pubs.acs.org/Langmuir © 2009 American Chemical Society
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Float and Compress: Honeycomb-like Array of a Highly Stable Protein Scaffold Arnon Heyman,†, Izhar Medalsy,‡, Or Dgany,† Danny Porath,‡ Gil Markovich,*,§ and Oded Shoseyov*,† †
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The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture and the Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel, ‡Physical Chemistry Department and Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem, Israel and §School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel. These authors contributed equally to this work. Received December 16, 2008. Revised Manuscript Received February 5, 2009
Organizing nano-objects, proteins in particular, on surfaces is one of the primary goals of bio/chemical nanotechnology. A highly stable protein scaffold (6His-SP1) was organized into a hexagonal 2D array by a new, versatile method. The protein was expelled from solution into the air/water interface and compressed in a Langmuir trough into a closely packed monolayer without the use of phospholipids or other surfactants at the interface. The 2D arrays formed at the air/water interface were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM).
Introduction Periodic, tight organization of nanoscale objects on surfaces is one of the main goals in nanotechnology. A promising step toward this goal is the exploitation of the vast potential of proteins as chemically sophisticated nanoscale building blocks for the formation of various types of ordered scaffolds and more complex nanostructures. The potential applications of ordered protein arrays range from nanoelectronic devices such as memory arrays or chemical sensors to fundamental studies of novel electronic/optical properties.1,2 Another important research direction for which surface patterning by proteins is of vast importance is nanolithography. As conventional photolithography techniques are being pushed to their limits, new approaches to fabricating periodically ordered nanostructures are beginning to emerge, such as the use of block copolymers, nanoparticle-based self-assembled monolayers, and micelles.3-6 At the same time, the self-assembly of biomolecules on surfaces is also being intensively studied.7,8 Nature’s ability to create ordered nanostructures can be exploited to create predesigned surface coverage (e.g., by the use of DNA molecules for the deposition of nanoparticles9 or *Corresponding authors. E-mail:
[email protected] and shoseyov @agri.huji.ac.il. (1) Markovich, G.; Collier, C. P.; Henrichs, S. E.; Remacle, F.; Levine, R. D.; Heath, J. R. Acc. Chem. Res. 1999, 32, 415–423. (2) Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Annu. Rev. Mater. Sci. 2000, 30, 545–610. (3) Guarini, K. W.; Black, C. T.; Zhang, Y.; Kim, H.; Sikorski, E. M.; Babich, I. V. J. Vac. Sci. Technol., B 2002, 20, 2788–2792. (4) Fu, J.; Feng, X.; Han, Y.; Pan, C.; Yang, Y.; Li, B. Macromol. Rapid Commun. 2003, 24, 487–491. (5) Lazzari, M.; Lopez-Quintela, M. A. Adv. Mater. 2003, 15, 1583–1594. (6) Boyen, H. G.; Kastle, G.; Zurn, K.; Herzog, T.; Weigl, F.; Ziemann, P.; Mayer, O.; Jerome, C.; Moller, M.; Spatz, J. P.; Garnier, M. G.; Oelhafen, P. Adv. Funct. Mater. 2003, 13, 359–364. (7) McMillan, R. A.; Paavola, C. D.; Howard, J.; Chan, S. L.; Zaluzec, N. J.; Trent, J. D. Nat. Mater. 2002, 1, 247–252. (8) Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H. Science. 2003, 301, 1882–1884. (9) Pinto, Y. Y.; Le, J. D.; Seeman, N. C.; Musier-Forsyth, K.; Taton, T. A.; Kiehl, R. A. Nano Lett. 2005, 5, 2399–2402.
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DOI: 10.1021/la804132z
by proteins that spontaneously self-assemble to form large 2D monolayers and offer an attractive alternative approach, such as s-layer10,11 and purple membrane proteins12). Well-ordered protein arrays enable the formation of much more accurate and long-range periodicity than most of the aforementioned conventional methods. Moreover, proteins can be engineered to display functional domains or amino acids for the targeted assembly of nanoscale structures7 and for surface patterning that could be further utilized to alter the surface’s chemical and physical properties. We have recently utilized a stable protein named SP113-15 to display different functional domains,16,17 bind nanoparticles, and spontaneously assemble in 2D arrays.18 The latter array assembly was achieved when the protein was trapped in a phospholipid/water interface. This led to the adsorption of phospholipids on the protein or between the surface and the protein. The aforementioned phospholipids technique has limited practical future applicability because it disrupts the direct connection of the protein to the surface. (10) Sleytr, U. B.; Sara, M.; Pum, D.; Schuster, B. Prog. Surf. Sci. 2001, 68, 231–278. (11) Sleytr, U. B.Sara, M.Pum, D.Crystalline Bacterial Cell Surface Layers (S-Layers): a Versatile Self-Assembly System. In Supramolecular Polymers; Ciferri, A., Ed.; Marcel Dekker: New York, 2000, pp 177-213. (12) Liang, H.; Whited, G.; Nguyen, C.; Stucky, G. D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8212–8217. (13) Wang, W. X.; Pelah, D.; Alergand, T.; Shoseyov, O.; Altman, A. Plant Physiol. 2002, 130, 865–875. (14) Dgany, O.; Gonzalez, A.; Sofer, O.; Wang, W.; Zolotnitsky, G.; Wolf, A.; Shoham, Y.; Altman, A.; Wolf, S. G.; Shoseyov, O.; Almog, O. J. Biol. Chem. 2004, 279, 51516–51523. (15) Wang, W.; Dgany, O.; Wolf, G. S.; Levy, I.; Algom, R.; Pouny, Y.; Wolf, A.; Marton, I.; Altman, A.; Shoseyov, O. Biotechnol. Bioeng. 2006, 5, 161–168. (16) Heyman, A.; Levy, I.; Altman, A.; Shoseyov, O. Nano Lett. 2007, 7, 1575–1579. (17) Heyman, A.; Barak, Y; Caspi, J.; Wilson, D. B.; Altman, A.; Bayer, E. A.; Shoseyov, O. J. Biotechnol. 2007, 131, 433–439. (18) Medalsy, I.; Dgany, O.; Sowwan, M.; Cohen, H.; Yukashevska, A.; Wolf, S. G.; Wolf, A.; Koster, A.; Almog, O.; Marton, I.; Pouny, Y.; Altman, A.; Shoseyov, O.; Porath, D. Nano Lett. 2008, 8, 473–477.
Published on Web 3/4/2009
Langmuir 2009, 25(9), 5226–5229
Heyman et al.
Article
In the present work, we combine two methodologies in order to better control the 2D assembly of soluble proteins with nanometric precision. The first method was developed in the mid 1990s by Yoshimura and co-workers19 and involves the injection of the protein into a buffer solution with high glucose and cadmium ion concentrations to expel the protein molecules from the solution into the air/water interface. Using this method, the protein molecules form a film at the air/water interface. The second method is the Langmuir-Blodgett technique,20,21 which allows better control of the closely packed monolayer’s formation through monitoring the surface tension in real time and compressing the protein layer into the desired array. This technique, aided by the high stability of the SP1 protein, uniquely allowed us to obtain an organized thin film of the intact protein at the air-water interface without the need to use an auxiliary amphiphile monolayer. Such lipid monolayers that have been often used to capture biomolecules at the air-water interface22 may interfere with some of the potential applications of these films. Houmadi and co-workers have recently used the Langmuir-Blodgett technique to study hydrophobin layers created at the air-water interface.23 We demonstrate a protein film with a 2D arrangement using a polyhistidine-tagged SP1 (named 6His-SP1), where the highly stable 6His-SP1 protein is originally water-soluble until the subphase conditions are changed to expel the protein into the air-water interface. This approach led to a highly ordered protein film.
Experimental Section Protein Expression and Purification. 6His-SP1 was expressed and purified as previously described.18 Briefly, E. coli BL21 (DE3) bacteria were used for expression at 37 °C with 0.1 mM IPTG added to the media at 0.8 O.D. Cells were finally sonicated, and the protein was purified from the bacterial soluble fraction using HIS-select nickel affinity gel (Sigma product no. P6611). Langmuir-Blodgett Setup. The 6His-SP1 protein was added to the subphase solution containing 10 mM MES at pH 6.5, 150 mM NaCl, and 10 mM CdSO4, followed by 5 min of incubation. Then, glucose solution was added to a final concentration of 2% (w/v), followed by another 5 min of incubation. Trough barriers compressed the film at 10 cm2/min. Transmission Electron Microscopy (TEM) Studies. Protein was sampled from the solution surface using glow-discharged, carbon-coated copper 400-mesh grids and was stained with 2% (w/v) uranyl acetate. The images were visualized on an FEI Tecnai-12 microscope. Images were recorded with a SIS Megaview III camera (Electron Microscopy Unit, Weizmann Institute of Science, Rehovot, Israel). AFM Imaging. A Dulcinea AFM system (NanoTec Electronica, Madrid, Spain) was used under ambient conditions, with Multi75B soft tapping mode AFM tips (Budget Sensor, Sofia, Bulgaria), with a nominal spring constant of ∼3 N/m, resonance frequency of ∼65 KHz, and tip apex radius
