pubs.acs.org/Langmuir © 2009 American Chemical Society
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Self-Assembly of F0F1-ATPase Motors and Ghost Ning Tao,†, Jie Cheng,†,‡, Ling Wei,§ and Jiachang Yue*,† †
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The National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, ‡Graduate School of the Chinese Academy of Sciences, Beijing 100101, China, and §College of Life Science, South China Normal University, Guangzhou 510631, China. These authors contributed equally to this work Received December 11, 2008. Revised Manuscript Received February 19, 2009 F0F1-ATPase motors have unique mechanical properties, making them attractive building blocks in the field of nanotechnology. However, their organization into well-defined structures with practical functions remains a critical challenge. Here, we describe a self-assembling complex formed by F0F1-ATPase and a ghost which is ordered. Formation of the complex includes two steps: the molecular motors first self-assemble into filaments and then attach to the ghost. The ghost and attached filaments then aggregate into large self-assembled complexes. On illumination, these complexes disassemble because of the rotation force of the molecular motors. The complexes are macroscopic, having a diameter greater than 1 mm. Such complexes of a flexible biomaterial (ghost) self-assembled with a dynamic biomaterial (F0F1-ATPase molecular motor) have several advantages, including flexibility, stability, and ability to be controlled by light, and could be used as controllable rotational molecular machines.
Introduction The spontaneous self-organization of complex structures from simple components is perhaps one of the most intriguing phenomena in the fields of chemistry, materials, and bioscience.1-5 Biomolecular self-assembly has a number of advantages in biology, and this feature has been designed and incorporated into desired structures. Erythrocyte membranes, or ghosts, are promising bioactive materials with applications in different fields and have been subjected to a remarkable degree of interest in recent years.6-10 The ghost is a kind of flexile membrane, composed of a lipid bilayer and cytoskeleton. It has a special bioconcave shape, where the diameter is about 7.3-8.5 μm and the thickness is about 1.7 μm.11 Ghosts loaded with drugs or other therapeutic agents have been exploited extensively, owing to their remarkable degree of biocompatibility, biodegradability, and a series of other potential advantages. These properties prompted us to design a novel self-organized material using ghosts. One of the most remarkable motor enzymes is F0F1-ATPase which catalyzes the production of adenosine 50 -triphosphate (ATP). It is commonly known as the ATP motor, and its mechanism of action in this process has been described.12-15 *Corresponding author. Telephone: +861064888576. Fax: +861064871293. E-mail:
[email protected]. (1) Rajagopal, K.; Schneider, J. P. Curr. Opin. Struct. Biol. 2004, 14, 480. (2) Yin, P.; Choi, H. M.; Calvert, C. R.; Pierce, N. A. Nature 2008, 451, 318. (3) Jin, Y.; Xin, R.; Ai, P.; Chen, D. Int. J. Pharm. 2008, 350, 330. (4) Schreiber, F. Prog. Surf. Sci. 2000, 65, 151. (5) Wu, J.; Huang, L.; Liu, J.; Ming, M.; Li, Q. G.; Ding, J. D. Chin. J. Chem. 2005, 23, 330. (6) Vertessy, B. G.; Steck, T. L. Biophys. J. 1989, 55, 255. (7) Mehrdad, H.; Adbolhossein, Z.; Mahshid, F.; Soliman J. Controlled Release 2007, 118, 145. (8) Rossi, L.; Serafini, S.; Piergie, F.; Antonelli, A.; Cerasi, A.; Fratemale, A.; Chiarantini, L.; Magnani, M. Expert Opin. Drug Delivery 2005, 2, 311. (9) Hamidi, M.; Tajerzadeh, H. Drug Delivery 2003, 10, 9. (10) Magnani, M.; Rossi, L.; Fratemale, A.; Bianchi, M.; Antonelli, A.; Crinelli, R.; Chiarantini, L. Gene Ther. 2002, 11, 749. (11) Mohandas, N.; Gallagher, P. G. Blood 2008, 112, 3939. (12) Boyer, P. D. Annu. Rev. Biochem. 1997, 66, 717. (13) Noji, H.; Yasuda, R.; Yoshida, M.; Kinosita, K. J. Nature 1997, 386, 6622. (14) Soong, R. K.; Bachand, G. D.; Neves, H. P.; Olkhovets, A. G.; Craighead, H. G.; Montemagno, C. D. Science 2000, 290, 1555. (15) Montemagno, C. D.; Bachand, G. Nanotechnology 1999, 10, 3.
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The F0F1-ATPase is embedded in the chromatophore, which is a vesicle of lipid-protein complex with an average diameter of 70 ( 15 nm (see the Supporting Information). Besides F0F1-ATPase, there are also some light-harvesting complexes (LH1 and LH2), cytochrome bc1, and the reaction center (RC) complexes on the chromatophore, which convert the light energy into proton motive force (PMF).16 The F0F1-ATPase consists of two rotary motors attached to a common shaft. The F1 motor generates power using ATP as its fuel while the F0 motor uses the proton motive force (PMF) from rotational diffusion. Integrated ATP biomolecular motors have received intense scrutiny because of their potential applications in medicine; however, since the force that a single motor can produce is insufficient to carry out certain tasks, it is recognized that the potential practical applications reported thus far are merely proof-of-principle demonstrations.17 In Escherichia coli, the F1 and F0 motors are composed of R3β3γδε and ab2cn subunits, respectively.18 These two parts of the enzyme are structurally linked by two stalks: a central stalk of the c and ε subunits that links to the c subunit ring, and an outer stalk of the δ b2 subunits linking the R3β3 subunits to the a subunit. If the δ subunit is removed, only one central “stalk” (ε, γ) remains in the F0F1-ATPase and the “rotator” and “stator” will be reorganized (see the Supporting Information). In this δ-free F0F1-ATPase motor, the stator will consist of the b and a subunits, and the rotator will consist of the c ring and the γ, ε, and R3β3 subunits.19 Thus, PMF converted from light could drive the new rotator easily with a single central “stalk”. The δ-free F0F1-ATPase motor20,21 is a good model for construction of second-generation light-driven molecular motors.
(16) Feniouk, B. A.; Cherepanov, D. A.; Voskoboynikova, N. E.; Mulkidjanian, A. Y.; Junge, W. Biophys. J. 2002, 82, 1115. (17) van den Heuvel, M. G. L.; Dekker, C. Science 2007, 317, 333. (18) Oster, G.; Wang, H. G. Biochim. Biophys. Acta 2000, 1458, 482. (19) Zhang, Y. H.; Wang, J.; Cui, Y. B.; Yue, J. C.; Fang, X. H. Biochem. Biophys. Res. Commun. 2005, 331, 370. (20) Su, T.; Cui, Y. B.; Zhang, X. A.; Liu, X. L.; Yue, J. C.; Liu, N.; Jiang, P. D. Biochem. Biophys. Res. Commun. 2005, 350, 1013. (21) Liu, X. L.; Zhang, X. A.; Cui, Y. B.; Yue, J. C.; Li, Z. Y.; Jiang, P. D. Biochem. Biophys. Res. Commun. 2006, 347, 752.
Published on Web 4/20/2009
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Here, we present a novel type of self-organized complex consisting of filaments of chromatophores with δ-free F0F1ATPases and ghost, the detailed structure of which was observed by fluorescence microscopy and confocal microscopy. In the absence of light, and using streptavidin, biotin-labeled chromatophore-embedded F0F1-ATPases joined together to form filaments. These filaments then attached to ghost like rods, with the F1 part facing away from the ghost. The ghost with attached filaments then aggregated into larger self-assembled complexes of two or three layers, held together by head to head links between the rotary F1’s. On illumination, this complex disassembled using a ziplike mechanism, as rotary F1’s were activated by light. These macroscopic complexes had a diameter greater than 1 mm and were able to function as rotation-controllable machines. The complex has several advantages, including flexibility, stability, and the ability to be controlled by illumination. Selfassembly and disassembly of the complex can be induced because F0F1-ATPases function as links holding the ghost together. They can therefore be used as controllable rotational molecular machines and will have extensive applications in medicine in the future.
Figure 1. Self-assembled filaments of F0F1-ATPase motors observed by fluorescence microscopy (A) and schematic view of this process (B). Single small green balls represent individual F0F1ATPase motors within chromatophores. Green lines represent chromatophores linked through biotin-streptavidin-biotin into filament structures. Scale bar represents 10 μm.
self-assembly and disassembly process was observed with an Olympus IX71 fluorescence microscope and recorded with a digital CCD camera (iXon CCD, ANDOR Technology). Confocal microscopy (Olympus FV500, optical scanning confocal microscope) was used during the scanning process of multiple layers of self-assembled complexes. FV1000 software was used to reconstruct the three-dimensional images.
Materials and Methods Ghost Preparation. Fresh pig’s blood was washed three times with cold 0.15 M NaCl buffer, pH 8.0, and plasma and leukocytes were discarded. Ghosts were obtained by hypotonic lysis. Red blood cells were obtained from fresh blood and washed three times with PBS buffer (isotonic phosphate-buffered saline, pH 8.0). The washed cells were added to 40 volumes of ice-cold 5P8 buffer (5 mM sodium phosphate, pH 8.0) and left at room temperature for 20 min before centrifuging at 20 000g for 1 h at 4 °C. The pale ghost layer was collected and washed three more times with lysis solution.22 Reconstitution of δ-Free ATPase. δ-free ATPase was reconstituted from F1 purified from PS3 bacillus and F1-deleted chromatophores purified from Rhodospirillum rubrum as previously described,19 and the ATP hydrolysis activity of the reconstituted δ-free ATPase was measured. Chromatophores were incubated with N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (F-DHPE) at room temperature for 30 min and then washed three times with 0.5 M Tricine buffer (pH 6.5) to remove free probe. Chromatophores were then labeled with lipid-biotin by incubating them together for 1 h at 37 °C, while ghosts were incubated with biotin for 1 h at 37 °C. Free biotin was removed by washing three times with 0.5 M Tricine buffer (pH 6.5). In order to link chromatophores and ghosts, streptavidin was added to the lipid-biotin-labeled chromatophores for 30 min at 37 °C. Free streptavidin was removed by washing three times with 0.5 M Tricine buffer (pH 6.5). Biotinylated ghosts were then incubated with chromatophores labeled with lipid-biotin-streptavidin for 1 h at 37 °C. Observation of Self-Assembly. Ghost linked with chromatophores were resuspended in 40 volumes of ice-cold buffer (0.5 mM sodium phosphate, pH 7.6) for 1 h. Addition of 5 mM NaN3, 2 mM MgCl2, 50 mM KCl, and 2 mM adenosine 50 -diphosphate (ADP) to the buffer created conditions for lightdriven rotation of δ-free ATPase within the ghosts. The complexes were put into a cell where they were then illuminated by light (passed through a 570 nm light filter) for 30 min at 4 °C. This illumination initiated proton transfer across the membrane of the chromatophores, and the rotor δ-free F0F1-ATPase was then driven by the PMF. Once proton transfer was initiated, the cell was incubated at 37 °C throughout the whole experiment. The (22) Steck, T. L.; Weinstein, R. S.; Straus, J. H.; Wallach, D. F. Science 1970, 168, 255.
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Results and Discussion Self-Assembly of F0F1-ATPase Motors with Ghosts. Figure 1 shows the filaments formed by chromatophores, labeled with fluorescent F-DHPE, and F0F1-ATPase motors through biotin-streptavidin-biotin links. It is clear that many chromatophores self-assembled in a linear arrangement about 2-4 μm long, with some filaments reaching a length greater than 10 μm, suggesting that most filaments consisted of about 20-40 chromatophores, since the diameter of each vesicle was about 80 nm.14 A similar phenomenon of self-assembly in a linear arrangement also occurred in other nanoparticles.23 Fluorescent filaments self-assembled with ghosts were observed by fluorescence microscopy and are shown in Figure 2A. Figure 2B shows self-assembled structures of ghosts and filaments observed by visible light microscopy, with shape changes in ghosts being indicated by arrows. Pressure affected the shape of ghosts; some were abnormal shapes (arrow 1), some were sickleshaped (arrow 2), some were triangular (arrow 3), some were compressed into a sunken shape (arrow 4), and some were flattened (arrow 5). Figure 2A and B clearly shows that large compact complexes are multilateral in shape. Variation in shape was induced by compact connections between the F0F1-ATPase molecules (Figure 2). It is of note that ghosts with fluorescence-labeled F0F1-ATPase motors linked together and self-assembled into a large structure (Figure 3), about 0.6 mm 1.2 mm in size, which was stable for more than 1 week. As discussed above, this large structure arose due to compact connections between ghosts, with F0F1-ATPase motors acting as junctions (Figure 2C). This head to head junction of F1 parts was helpful not only for assembly but also for disassembly when light drove the rotary F1 part. The selfassembled complex reported here is interesting in that the F0F1ATPase motor is a rotary motor controlled by light. Scanning confocal fluorescence microscopy was used to confirm the detail of the self-assembled structures, and results are shown in Figure 4. Thirty scans were conducted at 0.1 μm intervals from the top (1st) to the bottom (30th), giving a total thickness of about 3-4 μm. (23) Carrico, I. S.; Kirshenbaum, K. Nat. Nanotechnol. 2009, 4, 14.
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Figure 2. Self-assembled structure of ghosts and filaments. (A) Self-assembled structure of ghosts and filaments were observed by fluorescence microscopy. (B) The structure was observed under visible light, and shape changes in ghosts are indicated by arrows. (C) Schematic diagram of this procedure. In order to understand the self-assembly, a diagram was constructed based on experimental data. The broken line indicates the local detail of the connection between ghosts. At the connection point between complexes, the F1 part acted as a junction through head to head connections. Scale bar represents 10 μm. Langmuir 2009, 25(10), 5747–5752
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In Figure 5, chromatophores labeled with F-DHPE can be seen mainly in a round circle, indicating that they are linked around the edge of the ghost. Fluorescence in the middle of the layer indicated that some chromatophores were distributed on the concave surface of the ghost. These were linked in a horizontal direction, while those on the surface of ghosts were linked in a vertical direction. A three-dimensional reconstruction in which more detail of the shape of self-assembled complex can be clearly seen is shown in Figure 5. From the side view (Figure 5A), the thickness was
Figure 3. View of the self-assembled structures. Scale bar represents 100 μm.
3.5 μm, equivalent to that in Figure 4. It was interesting to see that most of the fluorescent filaments were in an upright direction in a vertical shape around the ghost (arrows indicated). From an apical view (Figure 5B), the shape resembled a flat boat, and fluorescent filaments could be seen along the surface of the ghost. Results from the full three-dimensional reconstruction showed that two ghosts had stuck together and the thickness was about 3.5 μm, and that fluorescent filaments were attached vertically to the ghost. A schematic view of a single self-assembled system is shown in Figure 5C. Disassembly by the Rotation Force of the F0F1-ATP Motor. Although self-assembled complexes could be stable for more than 1 week, the supramolecular system relying on interactions was fragile. Supramolecular assemblies could be disassembled by the external perturbation force produced by the rotation of the F0F1-ATPase motor. This meant that the rotary force of the ATPase motor could accumulate to overcome the weak interaction force between the assembled ghosts. Testing this hypothesis, however, is difficult because there are no appropriate tools for observing the mechanism of assembly. The F1 part, which faces away from the ghost, links the ghosts together and gives rise to the tight structure of the complex. When molecular motors were illuminated on ice, the stored proton potential energy caused the complex to disassemble. Disassembly of the supramolecular system can be seen in Figure 6. In Figure 6A1, there are many ghosts with visible F0F1-ATPase motors. The compact self-assembly structure was the same as that in Figure 2A (amplification in Figure 2A was 1000; 100 in Figure 6A1). After illumination on ice for 1 h, the PMF energy was stored in the chromatophore and did not dissipate. When the temperature was raised to 37 °C for about
Figure 4. Image processing by confocal microscopy in one ghost. Each step was 0.1 μm. Most chromatophores were situated around the ghost, with a few in the middle of the ghost.
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Figure 5. Three-dimensional reconstruction of a self-assembled complex (scale bar represents 2 μm) observed by confocal microscopy. (A) Side view confocal image of one ghost (arrows show the fluorescent filaments were in an upright direction around the ghost). (B) Apical view of fluorescent filaments across the surface of the ghost (arrows show fluorescent filaments around the ghost). (C) Schematic view of the self-assembly. In order to understand the self-assembly, a diagram was constructed based on experimental data. The two ghosts were stuck together, with fluorescent filaments surrounding the ghost vertically.
Figure 6. Disassembly by the rotation force of the F0F1-ATPase motor. (A) After disassembly, most of the ghosts moved away, and only a few that did not move were observed by fluorescence microscopy; arrows indicate the disassembled ghosts and their molecular motors. (A1) Before illumination and (A2) after illumination at 4 °C for 30 min; the temperature was raised to 37 °C, and the complexes disassembled rapidly. (B) Schematic view of the disassembly process. (B1) shows the compact structure of the self-assembly. (B2) shows disassembly after illumination. Scale bar represents 50 μm.
5 min, molecular motors began to rotate and the complexes disassembled rapidly (Figure 6A2).
Conclusion Our experiments on the self-assembly and disassembly of ghosts with F0F1-ATPase motors provided information on the assembly process. Filaments of chromatophores first self-assembled using the biotin-streptavidin-biotin system (Figure 1). Filaments then self-assembled with ghosts into a regular structure, with the F1 Langmuir 2009, 25(10), 5747–5752
part facing away from the ghost, because the surface of ghosts was usually negatively charged and filaments were positively charged (see the Supporting Information) (Figure 5). Ghosts with filaments were then able to extend into large self-assembled complexes in planar form with two or three layers (Figures 4 and 5). The two or three layers of ghosts could then extend into a compact, triangular, or multilateral complex (Figures 2 and 3). Finally, these complexes could be disassembled by illumination, dividing into single ghosts with F0F1-ATP motors (Figure 6). DOI: 10.1021/la804083f
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Although the self-assembly and disassembly of the complexes described here was observed under laboratory conditions, there is clear potential for using these complexes as novel functional materials and novel nanodevices in various fields in the future, as the complexes were quite stable and disassembly was controllable by light. Acknowledgment. We thank Prof. Lin Kechun (Medical Science Center, Peking University) for critical reading
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of this manuscript. This work was supported by the National Basic Research 973 Program (No. 2007CB935901), National Natural Science Foundation of China (No. 30800289; 20873176), and Chinese Postdoctoral Research Award (20070420075). Supporting Information Available: Structure of δ-free F0F1-ATPase motor and ghost. This material is available free of charge via the Internet at http://pubs.acs.org.
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