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Highly Selective Directed Assembly of Functional Actomyosin on Au Surfaces Pradeep Manandhar,†,‡ Ling Huang,†,‡ Justin R. Grubich,§,⊥ John W. Hutchinson,§,# P. Bryant Chase,§,‡ and Seunghun Hong*,| Physics, MARTECH, Biological Science, and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, and Physics and NANO Systems Institute, Seoul National University, Shilim-Dong, Kwanak-Gu, Seoul, Korea 151-747 Received November 11, 2004. In Final Form: January 30, 2005 The capability of assembling biomotors onto specific locations of solid substrates is a key for development of biomotor-based nanomechanical systems. We developed a method to direct the assembly of the heavy meromyosin fragment from rabbit skeletal muscle myosin onto specific locations of Au substrates utilizing surface molecular patterns. In this strategy, chemically directed patterns of streptavidin were achieved to direct highly specific assembly of biotinylated heavy meromyosin on the substratessa strategy applicable for patterning a variety of biotinylated moleculesswhile BSA was utilized to avoid nonspecific adsorption. In vitro motility assays of filament sliding were used to confirm functionality of assembled actomyosin.
Nanoscale motor proteins, which generate force and motion in biological systems by hydrolyzing adenosine triphosphate (ATP) as a fuel, are drawing increasing attention as a component for nanoscale electromechanical systems (NEMS).1-4 In the case of actomyosin, myosin heads bind to an actin filament after hydrolyzing ATP and undergo a conformational change that generates displacement of the actin filament by 5-10 nm with a force of at least 1 pN.5-7 The small dimensions of these proteins and their high fuel efficiency make them ideal candidates for hybrid nanomechanical systems such as nanoactuators, nanoscale engines, and so forth.4-7 A key technology in building protein motor-based nanomechanical devices is assembling these motor proteins onto specific locations of solid structures while maintaining their biomechanical functions. Previous reports show successful guiding of the motion of actin filaments or microtubules on solid surfaces.8-10 However, it is still very difficult to achieve a very specific nanoscale assembly of motor proteins onto solid substrates, and most of previous successful actin guiding experiments utilized physical guiding mechanisms. Herein, we present a successful * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: 82-2-880-1343. Fax: 82-2-884-3002. † Physics, MARTECH, Florida State University. ‡ Institute of Molecular Biophysics, Florida State University. § Biological Science, Florida State University. | Seoul National University. ⊥ Present Address: The Field Museum of Natural History, Chicago, IL 60605. # Present Address: School of Law, Case Western Reserve University, Cleveland, OH 44106. (1) Soong, R. K.; Bachand, G. D.; Neves, H. P.; Olkhovets, A. G.; Craighead, H. G.; Montemagno, C. D. Science 2000, 290, 1555. (2) Jia, L. L.; Moorjani, S. G.; Jackson, T. N.; Hancock, W. O. Biomed. Microdevices 2004, 6, 67-74. (3) Patolsky, F.; Weizmann, Y.; Willner. I. Nat. Mater. 2004, 3, 692. (4) Hess, H.; Vogel, V. Rev. Mol. Biotechnol. 2001, 82, 67. (5) Vale, R. D.; Milligan, R. A. Science 2000, 288, 88. (6) Veigel, C.; Bartoo, M. L.; White, D. C. S.; Sparrow, J. C.; Molloy, J E. Biophys. J. 1998, 75, 1424. (7) Schliwa, M.; Woehlke, G. Nature 2003, 422, 759. (8) Clemmens, J.; Hess, H.; Howard, J.; Vogel, V. Langmuir 2003, 19, 1738. (9) Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Bo¨hringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Langmuir 2003, 19, 10967. (10) Jaber, J. A.; Chase, P. B.; Schlenoff, J. B. Nano Lett. 2003, 3, 1505.
assembly of actomyosin onto specific locations of Au substrates. Figure 1 shows our strategy to construct patterns of heavy meromyosin (HMM) from rabbit skeletal muscles a soluble, proteolytic digest fragment of myosin that contains two motor domainsson Au substrates utilizing specific streptavidin-biotin interactions. In this process, specific regions of an extremely thin Au surface are first functionalized with carboxyl (-COOH) groups by patterning 16-mercaptohexadecanoic acid (MHA) self-assembled monolayers (SAMs), while the remaining regions are coated with methyl (-CH3)-terminated 1-octadecanethiol (ODT; Figure 1C). It should be noted that 5.6nm-thick Au/Ti films (4.2-nm Au on 1.4-nm Ti) transmit ∼75% of light (Figure 1I), which allows us to use fluorescence microscopy and conventional motion analysis tools. MHA-coated regions are then functionalized with biotin-CGGSGGHHHHHH-OH (biotinylated his-tag; Figure 1D) which localizes streptavidin binding (Figure 1E). In this case, his-tag binds selectively to negatively charged MHA regions. Each streptavidin has four biotin binding sites, which allows the further assembly of biotinylated HMM. Because HMM can also assemble onto hydrophobic surfaces (ODT area), the ODT surface is passivated with bovine serum albumin (BSA; Figure 1F) to block HMM adsorption to these regions. When biotinylated HMM solution is applied to the surface, motor proteins bind to the streptavidin patterns (Figure 1G). Then, the function of the motor proteins is verified by observing transport of actin filaments on HMM patterns (Figure 1H). Figure 2 shows a series of movie frames of a RhPhlabeled actin filament (red line) moving on HMM patterns (green region). The red and green images were recorded using an intensified black-and-white charge-coupled device camera through separate channels of a fluorescence microscope, colorized and merged. The green stripes correspond to the regions of fluorescently labeled streptavidin (with bound biotinylated HMM), while dark areas represent BSA-coated ODT regions. The time series show that actin filaments are actively transported along the HMM region, demonstrating that biotinylated HMM on the surface is functional. Significantly, actin filaments remain in the HMM-coated region when they encounter a boundary. Unlike relatively rigid microtubules that usually cross boundaries and leave such chemically defined
10.1021/la047227i CCC: $30.25 © 2005 American Chemical Society Published on Web 03/17/2005
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Figure 1. (A-H) Schematic diagram depicting the surface-templated assembly process for biotinylated HMM via streptavidinbiotin interactions. (I) Transmission coefficient data of nanometer-thick transparent Au film (4-nm-thick Au on 1-nm-thick Ti) which can be utilized as a substrate for mobility experiments using fluorescence microscopy.
tracks,9 flexible actin filaments can turn back at the boundary (Figure 2C). The motion of actin (by myosin) or microtubules (by kinesin) can be effectively guided by physical barriers, with guidance occurring over a wide range of angles for filaments approaching a boundary.8-10,20,21 Guidance of microtubules within chemically (11) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (12) Xia, Y.; Tien, J.; Qin, D.; Whitesides, G. M. Langmuir 1996, 12, 4033. (13) Kane, R. S.; Takayama, S.; Ostuni, E.; Engber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363. (14) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661. (15) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702. (16) Hong, S.; Mirkin, C. A. Science 2000, 288, 1808. (17) Kron, S. J.; Toyoshima, Y. Y.; Uyeda, T. Q. P.; Spudich, J. A. Methods Enzymol. 1991, 196, 399. (18) Ko¨hler, J.; Chen, Y.; Brenner, B.; Gordon, A. M.; Kraft, T.; Martyn, D. A.; Regnier, M.; Rivera, A.; Wang, C. K.; Chase, P. B. Physiol. Genomics 2003, 14, 117. (19) Kunioka, Y.; Ando, T. J. Biochem. 1996, 119, 1024. (20) Wright, J.; Pham, D.; Mahanivong, C.; Nicolau, D. V.; Kekic, M.; dos Remedios, C. G. Biomed. Microdevices 2002, 4, 205. (21) Bunk, R.; Klinth, J.; Montelius, L.; Nicholls, I. A.; Omling, P.; Tagerud, S.; Månsson. A. Biochem. Biophys. Res. Commun. 2003, 301, 783.
defined patterns of kinesin has been demonstrated to be effective, provided that microtubules approach the boundaries at shallow angles,9 but chemical guidance of actin motility has only been demonstrated previously on poly(methyl methacrylate) tracks with relatively low selectivity.22,23 For NEMS applications, it is critical to assemble motor proteins onto the desired regions of general solid surfaces with high selectivity. We did not observe any motility outside the streptavidin pattern regions, implying highly selective assembly of biotinylated HMM only onto streptavidin regions. We also examined the translocation of RhPh-labeled F-actin by unmodified and biotinylated HMM adsorbed onto various molecular surfaces on Au substrates (Figure 3). Because SAMs on Au surfaces provide versatile molecular interfaces, we can perform systematic studies of this motor function of HMM on various chemical groups.11 Overall, the speed of F-actin translocation by unmodified HMM deposited on positively charged [e.g., cysteamine and 2-mercaptobenzothiazole (MBT)] or neutral (e.g., ODT) (22) Suzuki, H.; Yamada, A.; Oiwa, K.; Nakayama, H.; Mashiko, S. Biophys. J. 1997, 72, 1997. (23) Nicolau, D. V.; Suzuki, H.; Mashiko, S.; Taguchi, T.; Yoshikawa, S. Biophys. J. 1999, 77, 1126.
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Figure 2. Time series of fluorescence micrographs showing motion of RhPh-labeled actin (red line) on a patterned HMM track. The green regions represent 20-µm-wide Alexa Fluor 488-labeled streptavidin patterns with bound biotinylated HMM (HMM track), while the dark area is a BSA-covered ODT region (no HMM).
Figure 3. Motility of unmodified HMM adsorbed onto various SAM patterns on Au and conventional surfaces (nitrocellulose). The terminal chemical groups of each molecular species are indicated in parentheses.
surfaces was comparable to that for biotinylated HMM adsorbed to streptavidin-coated surfaces (Figures 2 and 3), although some (particularly imidazole) were slower than HMM adsorbed to commonly used nitrocellulose on glass (Figure 3).18 However, HMM adsorbed to a negatively charged MHA surface does not support motility (Figure 3). These results confirm that surface properties can modulate the function of adsorbed proteins. In addition, it should be noted that HMM adsorbed and supported motility of actin filaments on hydrophobic surfaces (direct binding of HMM to ODT surface in Figure 3), presumably due to hydrophobic interactions.23 More detailed analysis regarding the density of bound HMM on various surfaces is yet to be done in future experiments, such as K+ethylenediaminetetraacetic acid ATPase activity assays or high resolution imaging. However, we did not observe significant differences in the number densities of actin filaments bound to HMM on various surfaces even with different motility results, indirectly indicating that there were no drastic variations in HMM densities on various surfaces.
Until now, most motility experiments have been limited to transparent surfaces such as glass because motion tracking typically relies on optical (fluorescence) microscopy. As far as the authors know, this is the first successful study of biological motor protein-based motility on metallic and electrically conductive surfaces, which opens new opportunities for detection and control. In our strategy, nanometer-thick Au films allow us to use conventional microscopy methods for motility experiments overcoming the surface limitation. The same strategy can be possibly applied to other surfaces. Even though we can achieve highly selective assembly of biotinylated HMM utilizing streptavidin-biotin biomolecular interactions, we also tried to selectively assemble unmodified HMM directly onto the molecular patterns with specific chemical groups. However, we found that unmodified HMM binds to most of the chemical groups tested with little selectivity and, therefore, we could not achieve the same degree of patterning of unmodified as with biotinylated HMM. Similar nonspecific adsorption has been reported in other protein motor assembly research.21,23 This result implies that, due to the versatile chemical groups on motor proteins, it is not always appropriate to rely on chemical functional groups alone to achieve specific localization of HMM. In addition to patterning HMM, we achieved a limited success in patterning actin filaments using chemical patterns. However, adsorbed actin was often fragmented and its function remains to be tested. In conclusion, we have developed a method to assemble functional motor proteins onto desired locations on Au substrate with high selectivity. For future NEMS applications one has to be able to assemble motors onto various transparent and nontransparent surfaces. We achieved highly specific assembly of protein motors utilizing streptavidin-biotin interactions. In addition, utilizing extremely thin Au films, we can still apply conventional motility analysis protocols to confirm the motility of assembled biomotor patterns. As far as the authors know, this is the first successful demonstration of highly selective assembly of functional actomyosin on metallic substrates. The results can be a key for the future development of biomotor-based NEMS applications.
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Experimental Section Au Film Preparation. Thin Au films (4.2-nm thick) were prepared with Ti (1.4-nm thick) as an adhesion layer on microscope coverslips (130-µm thick, Fisher Scientific) utilizing thermal evaporation (base pressure of 2 × 10-7 Torr). The Ti adhesion layer allows one to deposit stable Au layers on solid surfaces. The thickness of Au films was measured using a quartz crystal film thickness monitor. SAM Patterning. Various SAM molecules such as ODT, imidazole, cysteamine, MHA, and MBT were purchased from Sigma and tested for actomyosin adsorption. We utilized microcontact printing and dip-pen nanolithography (DPN) to pattern Au surfaces with organic molecules. Microcontact printing utilizes poly(dimethylsiloxane) stamps to deposit organic molecules onto specific regions of the surfaces.12,13 In the stamping process, we first applied the solution of organic molecules (∼5 mM in ethanol) on a stamp and then pressed the stamp onto the substrate for ∼8 s. DPN utilizes a moleculecoated atomic force microscope (AFM) tip to generate nanoscale organic molecular patterns.14-16 The DPN process was performed using a CP Research AFM system (Digital Instruments) that is equipped with lithography software (NanoInk, Inc.) and a closedloop scanner. A typical procedure to pattern organic molecules via DPN is first dipping an AFM tip (Si3N4 tip) in the solution of ink molecules (∼3 mM in ethanol) for 5 min and then sweeping the tip in contact with substrates (contact force ∼ 1 nN). After patterning the first molecular species via stamping or the DPN method, the remaining area is often backfilled with the second molecular species by dipping the patterned surface in the solution of the second molecular ink (∼3 mM in ethanol) for 5 min. Streptavidin Patterning. MHA patterns were functionalized with streptavidin for specific assembly of motor proteins. First, the MHA patterns were placed in a solution of biotinylated histag (0.05 mg/mL in deionized water, Biosynthesis, Inc.) and then rinsed with AB buffer (25 mM imidazole-HCl, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM ethylene glycol bis(2-aminoethyl ether)N,N,N′,N′-tetraacetic acid in deionized water) to remove excess biotinylated his-tag. His-tag binds to the carboxyl groups of MHA molecules and covers the MHA patterns with biotin for streptavidin assembly. Then, the pattern was treated with 0.25 mg/mL streptavidin prepared in phosphate buffer (Sigma). A fluorescently labeled streptavidin (Alexa Fluor 488 conjugate, Molecular Probes) was used in these studies to confirm localization by fluorescence microscopy (Figure 2), although the fluorescent label is not an essential aspect of the method. Streptavidin provides selective and high-affinity binding sites for biotinylated HMM or other biotinylated molecules. The ODT surface was passivated by 0.5 mg/mL BSA (Sigma) prepared in AB buffer to prevent nonspecific adsorption of HMM.
Letters Assembly of HMM and Motility Analysis. Unmodified or biotinylated HMM was assembled onto SAM or streptavidin patterns, respectively. The bioactivities of assembled actomyosin systems were confirmed by in vitro motility analysis in flow cells as previously described.17,18 HMM17,18 and biotinylated HMM19 were prepared according to earlier-reported protocols. When the HMM solution was infused into flow cells containing SAM or streptavidin patterns, unmodified HMM was adsorbed onto SAM surfaces via noncovalent interactions, while biotinylated HMM bound to streptavidin to give a very specific and selective region of HMM. After rhodamine-phalloidin (RhPh; Molecular Probes)labeled actin was added to the flow cell, motility buffer (AB plus 40 mM dithiothreitol, 1400 units/mL catalase, 3.75 mg/mL D-glucose, 35 units/mL glucose oxidase, 2 mM ATP, 0.3% methylcellulose) was infused into the flow cell to initiate motility. This flow cell was imaged by fluorescence microscopy at 30 °C; the temperature was maintained by circulating water from a temperature-controlled water bath through a copper coil wrapped around the objective.18 The motion of actin filaments was captured using a Diastar upright fluorescence microscope (Leica) with a silicon-intensified target (SIT) camera (model VE 1000, DageMTI) and was recorded on videocassettes with an added timedate generator signal (model WJ-810, Panasonic). The images were then analyzed using custom motion analysis software (written by Thomas Asbury at Florida State University).
Acknowledgment. We thank Victor Miller, Shanedah Williams, and Lori McFadden in Department of Biological Science, Florida State University, for their help in preparing proteins. We gratefully acknowledge support from the NSF (NIRT ECS-0210332) and from the Defense Advanced Research Projects Agency (DARPA) Defense Sciences Office (DSO) under the auspices of Drs. Alan Rudolph and Anatha Krishnan through the Space and Naval Warfare Systems Center (SPAWAR), San Diego, Contract No. N66001-02-C-8030. S.H. acknowledges the financial support from the NANO-Systems InstituteNational Core Research Center (NSI-NCRC) program of the Korea Science and Engineering Foundation (KOSEF) and from the BK21 project at Seoul National University. Supporting Information Available: Fluorescence microscope movie showing the actin filament moving on myosin patterns. This material is available free of charge via the Internet at http://pubs.acs.org. LA047227I
