CRYSTAL GROWTH & DESIGN
Epitaxial Growth of Lysozyme on Fatty Acid Thin Films
2007 VOL. 7, NO. 2 416-419
T. Kubo, H. Hondoh, and T. Nakada* Department of Physical Sciences, Faculty of Science and Engineering, Ritsumeikan UniVersity, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan ReceiVed April 22, 2006; ReVised Manuscript ReceiVed October 24, 2006
ABSTRACT: Orientations of hen egg white lysozyme crystals nucleated on fatty acid thin films were investigated by atomic force microscopy and optical microscopy. It is found that lysozyme crystals with their {110} planes parallel to fatty acid thin films formed on mica substrates, tend to arrange along the three directions coinciding with the 6-fold symmetry of the mica surface. In addition, it was clearly shown that the length of the fatty acid molecules is crucial for inducing the epitaxial growth of lysozyme crystals. The mechanism to induce this epitaxial growth on fatty acid films is discussed. Introduction For decades, hetero-epitaxial growth of organic materials has been paid much attention because of its importance and unique characteristics.1-5 For example, lattice mismatch is often insignificant in the epitaxial growth of organic materials, yet for inorganic materials, this mismatch is crucial. However, irrespective of the numerous epitaxial studies on chain and planar organic molecules, very few epitaxial growth studies of complex, three-dimensional molecules, such as proteins, have been attempted. In fact, to the best of our knowledge, since McPherson et al. first published their paper in 1988,6 all attempts to control the orientation of protein crystals on inorganic substrates have proven unsuccessful. On the other hand, it was recently reported that oriented lysozyme crystals can be nucleated on organic thin films.7-10 These results suggest that the epitaxial growth of protein crystals is possible if appropriate organic substrates are employed. The elucidation of the mechanism of epitaxial growth of protein crystals could yield the further understanding of epitaxial growth of other organic crystals. Here, we report the first demonstration of epitaxial growth of protein crystals on fatty acid thin film substrates. Due to its commercial availability as a high-purity sample, hen egg white lysozyme (HEWL) is used as a model protein. We used fatty acids because it is easy to fabricate the well-ordered crystalline films by physical vapor deposition. The influence of fatty acid carbon chain lengths on the epitaxial growth of lysozyme is also presented. Experimental Procedures Myristic acid (CH3(CH2)12COOH), stearic acid (CH3(CH2)16COOH), and behenic acid (CH3(CH2)20COOH) samples (99% purity, Nacalai Tesque) and cerotic acid (CH3(CH2)24COOH) samples (98% purity, ACROS) were evaporated on the surfaces of air-cleaved mica substrates under high vacuum (10-5 Torr). The deposition conditions for preparing fatty acid thin films are summarized in Table 1. The structures and coverage of the as-formed fatty acid films were determined by atomic force microscopy (AFM; Nanoscope E, Digital Instruments Inc.), using microfabricated triangular Si3N4 cantilevers (spring constant 0.12 N/m) for observation. The structures of fatty acid thin films fabricated by physical vapor deposition are strongly influenced by the deposition conditions, such as deposition rate, substrate temperature, and substrate materials.11-13 In this study, monolayer fatty acid film substrates prepared using * Corresponding author. Tel: +81-77-561-3974. Fax: +81-77-561-3994. E-mail:
[email protected].
Table 1. Deposition Conditions for Preparing Fatty Acid Thin Filmsa
a
substance
furnace temperature (°C)
deposition time (s)
myristic acid stearic acid behenic acid cerotic acid
61 67 84 83
85 70 88 107
Substrate temperature was fixed at room temperature.
previous methods9,10 exhibit nearly uniform coverage (behenic acid and cerotic acid, 81% ( 4%; myristic acid and stearic acid, ∼100%) and are used here in order to eliminate the influence of substrate structural changes on nucleated lysozyme crystals. Tetragonal lysozyme crystals were prepared using a conventional batch technique with crystallization conditions as described elsewhere.9 The crystal orientation of hen egg white lysozyme crystals (Seikagaku Kogyo, Co. Ltd.) is readily determined from the corresponding optical micrographs. Hereafter, for brevity, crystals with {hkl} faces parallel to the substrate are described as “{hkl}-oriented crystals”. In this study, the observed aggregates and irregular-shaped (e.g., spherulitic) crystals were ignored, and only the faceted lysozyme crystals nucleated on the substrate were considered.
Results and Discussion Figure 1 shows an optical micrograph of lysozyme crystals nucleated on a stearic acid monolayer film. Here, the {110}oriented lysozyme crystals are preferentially nucleated on the film in the same manner as those formed on behenic acid films,9,10 which is in stark contrast to those on bare mica, where no nucleation was observed. As clearly seen in this figure, the c-axes of the {110}-oriented crystals tend to arrange along the three directions associated with the (001) plane of mica. This result implies that the epitaxial growth of lysozyme is induced by stearic acid molecules adsorbed on mica. Moreover, this same epitaxial growth is also observed on fatty acid monolayer films prepared using molecules with different carbon chain lengths. The total number and the number of oriented crystals nucleated on four kinds of fatty acids films are shown in Figure 2. The total numbers are almost constant on any fatty acid thin films. Moreover, the number of {110}-oriented crystals nucleated on stearic acid, behenic acid, and cerotic acid are almost the same. In contrast, the number of {110}-oriented crystals nucleated on myristic acid monolayer films is much smaller than that nucleated on stearic acid, behenic acid, and cerotic acid. Figure 3 shows the number ratio of the crystals nucleated on fatty acid films as a function of the angle between the c-axis of the {110}oriented crystals and the b-axis of mica. The three peaks clearly
10.1021/cg060239c CCC: $37.00 © 2007 American Chemical Society Published on Web 01/12/2007
Epitaxial Growth of Lysozyme
Crystal Growth & Design, Vol. 7, No. 2, 2007 417
Figure 1. Optical micrograph of lysozyme crystals nucleated on a stearic acid monolayer film. The arrow indicates the direction of the b-axis of mica. The three directions indicating the c-axes of the {110}oriented lysozyme crystals are represented by lines, dotted lines, and broken lines.
Figure 2. Dependence of the number of lysozyme crystals nucleated on fatty acid thin films on the length of carbon chains of fatty acids. The numbers in parentheses stand for the carbon numbers of fatty acid molecules.
Figure 3. Relationship between the angle of the c-axis of {110}oriented lysozyme crystals relative to the b-axis of the mica substrate, and the ratio of the number of {110}-oriented lysozyme crystals nucleated on (a) myristic acid, (b) stearic acid, (c) behenic acid, and (d) cerotic acid monolayer films.
observed are present at 0° and (60° relative to the b-axis of mica except for the case of myristic acid. The three directions coincide with the 6-fold symmetry of the cleaved mica surface, which is thought to be composed of (SiO4) tetrahedrons. In order to clarify the orientations of the lysozyme crystals in detail, the autocorrelation functions of these histograms were calculated, as shown in Figure 4. There is no clear peak for myristic acid (2.1 nm in length). The difference between the maximums and minimums for stearic acid (molecular length is about 2.6 nm) is slightly larger than that for behenic acid (3.1 nm) and cerotic acid (3.6 nm), suggesting that the induced epitaxial growth of lysozyme is dependent on the molecular length of the fatty acid. Except for the case of myristic acid, the nucleation rate of the {110}-oriented lysozyme crystals did not change significantly, but decreased slightly with respect to the length of the fatty acid molecules in the film. As such, the mean densities of the {110}-oriented lysozyme crystals on stearic acid, behenic acid, and cerotic acid thin films were 5.1 ( 0.6, 4.1 ( 0.4, and 3.4 ( 0.5 mm-2, respectively. On
myristic acid films, the density of the {110}-oriented crystals was significantly smaller (1.6 ( 1.1 mm-2), suggesting a lower limit for the length of the fatty acid molecules capable of promoting nucleation and inducing epitaxial growth of {110}oriented lysozyme crystals. It is worth noting that these results resemble the nucleation of alcohol crystals on fatty acid thin films; alcohol crystallization was only promoted when the length of the fatty acid molecules used was the same as or slightly longer than that of the alcohols studied.13 In contrast, the densities of the {101}-oriented and crystals were independent of the lengths of the molecules in the film. Furthermore, we found no detectable difference in the morphology and crystal structure of the {110}-oriented lysozyme crystals nucleated on one or all of the monolayer fatty acid thin films used. As mentioned in our previous work,9,10 the carbon chain axes of the behenic acid molecules in the corresponding thin film are nearly perpendicular to the mica surface in air (Figure 5a). However, in aqueous solution, the structure of these films is noticeably different. In order to determine the structure of the
418 Crystal Growth & Design, Vol. 7, No. 2, 2007
Figure 4. Normalized autocorrelation function, as a function of the fatty acid carbon number. Symbols (2), (9), (O), and (]) indicate the myristic acid, stearic acid, behenic acid, and cerotic acid monolayer films, respectively.
Figure 5. A model for the structure of fatty acid monolayer films in (a) air and (b) solution. Circles and lines in the schematic drawings indicate hydrophilic carboxylic groups and hydrophobic carbon chains of a behenic acid molecule, respectively.
behenic acid films in solution, we performed in situ AFM observations of mica substrates partially covered with behenic acid monolayers (∼70%). Here, it was observed that the fatty acid surface in air shows a continuous layer, albeit with a number of pits (of monolayer thickness). In solution, there exists a relatively large flat area, which is not bare mica substrate and small amounts of islands. Therefore, we conclude that the behenic acid film in solution has an entirely different structure from that formed in air. As is well-known, fatty acid thin films fabricated by physical vapor deposition often show two types of molecular orientation, normal growth films in which the carbon chain axes of the molecules are perpendicular to the substrate and lateral growth films in which the chain axes are parallel to the substrate.11 The islands in solution are comparable to the lengths of two behenic acid molecules. This indicates that the structure of the islands is almost identical to the structure of normal growth films. Meanwhile, it was revealed that the {110}-oriented crystals were not nucleated on the normal growth films.9 Thus it is most likely that the lysozyme crystals epitaxially nucleate and grow on the flat area. Interestingly, the behenic film on flat area in solution has a similar structure to a normal growth film, which is a little odd when one considers that the pits are expected to be larger due to the films being composed of dimers. Moreover, in solution, it is likely that the hydrophilic carboxyl groups of behenic acid interact with both the surrounding water molecules and the hydrophilic mica surface. We previously reported that behenic acid monolayers and sodium acetate synergistically promote the nucleation of {110}-oriented lysozyme crystals, where both behenic acid and acetic acid have carboxyl groups.10 Based on the above
Kubo et al.
Figure 6. A model for the arrangement of lysozyme molecules on fatty acid thin films. As shown in Figure 5, circles and lines in the schematic drawings indicate hydrophilic carboxylic groups and hydrophobic carbon chains of a behenic acid molecule, respectively. A single molecule of lysozyme is enclosed by a circle.
observations, it is likely that well-ordered fatty acid molecules adsorb parallel to the mica substrate (Figure 5b) and have molecular lengths very close to that of lysozyme (about 3 nm); namely, periodically arranged carboxyl groups accelerate nucleation and promote the epitaxial growth of {110}-oriented lysozyme crystals (Figure 6). As one can see in Figure 3, the directions of c-axes of the lysozyme crystals are not completely consistent with the b-axis of mica. This implies that the periodicities of the carbon chains fluctuate along the 6-fold symmetry of the mica surface. It is probably because the interaction between the carbon chains of fatty acids and the mica surface is relatively weak, because the chains are hydrophobic and the mica surface is hydrophilic. It should also be noted that the nucleation rate decreased slightly with respect to the length of the molecules (stearic acid, behenic acid, and cerotic acid) used in the fatty acid monolayer films. The mean density ratios of the {110}-oriented lysozyme crystals formed on behenic acid (1.2) and stearic acid (1.5) thin films are slightly larger with respect to those formed on cerotic acid thin films. When the carbon chain axes of the fatty acids in solution are parallel to the mica surface, the area density ratio of the carboxyl groups on behenic acid (1.2) and stearic acid (1.4) are also slightly larger with respect to the cerotic acid thin films. Although there are large standard deviations in the mean densities of the {110}oriented crystals nucleated on these fatty acid films, their tendency is quite similar. So far, conclusive evidence to corroborate these results has been hampered by the difficulty in observing this phenomenon in situ at the molecular level; however, we believe the observed similarity supports our hypothesis. Conclusion We successfully demonstrated the epitaxial growth of lysozyme crystals on fatty acid monolayer films. The c-axes of the {110}oriented lysozyme crystals nucleated on stearic acid, behenic acid and cerotic acid thin films are oriented with respect to the three directions corresponding to the 6-fold symmetry of the mica surface. On the other hand, almost no {110}-oriented lysozyme crystals were observed on the monolayer film formed from myristic acid. These results indicate that monolayer films of fatty acid molecules with an appropriate carbon chain length
Epitaxial Growth of Lysozyme
promote the epitaxial growth of lysozyme. In addition, a similar relationship between the mean densities of the {110}-oriented crystals and the carboxyl groups of the fatty acid film substrates was observed. These results suggest that the carboxyl groups of the fatty acids promote the nucleation and epitaxial growth of {110}-oriented lysozyme crystals. Acknowledgment. This work was partially supported by a Grant-in-Aid (No. 17560016) from the Scientific Research of the Ministry of Education, Science and Culture of Japan. References (1) Mauritz, K. A.; Bear, E.; Hopfinger, A. J. J. Polym. Sci., Polym. Phys. Ed. 1973, 11, 2185. (2) Irie, S.; Isoda, S.; Kuwamoto, K.; Miles, M. J.; Kobayashi T.;
Crystal Growth & Design, Vol. 7, No. 2, 2007 419 Yamashita, Y. J. Cryst. Growth 1999, 198/199, 939. (3) Schmitz-Hu¨bsch, T.; Sellam, F.; Staub, R.; To¨rker, M.; Fritz, T. Ku¨bel, Ch.; Mu¨llen, K.; Leo, K. Surf. Sci. 2000, 445, 358. (4) Mannsfeld, S. C. B.; Fritz, T. Phys. ReV. B 2004, 69, 075416. (5) Mannsfeld, S. C. B.; Leo, K.; Fritz, T. Phys. ReV. Lett. 2005, 94, 056104. (6) McPherson, A.; Shlichta, P. Science 1988, 230, 385. (7) Tsekova, D.; Dinitova, S.; Nanev, C. N. J. Cryst. Growth 1999, 196, 226. (8) Rong, L.; Komatsu, H.; Yoda, S. J. Cryst. Growth 2002, 235, 489. (9) Kubo, T.; Uchiyama, Y.; Mizushima, T.; Hondoh, H.; Nakada, T. J. Cryst. Growth 2004, 269, 535. (10) Kubo, T.; Uchiyama, Y.; Mizushima, T.; Hondoh, H.; Nakada, T. J. Cryst. Growth 2005, 275, e1431. (11) Matsuzaki, F.; Inaoka, K.; Okada, M.; Sato, K. J. Cryst. Growth 1984, 69, 231. (12) Takiguchi, H. Doctoral Thesis, Hiroshima University, 1998. (13) Takiguchi, H.; Iida, K.; Ueno, S.; Yano, J.; Sato, K. J. Cryst. Growth 1998, 193, 641.
CG060239C
