatically controlled to 20 °C by a
temperature controller (TAITEC, Saitama, Japan). The compression rate was fully controlled by the trough system
using a microcomputer. A monolayer of Chromoionophore I was spread on the water subphase (Milli Q water) of the
handmade trough buffered with Tris-HCl adjusted to the desired pH and salt concentrations, the temperature of which
was automatically controlled to 20 °C by a temperature controller (TAITEC). The compression rate was controlled so as
to be 2.5 × 10-4 nm2/molecule s by the trough operation system using a microcomputer.
Ferritin and catalase were used as model proteins in the study. Ferritin from equine spleen was purchased from
Sigma-Aldrich Corp. (St. Louis, MO) and catalase from beef liver was purchased from Boehringer Mannheim
(Germany). They were further purified by gel filtration HPLC (Tosoh TSKgel G3000SWxl ) before the each adsorption
experiment at the air/water interface. Ferritin was dissolved in 20 mM Tris-HCl, 100 mM NaCl, pH 7.1, and the
concentration was finally adjusted to 20 μg/ml so that it can be used as a subphase for the experiment with C18CD.
Conversely, for the experiment with Chromoionophore I, ferritin was dissolved in 20 mM Na-Phosphate, 10 mM NaCl,
pH 5.9, and the solution was further diluted when used as a subphase. Catalase was dissolved in 20 mM Tris-H2SO4, 10
mM NaCl, 10 mM CdSO4, at either pH 6.1 or pH 7.1 with the concentrations of 20 μg/ml used as subphase.
The procedure for the protein crystalline array formation at the air/water interface was as follows. The protein
solution was introduced to the small Langmuir trough of which diameter and depth were 10 mm and 2 mm,
respectively. Onto the surface of each subphase, the chloroform solution containing each amphiphilic compound
(C18CD or Chromoionophore I) for adsorption template was gently spread with a Hamilton syringe, with adsorption
taking place at room temperature for a desired period.
The specimen grid covered with a carbon support film was placed carefully onto the supporting monolayer formed
on the subphase after a desired incubation time. As the adsorption of protein molecules to each supporting monolayer
from the bulk subphase proceeds, the bound protein molecules were expected to be closely packed by the lateral
migration due to the fluidity of the support monolayer. The specimen grid, carefully detached from the surface, was
blotted and immediately negatively stained with 1 % uranyl acetate, and then observed on a FEI Tecnai F20 (FEI
Company, the Netherlands) operating at an accelerating voltage of 120 kV. TEM images were recorded by making use
of the slow scan CCD camera (Gatan Retractable Multiscan Camera) under low electron dose condition at
magnification of 11,500×, 25,000×, and 50,000×.
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a)
Fig. 2 Electron micrographs of negatively
stained ferritin molecules adsorbed on
C18CD monolayer film (a), and enlarged
image (b). Scale bars, (a) 100 nm, and (b)
50 nm.
b)
3. Results and discussion
3.1 Two-dimensional array formation of ferritin by use of C18CD
The structure of β-CD derivative, C18CD is shown in Fig. 1(a). The π-A isotherms recorded for the C18CD derivative
at 20 °C is shown in Fig. 1(b) [8]. Following the evaporation of the solvent (5 min), the monolayer was compressed at a
rate of 2.1 × 10-3 nm2/molecule s. The isotherm indicates that the C18CD derivative is capable of forming monolayers at
the air/water interface. The modification of water-soluble CD molecule with the C18 alkyl chains is sufficient to obtain
amphiphilic properties to form monolayers. The limiting area per molecule (extrapolation of the linear part on the
isotherms to the abscissa) of C18CD is 2.20 nm2. For reference, the area of the secondary face of native β-CD is 1.86
nm2 and the calculated area per molecule of native β-CD in the hexagonal closest packed structure is 2.10 nm2 [12 - 14].
In Figure 1(c), the plain line shows the surface pressure change when an aliquot of C18CD in chloroform was spread
onto the buffered subphase containing ferritin molecules. The broken line is the control without the protein in the
subphase [11]. Once there is full coverage on the water surface by the C18CD derivative (surface pressure, 11.5 mN/m),
the surface pressure decreased slightly for a while followed by gradual increase to a value of 14.5 mN/m. The
adsorption of ferritin molecules (440 kDa) from the subphase to the C18CD monolayer is recognizable due to the
pressure increase. There are two possible reasons why the surface pressure increases with the adsorption of ferritin
molecules onto the C18CD supporting monolayer. One is that the binding of ferritin molecules stabilizes the structural
orientation of C18CD molecules in the monolayer [15]. The other one is due to the intermolecular interaction and/or
repulsion between the ferritin molecules as their density increases on the supporting monolayer. In the absence of
ferritin molecules in the subphase (broken line in Fig. 1(c)), the spread C18CD monolayer was observed as follows; The
surface pressure of 11.5 mN/m decreased quickly to the level of 9.0 mN/m and remained at that value.
Figure 2 shows electron microscopic images under negative staining of ferritin monolayer arrays adsorbed to the
C18CD supporting monolayer after 6-hour incubation at 25 °C. The subphase pH was 7.1. The proteinaceous
polypeptide shell of ferritin molecules is seen like a doughnut white surrounding the dark colored core consisting of
iron oxide in the molecule. Neither dissociation into subunits, nor aggregates were observed by TEM. This means the
C18CD derivative forms a better supporting monolayer for subsequent construction of protein monolayer assemblies.
The adsorption method using the C18CD as a supporting monolayer would be a promising technique for preparing twodimensional crystalline arrays of proteins, once key-parameters of the preparation conditions such as initial protein
concentration, pH and ionic strength (buffer condition) in the subphase, incubation period, temperature, and so forth are
optimized for each protein of interest.
The synthesized amphiphilic CD derivative, C18CD, revealed that it can be assembled as preformed monolayers for
the adsorption of proteins or enzymes. The CD molecules on the water surface interacted favorably with biological
macromolecules and provide specific adsorption sites for them. The advantage of using the amphiphilic CD molecules
is that they are possibly less toxic and have little denaturation tendency to biological compounds upon adsorption
because CD molecules are natural compounds.
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Fig. 3
(a) The structure of
Chromoionophore I. (b) π-A
isotherms of Chromoionophore I
spread on a pure water phase
containing 20 mM Tris-HCl, 10
mM NaCl at pH 7.0 (at 20 °C).
Following evaporation of the
solvent (10 min), the monolayer
was compressed at a rate of 2.5 ×
nm2/molecule
s.
(c)
10-4
Relaxation of Chromoionophore I
on a pure water subphase buffered
with 20 mM Tris-HCl, 10 mM
NaCl, pH 7.0 (at 20 °C).
Following 10 min after spreading
the solution of Chromoionophore
I, the monolayer was compressed
at a rate of 2.5 × 10-4
nm2/molecule s. Once the surface
pressure reached 20 mN/m the
barrier was fixed and then the
surface pressure was continuously
monitored as a function of time.
3.2 Two-dimensional array formation of ferritin by use of Chromoionophore I
Next, we have examined whether an amphiphilic Chromoionophore I (ETH5294) can be assembled as a preformed
monolayer for the adsorption of proteins. The chemical structure of Chromoionophore I used through the study is
shown in Fig. 3(a). Therefore, if a monolayer film of Chromoionophore I can be achieved as a Langmuir film on the
water surface, it would be expected to work as a supporting film (or template) for the adsorption of a variety of
functional protein molecules and for the preparation of two-dimensional array assembly. In other words, a variety of
functional molecules such as protein enzymes without any long alkyl chains are expected to be incorporated in
Langmuir-Blodgett (LB) films with an ordered architecture. Such films could also be promising materials for
molecular-electronic and bio-electronic devices [16].
The π-A isotherms recorded for Chromoionophore I at subphase pH 7.0 (at 20 °C) is shown in Fig. 3(b). Following
the evaporation of the solvent (10 min), the monolayers were compressed at a rate of 2.5 × 10-4 nm2/molecule s. The
isotherms indicate that Chromoionophore I is capable of forming monolayers at the air/water interface. The
hydrophobicity of Chromoionophore I with C17 alkyl chain appears to be sufficient to obtain amphiphilic properties to
form monolayers. The limiting area per molecule of Chromoionophore I was 3.50 × 10-1 nm2 at pH 7.0. Figure 3(c)
shows the relaxation profile of the compressed monolayers of Chromoionophore I after the compression. After the
compression, the barrier was fixed once the surface pressure reached 20 mN/m. The surface pressure was then observed
continuously as a function of time.
The Chromoionophore I molecules on the water surface appeared to interact favorably with biological
macromolecules and provide adsorption sites. The advantage of using amphiphilic Chromoionophore I molecules is
their lower toxicity and in their property to reduce the tendency of biological compounds to denature upon adsorption
because its diethylamino moiety is similar to diethylaminoethyl residue, commonly used in the weak anion exchange
column chromatography. In addition, one can recognize that its benzo[a]phenoxazine moiety, which is rich in πelectrons, may interact with biological molecules through hydrophobic interaction, and the protonation of nitrogen
atoms may result in electrostatic interactions.
Figure 4(a) through (c) show electron microscopic images of a series of ferritin adsorptions to the Chromoionophore
I supporting monolayer after 6-hour incubation period at different magnifications. The subphase pH was 7.1. In TEM
images, densely packed monolayer arrays of ferritin molecules are dominant, and the iron oxide in the core of each
ferritin molecule is observed as dark black dots, surrounded by 24-subunit polypeptides in ferritin molecule. Although
the double layer architectures are recognized at around the peripheral regions in Fig. 4(a), neither broken molecules nor
unfolded aggregations were observed by TEM (Fig. 4(b), (c)). Therefore, Chromoionophore I is found to be a
supporting monolayer for proteins at the interface, suggesting that Chromoionophore I has the potential to act as a
template (supporting monolayer) for protein molecules to form a monolayer array at the air/water interface. Further
optimization of the experimental parameters is a prerequisite for fabricating extended two-dimensional densely packed
and/or crystalline arrays of ferritin using this supporting monolayer.
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a)
b)
c)
Fig. 4 Electron micrographs of negatively stained ferritin molecules adsorbed on Chromoionophore I monolayer film (a), and
enlarged images (b, c). Scale bars, (a) 200 nm, (b) 100 nm, and (c) 50 nm.
3.3 Two-dimensional catalase crystalline array formation by use of Chromoionophore I
Following the above procedure using ferritin, another enterprise using catalase as another candidate protein was
performed to form two-dimensional crystalline array formation of catalase by use of Chromoionophore I monolayer.
Catalase mediates decomposition of hydrogen peroxide into oxygen and water. Catalase is also a large rectangular
parallelepiped shaped protein (240 kDa) composed of four identical subunits. Figure 5 shows electron microscopic
images of a series of catalase adsorptions to the Chromoionophore I supporting monolayer after 3-hour incubation
period at different magnifications. The subphase pH was 6.1 for Fig. 5(a) and (b), and pH 7.1 for Fig. 5(c).
A close look at the TEM images (Fig. 5) shows that the quasi-rectangular lattice lines are recognized probably due to
the initial stage of crystallization and crystal growth phases. Needless to say, further optimization for the above
mentioned conditions is required to achieve fruitful results with two-dimensional catalase crystalline array formation.
3.4 Summary discussion
In the case of two-dimensional crystallization using amphiphilic monolayers at the air/water interface, measurements
for the isothermal surface pressure versus molecular area curve can be performed in order to determine if a monolayer is
in a fluid state. At the collapsing point, the collapse pressure is the critical pressure at which the monolayer becomes too
compact and unstable. At this surface pressure and higher, the monolayer at the interface collapses in the fluid state. It is
clear that the supporting monolayer at the fluid state is the most favorable condition for the growth of two-dimensional
protein crystals [17, 18].
a)
b)
c)
Fig. 5 Electron micrographs of negatively stained catalase molecules adsorbed on Chromoionophore I monolayer film (a), and
enlarged images (b, c). The subphase pHs, (a, b) 6.1, and (c) 7.1. Scale bars, (a) 200 nm, and (b, c) 100 nm.
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One must always keep in mind that the surface crystallization trails generally leads to a closely packed layer of
protein molecules, and are prone to produce hexagonally closely packed protein assembly which might be taken to be
spontaneously formed protein crystals when observed at low resolution. In order to tell the difference between true twodimensional crystals from the presence of hexagonally closely packed protein molecules, one can further investigate the
diffraction patterns.
The systematic study and meticulous observation of two-dimensional crystallization process using various kinds of
amphiphilic compounds, including diluting lipids exhibiting a variety of fluidity characteristics, will greatly improve
our understanding of the different steps involved in self-organization of protein interacting with a monolayer template
through adsorption. We have recently recognized the importance of finding a suitable mixture of functional chemical
compounds as an amphiphilic substrate and diluting lipids to provide the optimal fluidity properties for two-dimensional
protein crystallization.
4. Conclusion
Ferritin and catalase molecules were observed in a regularly close-packed arrangement after adsorption to the
amphiphilic C18CD or Chromoionophore I monolayer (template) which had been formed at the air/water interface. In
protein science, surface denaturation is known for many water-soluble proteins, where proteins can be unfolded and
adsorbed on a water surface making insoluble film. In such case the surface pressure increases with the protein
unfolding. Conversely, the surface pressure of ferritin solution was retained at 0 mN/m under our experimental
condition when the water surface was compressed with the barriers without the amphiphilic supporting monolayer (Ishii
and Kobayashi, unpublished observation). Observations with TEM indicate that there were not any broken molecules
which correspond to the direct evidence of unfolded or denatured protein molecules. Therefore, the increase in surface
pressure can be understood as being caused by the adsorption of protein molecules onto the supporting template without
unfolding.
A monomolecular layer of ferritin molecules was formed by adsorption from the subphase onto a Langmuir film
consisting of an amphiphilic β-CD derivative at the air/water interface. The course of the adsorption of ferritin
molecules was monitored, and the resultant two-dimensional crystalline arrays were observed by TEM. The results
show the potential of the amphiphilic β-CD derivative, C18CD, to work as a milder adsorbent for protein molecules to
dictate two-dimensional protein crystalline arrays at the air/water interface.
Next, monolayer formation and behavior on a water surface of Chromoionophore I were studied by analyzing the
surface pressure-molecular area (π-A) isotherms. Chromoionophore I formed a Langmuir film, and the stability of the
monolayer appeared to depend on the subphase pH as well as the degree of protonation in the Chromoionophore I
molecule. Adsorption experiments with ferritin and catalase have demonstrated the utility of Chromoionophore I
monolayer as a potential template although parameters which dictate the suitable conditions have to be further
optimized to obtain a high densely packed two-dimensional protein crystalline arrays.
Thus far, about one thousand different fold have been known on the basis of the stored protein structure elements
(Protein Data Bank (PDB)). And it is estimated that there exist mutually different folds of about a few thousand.
Structural biologists and crystallographers use tools that have the capacity to solve protein structures at the atomic level
e.g. X-ray crystallography of three-dimensional protein crystals and nuclear magnetic resonance (NMR) spectroscopy
for concentrated protein solutions. Thus, most of those structures solved are limited to water-soluble proteins [19].
Parallel to those, the two-dimensional crystallization technique has been successfully employed for a variety of different
proteins. These are mainly water-soluble proteins, but our recent studies in progress have shown that this technique is
also applicable to membrane proteins. Although membrane proteins are widely recognized as target molecules for
innovative drug development leading to pharmaceutical and clinical use, membrane proteins as well as large
proteinaceous macromolecular complexes are mostly excluded from recent “high-through put” approach for protein
structure determination [20, 21]. Two-dimensional membrane protein crystals obtained by this elegant technique on
monolayer substrate will be essentially useful in transmission electron crystallography and further in combination with
X-ray crystallography at high resolutions.
Acknowledgements The author would like to thank Dr. K. Kobayashi for providing the C18CD derivative and suggestions with the
initial stage results during the study and Dr. K.S. Kim for proofreading of the draft stage of the article.
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