ВЫСОКОМОЛЕКУЛЯРНЫЕ СОЕДИНЕНИЯ, Серия А, 2009, том 51, № 6, с. 1015-1025
УДК 541.64:539.2
NANO-BIOMACHINE FROM ACTIN AND MYOSIN GELS
© 2009 г. Yoshihito Osadaa, Jian Ping Gongb
a RIKEN, Advanced Science Institute, Hirosawa, Saitama351-0198, Japan b Hokkaido University, Biological Sciences, Graduate School of Science, Sapporo 060-0810, Japan
e-mail: osadayoshi@riken.ac.jp
Abstract—We introduce here an ATP (adenosine triphosphate)-fueled nano-biomachine constructed from actin and myosin gels. Various types of chemically cross-linked actin gel, which are tens of times larger in size than native actin filaments (F-actin), were formed by complexing with cation-polymers and placed on a chemically cross-linked myosin gel. By adding dilute solution of ATP, they moved along the myosin gel with a velocity as high as that of native F-actin by coupling to ATP hydrolysis. Formation mechanism and structure of actin complexes as well as those of myosin gels were studied in detail and elucidated with the specific characteristics of the motility. These results demonstrate that one can construct nano-biomachines fueled by chemical energy of ATP with controlled motility.
1. INTRODUCTION
The motion of a man-made machine, which itself is a systematic assembly of hard and dry materials such as metals, ceramics or plastics, is performed by the relative displacement of the constituent parts of the machine. Their energy efficiency fueled by electrical or thermal energy is around 30%. In contrast to this, the motion of a living organism, which is largely consisted of protein architecture, is performed by the protein deformation on a molecular level, which is then integrated to a macroscopic level through its hierarchical structure [1—3]. Since a biological motor is driven by the direct conversion of chemical energy to mechanical work, the efficiency is as high as 80—90% [4].
Over the past number of years, we have demonstrated several kinds of artificial soft machines which were constructed using synthetic polymer gels [5—11]. Examples include Gelooper (gel-looper), Gelf (gel golf), gel valves, chemical motor, etc. [5—8]. However, lack of the mechanical toughness and slow response of the gel have restricted further application of these machines as actuators.
Recently, we have developed ATP-fueled biomachine made of chemically cross-linked giant actin gel, which moves along a chemically cross-linked myosin gel with a velocity as high as that of native actins [12—15]. The mo-tility was closely related to the hierarchical structure of both actin and myosin gels. These results indicate that muscle proteins can be tailored into desired shape and size without sacrificing their bioactivities to build up biomachine systems with desired motility.
2. GIANT ACTIN GELS
We have found that the giant actin gel filaments, several tens of micrometers in length can be formed from scallop actin filaments (F-actin) by mixing with
cationic polymers in the presence of a cross-linking agent [13].
Figures 1a—1d show some examples of fluorescence microscope images of giant actin fibers obtained by mixing with poly-L-lysine hydrochloride (p-Lys) (Fig. 1a), x,y-ionene (x = 3 or 6; y = 3, 6, or 12) bromide polymers (x,y-ionene) (Fig. 1b—1d). Here, chemical cross-linking was undergone using glutaral-dehyde to prevent the backward dissociation of fibrous actin to globular one. One can see that large filaments with stranded and branched structure of 20—30 ^m in length are formed in the presence of p-Lys, 3,3-, 6,6-, and 6,12-ionene polymers and their morphological nature, both of size and shape, is totally different from that of native F-actin (Fig. 1e). The number-average length of the fluorescence image of F-actins is 2.1 ^m with the standard deviation of 0.1 ^m (average over 784 samples) in F-buffer which provides a condition to keep actins in a filamentous state rather than globular state.
Figure 2 shows time courses of the average length of the giant fibers grown in the presence of various kinds of cationic polymers.
Actin fibers grow with time and reach as large as 5—20 ^m within one or two hours, which is about 2—10 times larger than that of a native F-actin. Since F-actin is negatively charged under given buffer (the isoelectric point of actins is pH 4.7), the formation of a giant fiber is clearly attributed to the complexation with polycations through electrostatic interaction. In fact, negatively charged polymers such as poly L-glutamic acid sodium salt (p-Glu), deoxyribonucleic acid sodium salt (DNA), and neutral polymer-polyethylene glycol (PEG) do not grow at all (Fig. 2). The growth profiles depend on the chemical structure of the polycations. P-Lys brings about a relatively slow but large complex, while 3,3-ionene polymer gives the small one. The complicated x, y dependence for x,y-
Fig. 1. Fluorescence microscope images of the polymer—actin complexes formed by mixing F-actin and various cationic polymers at room temperature: p-Lys (a), 3,3-ionene (b), 6,6-ionene (c), 6,12-ionene (d), F-actin only (e). The molar ratio of the ammonium cation of polymer to monomeric actin was kept constant at 30 : 1 for x,y-ionene polymers and 100 : 1 for p-Lys, which correspond to the weight ratios [3,3-ionene]/[Actin] = 0.41 g/g, [6,6-ionene]/[Actin] = 0.61 g/g, [6,12-ionene]/[Actin] = 0.81 g/g, [p-Lys]/[Actin] = 0.35 g/g. The actin concentration was 0.001 mg/ml. Reproduced from ref. [13] with permission.
ionene polymers might be associated with their complementary effect between the charge density and hy-drophobicity. Both 3,3- and 6,10-ionene form short complexes, while 6,6-ionene gives the longest one. The decrease in the filament size after 100 min of mixing observed for 6,4- and 6,12-ionene complexes is not clear, but any rearrangement of the complex might have occurred.
Since the fiber formation by polymer—actin complexes is an equilibrium reaction, the morphological features of the product should depend on actin (Ca) and polymer (C^) concentrations. Figure 3a shows how the size of the complexes is changed when the ac-tin concentration is varied under the constant polyca-tion (p-Lys) concentration of 3.5 x 10-4 mg/ml. One can see that the complex size increases abruptly when the actin concentration exceeds 0.001 mg/ml, which is then equilibrated to a constant length around 15— 20 mm. If we define P as a length ratio of the complex obtained under given polymer concentration to the maximum one at the constant actin concentration of 0.001 mg/ml, and plot P as a function of polymer concentration, we get Fig. 3b. No actin growth at all is observed when the mixing ratio of p-Lys to actin is lower than 0.14 g/g, indicating that the polymer—actin complex does not form under such low concentration. However, when the mixing ratio of p-Lys to F-actin exceeds 0.21 g/g, the length of polymer—actin complexes abruptly increases, indicating that the complex formation is cooperative in nature. The similar cooperative behavior was also observed for the complex-ation with x,j-ionene polymers, and the critical mixing ratios of 3,3-, 6,4-, 6,6-, 6,10-, and 6,12-ionene to F-actin were 0.81, 0.054, 0.12, 0.024, and 0.022 g/g,
Average length, ^m
Time, min
Fig. 2. Time courses of the polymer—actin complexes growth observed with fluorescence microscope (open symbols) and transmission electron microscope (closed symbols). The molar ratio of the ammonium cation to monomeric actin was 30 : 1 for x,y-ionene polymers and 100 : 1 for p-Lys. The corresponding weight ratios were [3,3-ion-ene]/[Actin] = 0.41 g/g, [6,4-ionene]/[Actin] = 0.54 g/g, [6,6-ionene]/[Actin] = 0.61 g/g, [6,10-ionene]/[Actin] = = 0.74 g/g, [6,12-ionene]/[Actin] = 0.81 g/g, [p-Lys]/[Actin] = = 0.35 g/g, [p-Glu]/[Actin] = 0.36 g/g, [DNA]/[Actin] = = 0.77 g/g, [PEG]/[Actin] = 0.10 g/g. Actin concentration: 0.001 mg/ml. Reproduced from ref. [13] with permission.
respectively. Thus, the polymer-actin complexation is a cooperative process and one can get large complexes only when both F-actin concentration and the mixing ratio exceed certain critical values. Now, we can explain why we obtained the small complex in the case of 3,3-ionene in Fig. 1. In that case the experiment was performed at [3,3-ionene]/[actin] = 0.41 g/g, which was less than the critical value of 0.81 g/g.
From Fig. 3b we can obtain the apparent binding constant (K), cooperativity parameter u and other thermodynamic parameters of the actin-polymer complexation by the following equation [16—18]:
K = K 0U = 1 / ( & )0.5, where K0 is the binding constant of the cationic polymer initially connected to an isolated binding site on the actin filament (initiation process), (Cs)05 is the cationic polymer concentration at P = 0.5. The value of u, which tells the extra interaction energy between the binding sites (propagation process), can be calculated from the slope of the growing profile at the half-length point:
( dp/d ln Cs )05 = VU/4 (1)
K0 and u as well as the total binding energy (AFtoaal = = — RT lnK) and cooperative energy change (AFcoop= = — RTlnu) were calculated and the results summarized in Table 1. One can see large values of the cooperative parameter u and large cooperative energy (AFcoop) changes for p-Lys and 3,3-ionene, and the small value of the cooperative parameter (consequently, a large binding constant of the initiation process, K0) for 6,12-ionene.
3. POLYMORPHISM OF GIANT ACTIN GELS
The polymer—actin complexes exhibit a rich polymorphism in a wide range of polymer (Cp) and salt (Cs) concentrations [19]. There are five characteristic phases in the Cp—Cs phase diagram (Fig. 4). Phase I is the area where F-actin complex does not grow. In phase II the native F-actin and polymer—actin complex coexist. In this phase II, the fraction of native F-actins increases with an increase of Cs. The borderline between phase I and phase II shifts to a higher Cp with an increase of Cs. This can be explained by the screening effect of s
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