Two-dimensional crystalline array formations of proteins by use of the self-assembled monolayer at the air/water interface

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1 Two-dimensional crystalline array formations of proteins by use of the self-assembled monolayer at the air/water interface Noriyuki Ishii 1,2 1 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central-6, Higashi, Tsukuba, Ibaraki , Japan 2 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Naka-cho, Koganei, Tokyo , 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 mol/l. Chloroform (spectroscopy grade) was purchased from the Kishida Chemical Co., Ltd. (Osaka, Japan). Chromoionophore I (ETH 5294: [9-(diethylamino)-5- octadecanoilimino-5h-benzo[a]phenoxazine], molecular wt ) was purchased from Fulka (Buchs, Switzerland). Chromoionophore I was dissolved in chloroform solution at a concentration of mol/l. 929

2 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 nm 2 /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 cm 2 ) both with a Wilhelmy type microbalance using a platinum plate. The CD derivative, C18CD was dissolved in chloroform solution with a concentration of 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 nm 2 /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-H 2 SO 4, 10 mm NaCl, 10 mm CdSO 4, 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,

3 a) b) 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. 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 nm 2 /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 nm 2. For reference, the area of the secondary face of native β-cd is 1.86 nm 2 and the calculated area per molecule of native β-cd in the hexagonal closest packed structure is 2.10 nm 2 [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. 931

4 3.2 Two-dimensional array formation of ferritin by use of Chromoionophore I 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 nm 2 /molecule s. (c) 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 nm 2 /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. 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 nm 2 /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 nm 2 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 π- electrons, 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. 932

5 Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.) 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. 933

6 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. 934

7 References [1] Dietrich J, Vénien-Bryan C. Strategies for two-dimensional crystallization of proteins using lipid monolayers. London: Imperial College Press; [2] Fromherz P. Electron microscopic studies of lipid protein films. Nature. 1971; 231: [3] Uzgiris EE, Kornberg RD. Two-dimensional crystallization technique for imaging macromolecules, with application to antigenantibody-complement complexes. Nature. 1983; 301: [4] Furuno T, Sasabe H. Two-dimensional crystallization of streptavidin by nonspecific binding to a surface film: study with a scanning electron microscope. Biophysical Journal. 1993; 65: [5] Fujiyoshi Y. The structural study of membrane proteins by electron crystallography. Advances in Biophysics. 1998; 35: [6] Glaeser RM. Limitation to significant information in biological electron microscopy as a result of radiation damage. Journal of Ultrastructure Research. 1971; 36: [7] Bender ML, Komiyama M. Cyclodextrin Chemistry. New York: Springer-Verlag; [8] Kobayashi K, Kajikawa K, Sasabe H, Knoll W. Monomolecular layer formation of amphiphilic cyclodextrin derivatives at the air/water interface. Thin Solid Films. 1999; 349: [9] Wang K, Seiler K, Morf WE, Spichiger UE, Simon W, Lindner E, Pungor E. Characterization of potassium-selective optode membranes based on neutral ionophores and application in human blood plasma. Analytical Science. 1990; 6: [10] Jose J, Burgess K. Benzophenoxazine-based fluorescent dyes for labeling biomolecules. Tetrahedron. 2006; 62: [11] Kobayashi K, Ishii N, Sasabe H, Knoll W. Monomolecular layer formation of ferritin molecules on amphiphilic cyclodextrin derivative at the air/water interface. Bioscience, Biotechnology, and Biochemistry. 2001; 65: [12] Ling C-C, Darcy R. 6-S-hydroxyethylated 6-thiocyclodextrins: expandable host molecules. Journal of the Chemical Society, Chemical Communications. 1993; 1993: [13] Kawabata Y. Development of materials for molecular organizates. 7th Symposium on Future Electron Devices p [14] Jabbari A, Sadeghian H. Amphiphilic cyclodextrins, synthesis, utilities and application of molecular modeling in their design. In: Sezer AD, editor. Recent advances in novel drug carrier systems. Croatia: InTech; p [15] Petty MC. Langmuir-Blodgett films: An introduction. Cambridge: Cambridge University Press; [16] Ishii N. Monomolecular layer formation of amphiphilic Chromoionophore I at the air/water interface. Transaction of the Materials Research Society of Japan. 2009; 34:1-8. [17] Darst SA, Ahlers M, Meller PH, Kubalek EW, Blankenburg R, Ribi HO, Ringsdorf H, Kornberg RD. Two-dimensional crystals of streptavidin on biotinylated lipid layers and their interactions with biotinylated macromolecules. Biophysical Journal. 1991; 59: [18] Mosser G, Brisson A. Condition of two-dimensional crystallization of cholera toxin B-subunit on lipid films containing ganglioside GM1. Journal of Structural Biology. 1991; 106: [19] Ishii N. Crystallization, structure and functional robustness of isocitrate dehydrogenases. In: Chandrasekaran A, editor. Current Trends in X-Ray Crystallography. Croatia: InTech; p [20] Ishii N. Observation by transmission electron microscopy of organic nano-tubular architectures. In: Méndez-Vilas A, editor. Current Microscopy Contributions to Advances in Science and Technology. Spain: Formatex; p [21] Ishii N. Image analyses of two-dimensional crystalline arrays of membrane proteins and protein supramolecular complexes. In: Echon RM, editor. Advances in Image Analysis Research. New York, USA: Nova Science Publishers, Inc.; p