NANO LETTERS
Single-Molecule Force Spectroscopy on Curdlan: Unwinding Helical Structures and Random Coils
2003 Vol. 3, No. 8 1119-1124
Lu Zhang,† Chi Wang,† Shuxun Cui,† Zhiqiang Wang,*,‡ and Xi Zhang*,† Key Lab for Supramolecular Structure and Materials, College of Chemistry, Jilin UniVersity, Changchun 130023, P. R. China, and Department of Chemistry, Tsinghua UniVersity, Beijing 100084, P. R. China Received May 10, 2003; Revised Manuscript Received June 18, 2003
ABSTRACT Curdlan is an important biomacromolecule whose bioactivity is strongly depending on its supramolecular structures. We have demonstrated that atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is able to recognize triple- and double-helical structures and random coils in curdlan, which are controlled by the concentration of sodium hydroxide. We have obtained important information about the force-induced conformation transition and dynamics of supramolecular structures of curdlan at the molecular level.
Single-molecule force spectroscopy (SMFS), an atomic force microscopy (AFM)-based technique, has been widely used to interpret the nanomechanical properties of both biomacromolecules and synthetic polymers.1-16 Force spectroscopy on polysaccharides, a series of important natural building blocks of living organisms, has aroused increasing interest because SMFS is a good tool for investigating the forceinduced conformational transitions, the dynamics, and supramolecular structures of polysaccharides at the molecular level.17-26 Rief et al. first studied dextran, a liner R-(1,6)linked polysaccharide, by means of SMFS and revealed a special “shoulder-like” plateau at around 750 pN observed in the force profiles. They attributed the plateau to a twist of the C5-C6 bond in the pyranose ring.17 Marszalek et al. proved that it was the chair-boat conformational transition of the glucopyranose ring that governed the shoulder-like elongation.18 By comparing the R-(1,4)-linked and β-(1,4)linked polysaccharides, Li et al. found that only R-(1,4)linked glycan could yield the distinct elongation, and there were no such shoulders appearing in β-(1,4)-linked polysaccharides.19 The force-governed shoulder-like transition was thought to be the fingerprint property of R-(1,4)-linked glycan rings. Theoretically, Berthold et al. simulated the individual β-(1,4)-linked cellulose and R-(1,4)-linked amylose and confirmed that only R-glucose has a fingerprint shoulder.20 Marszalek et al. demonstrated that atomic levers governed the conformational transitions of pyranose rings. They regarded the glycosidic bonds as mechanical levers that drove * Corresponding authors. E-mail:
[email protected]. † Jilin University. ‡ Tsinghua University. 10.1021/nl034298d CCC: $25.00 Published on Web 07/03/2003
© 2003 American Chemical Society
the conformational changes of the pyranose ring.21 In subsequent studies using SMFS, they continued to identify the components of mixtures of polysaccharides by different fingerprints of the pyranose rings.22 Xu et al. used force spectroscopy on a series of carrageenans, which convinced us of another facet of the fingerprint of the force-induced chair-boat transition and provided experimental evidence that the oxygen bridge could inhibit the transition.23 Beyond the molecular level, the complex supramolecular structures of polysaccharides play an essential role in their biofunctions. The polysaccharides are also capable of existing in helical forms, which are similar to the double helix of DNA.29 Li et al. studied xanthan, which has a helical structure in its native state and showed that the during the process of denaturation xanthan lost its helical structure, as indicated by SMFS.24 Xu et al. studied another polysaccharide, carrageenan, which can undertake a coil-helix-gel transition depending upon the counterions and the concentration of salt.25 As indicated in Scheme 1, curdlan, one of the microbial polysaccharides, is a naturally occurring linear polysaccharide composed of β-(1,3)-linked D-glucose, and it adopts a triplehelical conformation as determined by X-ray fiber diffraction in the solid state.27 This natural polysaccharide is not soluble in water, but it can easily dissolve in alkaline aqueous solution because of the ionization of hydroxyl groups and in strong polar solvent such as dimethyl sulfoxide.28 Similar to the other bacterial branched and linear β-(1,3)-linked D-glucans, curdlan and its derivatives have biofunctions such as anti-HIV and antitumor activity.29 These bioactivities depend on the highly ordered structure of the polysaccharide
Scheme 1. Chemical Structure of Curdlan, a β-(1,3)-D-Glucose-Linked Polysaccharide Whose Suprastructure Depends on the Concentration of NaOH(aq)
in solution, but these activities cannot be maintained in its disordered state.30-33 By using a concentration of sodium hydroxide greater than 0.24 M, one may assume that curdlan has a random coil conformation, whereas in solutions more dilute than 0.19 M NaOH(aq) curdlan exists as helical structures due to intramolecular and intermolecular hydrogen bonding. A conformational transition from helix structures to random coils occurs at concentrations of NaOH between 0.19 and 0.24 M.30 A possible arrangement during the change is that a duplex structure may form with the third chain separating from the duplex that subsequently merges into the triplex end. In addition, curdlan can form reversible or irreversible gels by cooling and heating in aqueous solution. The reversible gel occurs by cooling after heating at 55-60 °C as a low-set gel. The irreversible gel is formed by heating above 80 °C as a high-set gel. Though the mechanism of gelation is not very clear, Tako et al. proposed that a hydrophobic interaction might take place between the methyl groups at C6 of D-glucosyl residues on different molecules of curdlan in its irreversible gel.34 However, many problems remain, such as the transition force of conformational change and enhanced hydrophobic interaction after thermal treatment, which cannot be addressed by conventional methods. In this paper, using SMFS, we attempt to obtain force spectroscopy measurements on curdlan and to distinguish the existence of suprastructures in different conditions. Our experiment is also aimed at understanding the mechanism and dynamics behind the conformational transition induced by sodium hydroxide in certain concentrations, thus bridging the relationship between force patterns and the structures of curdlan. In addition, for the irreversible gel of curdlan, we wanted to study the enhanced hydrophobic interaction by thermal treatment. Curdlan from Alcaligenes faecalis (Sigma, C7821, 99%) was dissolved in a 0.10 M NaOH(aq) , and the solution was sealed to protect it from carbon dioxide in the air. About 0.2 mL of the curdlan solution was dropped onto a clean, fresh glass slide (18 mm × 18 mm) and incubated at room temperature for approximately 35 min. The excess liquid was removed from the substrate, and then the substrate with polymer was ready for SMFS measurements. A series of aqueous NaOH solutions (0.01, 0.10, 0.20, and 0.50 M) were 1120
also sealed to protect them from the air before use as buffers. Deionized water was used for all solution preparations. The force measurements were carried out on a homebuilt SMFS in an AFM liquid cell with silicon nitride contact tips (PARK, Sunnyvale, CA) with spring constants of 12-60 pN/ nm, as determined from thermal excitation.35,36 The experimental details of SMFS have been described elsewhere.3,7,11,24 To obtain single-chain stretching, it is necessary to use a dilute polymer solution during sample preparation. Thus, the density of molecules at the solid/liquid interface will be lowered, and the intermolecular entanglement and knotting can be effectively avoided, which will simplify the explanation of the experimental data. An AFM contact tip is brought into contact with the polymer sample that was physisorbed on the glass slide, whereupon the macromolecular chain is absorbed on the tip, forming a polymer bridge between the tip and substrate. Upon the separation of tip and substrate, the polymer is stretched, and the stretching force is measured as a function of the separation distance, recorded as a forceextension curve. Once we confirm that a single molecule is stretched and then keep the stretching force lower than the rupture force, we can repeatedly stretch and relax the same polymer chain, obtaining the trace-retrace force-extension curves. We know that the loading rate affects the shape of the force-extension traces or the unbinding forces in some systems.6,37-40 In these cases, the force-induced transition or unbinding processes occur in a nonequilibrium state. In our experiment, the stretching rate is fixed between 2 and 4 µm s-1 if not otherwise specified. It is reasonable to compare force values under the same stretching rate. We use the modified freely jointed chain (M-FJC) model to describe single-stretching polymer chains semiquantitatively.41,42 The M-FJC model treats the macromolecule as a chain of statistically independent segments of length lk, Kuhn length, and the segments can be deformed under external stress. The M-FJC model is based on the Langevin function:
{ [ ] }[
X(F) ) coth
kBT Flk nF Lcontour + kBT Flk Ksegment
]
Here, F is the external force acting on the polymer chain, X is the extension of the polymer chain (end-to-end distance), Lcontour is the length of the polymer chain at maximal extension, n is the number of statistical segment being stretched in the filament, kB is Boltzmann’s constant, and T is temperature in Kelvin. The deformation of stretching segments is characterized by the segment elasticity, Ksegment. The elasticity of a modified FJC chain is dominated by either the entropic contribution in the low-force region or enthalpy in the high-force region. Random Coils of Curdlan. Using different cantilevers in different experiments, we obtained typical force extension curves of curdlan in 0.50 M NaOH(aq), in which curdlan is completely soluble, existing as random coils, as shown in Figure 1. From the Figure, we can see that all of the curves exhibit a similar deformation that is characteristic of the Nano Lett., Vol. 3, No. 8, 2003
Figure 2. Reversible manipulation of the individual curdlan filament in 0.50 M NaOH(aq), which indicates that the conformational transition is a reversible process. Figure 1. Several typical force-extension curves of curdlan in 0.50 M NaOH(aq). One of the force curves is fitted by the M-FJC model, noted as the dashed line. The normalized force curves are superimposed and plotted in the inset.
macromolecular chains: a sharp, monotonically rising force with increasing extension and a rapid drop in the force to zero upon rupture from the tip or the substrate. The force curves of curdlan have different stretching lengths because of its polydispersity on the substrate and uncontrolled stretching points. To compare the elasticity of the force curves under the same stretching length, we normalized the curves by dividing the corresponding lengths measured under the same force of 350 pN.43 Despite different stretching lengths, the normalized curves can be well superimposed as shown in the inset of Figure 1, which indicates the singlechain stretching in the experiment. Different from the force curves of R-(1,4)-linked polysaccharides, there are no shoulder-like plateaus in the force curves of curdlan because of the β-(1,3)-linked pyranose ring. The elongation induced by force reflects the mechanical properties of the main chain. All of the force curves were simulated and semiquantitatively described by the M-FJC model, giving a narrow distribution of lk ) 1.40 ( 0.10 nm and Ksegment ) 11 000 ( 1000 pN/nm (Figure 1).44 These facts also corroborate the fact that predominately individual curdlan chains was stretched and indeed the deformation of a single chain under tension was measured. Studies of SAXS have revealed, for the conformation of curdlan in alkaline aqueous solution, that only random coils exist in its highconcentration alkaline aqueous solution, and curdlan is believed to be completely dissolved.30 The deformation behavior of curdlan chains also represents the common characteristics of polymers and shows that there are no suprastructures as revealed in the force curves of curdlan in 0.50 M NaOH(aq), suggesting the random coils conformation in 0.50 M NaOH(aq). The trace-retrace force curves have been obtained by keeping the stretching force lower than the rupture force. Figure 2 shows a consecutive trace-retrace pair from the same curdlan chain. From the Figure, we observed no hysteresis between the stretching and relaxing traces, supporting the single-chain manipulation of random coils under equilibrium conditions. Helical Structure of Curdlan. Different from the force curves of random coils of curdlan in 0.50 M NaOH(aq), some force curves were obtained as shown in Figure 3 when the concentration was lowered to 0.10 M. Nano Lett., Vol. 3, No. 8, 2003
Figure 3. Several typical force-extension curves of curdlan in 0.10 M NaOH(aq), which show a plateau of about 60 pN. The normalized force curves are superimposed and plotted in the inset.
Although the force curves have different stretching lengths, they have a similar characteristic: as the stretching force increases with the extension, every force curve has a clear plateau, though the lengths of the plateau are different. To see these plateaus more clearly, three of these force curves, obtained at the same stretching rate, were dividually superimposed in Figure 3. We normalized and superimposed the three force curves, as shown in the inset of Figure 3. From the inset, we can find two main points. One is that all of the filaments show plateaus at the same force, measured to be about 60 pN, indicating that the plateaus originate from the same conformational change. The other is that in the highforce region all of the force-extension curves can be well superimposed. This fact suggests that after the conformational transition the stretched chains have the same elastic property, which indicates that they most likely belong to single-chain stretching if we consider the cleanness of the force curves and the sample preparation. To clarify the behavior of the force curves of curdlan in 0.10 M NaOH(aq) further, we compared the normalized force curves of curdlan in 0.10 and 0.50 M NaOH(aq), as shown in Figure 4. In the low-force region, the constant plateau of curdlan in 0.10 M was quite clear compared to the singlechain stretching of random coils of curdlan in 0.50 M NaOH(aq). Moreover, after the plateau, we have found that the two force curves in the high-force region can be well superimposed. This fact we have also inferred the reasonability of single-chain manipulation. The result indicates that at high force the force curves of curdlan in 0.10 and 0.50 M NaOH(aq) show the same elasticity. That a helix-coil transition of double-stranded DNA (dsDNA) has a distinct plateau in its force-extension curves and that the plateau is 1121
Figure 4. Comparison of the normalized force curves of curdlan in 0.10 and 0.50 M NaOH(aq). After the conformational transition, the two force curves can be superimposed well in the high-force region.
Figure 5. Subsequent deformation curves of the same individual curdlan chain in 0.10 M NaOH(aq). The force curves show hysteresis between the stretching and relaxation traces.
regarded as a major characteristic of dsDNA when stretched, corresponding to the process of a helix-coil transition, have been well studied. Many studies have shown that curdlan and its derivatives adopt a favored triple-helical structure at low concentrations of NaOH(aq). Similar to dsDNA, we attribute the observed plateaus of curdlan in 0.10 M NaOH(aq) to the transition of the helical structure, corresponding to the equilibrium state when the external force is equal to the transition force.45 When the external force is larger than transitional force, the triplex is completely transformed, and the force curves are the same as the stretching of singlestranded curdlan in 0.50 M NaOH(aq). Moreover, the comparison of the normalized force curves of curdlan in 0.10 and 0.50 M NaOH(aq) (as shown in Figure 4) may convince us that only one triple helix is unwound during structural transition. Therefore, the force of 60 pN correspondings to the transition force of one triplex chain under a certain stretching rate. Similar to the results of curdlan in 0.10 M NaOH(aq), we also observed long plateaus of 60 pN in the force-extension curves of curdlan in 0.10 M NaOH(aq), where the triplex structure of curdlan was also retained. These results can confirm the formation of helical structures in equal or less than 0.10 M NaOH(aq), and the unfolding force is about 60 pN. This transition force correlates reasonably with its melting temperature.46 Again, without breaking the polymer bridge, we manipulated the same triplex chain and recorded the deformation of its stretching- and relaxing-force curves continuously. Figure 5 shows one consecutive trace-retrace pair of the same triplex chain. From the Figure, we can see that a helixcoil transition occurred in the force-extension curves com1122
pared to the trace-retrace force curve pair, which was obtained from random coils of curdlan in 0.50 M NaOH(aq) as shown in Figure 2. Hysteresis between the stretching and relaxation traces of Figure 5 is detected, revealing the irreversible nature of the transition. As the triplex chain is stretched, helical structures are transformed. When the curdlan chain is relaxed, the molecule recombines to form the former sequence. However, compared to the stretching traces, a smaller force is needed when the molecule is relaxed and anneals to form its former sequence. Moreover, the triplex structure of curdlan in 0.10 M NaOH(aq) is not perfect as it is in its crystalline state, in which three chains of the triplex structure are strandseparated in some individual pitch. In our SMFS experiments, we also obtained some force curves without plateaus in the filaments under this condition, and they showed the same elastic property as that of force curves of curdlanin 0.50 M NaOH(aq). Structure Transition of Curdlan. As indicated above using [NaOH] > 0.24 M, the curdlan assumes a random coil conformation, but in [NaOH] < 0.19 M, curdlan molecules are in the triplex structure. However, a conformational change is observed as the concentration of NaOH, which is used to dissolve the curdlan, increased from 0.19 to 0.24 M. The possible arrangement during the change is that a duplex structure occurs while the third chain separates from the duplex, which subsequently merges into the triplex. To detect this arrangement of the change, we use SMFS to obtain the force curves of curdlan in 0.20 M NaOH(aq). We suppose that the force curve of a duplex should have a lower transitional force than that of the triplex. Because one chain is separated from the duplex because of the enhanced ionization of hydrogen bonds, the transition force of the suprastructure, formed mainly by hydrogen bonds, is weakened. Figure 6a shows the comparison of two force curves of curdlan in 0.10 M (I) and 0.20 M NaOH(aq) (II). We can clearly see that the two force curves have plateaus with different heights. From the analysis of more than 300 data points that were taken from 5 individual force curves, the mean value of the transitional force is 60 pN in 0.10 M NaOH(aq) (Figure 6b I) and 40 pN in 0.20 M NaOH(aq) (Figure 6b II). Compared with the transitional force of curdlan in 0.10 M NaOH(aq) favoring the triplex, the transitional force of curdlan in 0.20 M NaOH(aq) is lower, and it agrees well with our above prediction. Furthermore, one thing should be pointed outsthat force curves bearing a plateau of 60 pN obtained in 0.20 M NaOH(aq) can be also observed. This finding implies the complexity of the transition state that corresponds to the conformational transition from an ordered structure to a random coil. The random coil was believed to belong to the imperfect parts of the curdlan triplex chain, and the percentage of random coil in the force curves, which were taken in 0.10 M NaOH, is about 10%. Enhanced Hydrophobic Interaction by Thermal Treatment. Curdlan forms reversible and irreversible gels by cooling and heating in aqueous solution. The reversible gel occurs by cooling after heating to 55-60 °C as a low-set Nano Lett., Vol. 3, No. 8, 2003
Figure 6. (a) Comparison of the force curves of curdlanin 0.10 and 0.20 M NaOH(aq), noted respectively as (I) and (II). (b) Histograms for the transitional forces obtained from curdlan in 0.10 M (I) and 0.20 M (II) NaOH(aq). The mean values are 60 pN for (I) and 40 pN for (II).
gel. The irreversible gel is formed by heating above 80 °C as a high-set gel. However, the irreversible gel formed at high temperature can remain at ambient temperature, and it allows us to study the high-set gel using SMFS. The main structure of curdlan for all gel states is the triple helix, though other states exist in the gel.47 Figure 7a shows the comparison of curdlan in 0.10 M NaOH(aq) before (I) and after (II) thermal treatment. On the basis of more than 200 data points, the mean value of the transitional force is 60 pN (Figure 7b I) before thermal treatment and 90 pN after thermal treatment (Figure 7b II). The transition force with thermal treatment consists of two parts: one is hydrogen bonding of the helix of curdlan, which is the main contribution of the transition force before thermal treatment and the other is hydrophobic interaction, which plays a dominant role in intramolecular association. We think that a reasonable explanation of the increase in the transition force after thermal treatment is due to the enhancement of hydrophobic interaction.34 In conclusion, we have obtained the full landscape of the force spectroscopy on curdlan, from triple helix via double helix to random coil, which is controlled by the concentration of sodium hydroxide. The force-extension curves of curdlan in 0.10 M NaOH(aq) show a flat plateau at around 60 pN, which is ascribed to the unwinding of the triple helix of Nano Lett., Vol. 3, No. 8, 2003
Figure 7. (a) Comparison of the force curves of curdlanin 0.10 M NaOH(aq) before (I) and after (II) thermal treatment. (b) Histograms for the transitional forces obtained from curdlan in 0.10 M NaOH(aq) before (I) and after (II) thermal treatment. The mean values are 60 pN for (I) and 90 pN for (II).
curdlan. However, in the case of [NaOH] > 0.24 M, we have obtained only monotonic force-extension curves, indicating the existence of random coils. When the concentration of sodium hydroxide is between 0.19 and 0.24 M, we have obtained force-extension curves with flat plateau, but the height of the plateau decreases to about 40 pN, suggesting the unwinding of the duplex structure of curdlan. Upon thermal treatment of curdlan, we have obtained forceextension curves with plateau at about 90 pN. Compared to curdlan before thermal treatment, the increase in the transition force is the result of the enhancement of hydrophobic interaction. Acknowledgment. This study was supported by the National Natural Science Foundation of China (50073009) and the Major State Basic Development Program (grant G2000078102). References (1) Binning, G.; Quate, C. F.; Gerber, Ch. Phys. ReV. Lett. 1986, 56, 930. (2) Radmacher, M.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Phys. ReV. Lett.1994, 265, 1577. 1123
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NL034298D
Nano Lett., Vol. 3, No. 8, 2003
