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Cite This: Macromolecules 2019, 52, 5006−5013

Single-Molecule Studies Reveal That Water Is a Special Solvent for Amylose and Natural Cellulose Lu Qian,† Wanhao Cai,† Duo Xu,‡ Yu Bao,† Zhong-yuan Lu,‡ and Shuxun Cui*,† †

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Key Laboratory of Advanced Technologies of Materials (Ministry of Education), Southwest Jiaotong University, Chengdu 610031, China ‡ State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, Jilin University, Changchun 130023, China S Supporting Information *

ABSTRACT: It is generally accepted that water is deeply involved in the structures and functions of DNA and proteins. For polysaccharides, however, the role of water remains poorly understood. Amylose and natural cellulose (NC) are two polysaccharides with similar molecular structures but different linkages (α or β) between the pyranose rings. In this study, the effects of H-bonds on the single-molecule mechanics and affinity for water of amylose and NC are explored by single-molecule atomic force microscopy (AFM) and molecular dynamics (MD) simulations, respectively. The experimental results show that the single-molecule mechanics of both amylose and NC are dependent on the solvent polarity. Accordingly, the status of H-bonds of each polysaccharide can be inferred. We find that the two polysaccharides present the same status of H-bonds in a given organic solvent: the intrachain H-bonds can be formed in a nonpolar solvent (nonane), while they are completely prohibited in a highly polar solvent (dimethyl sulfoxide, DMSO). However, the statuses of H-bonds differ largely in water, where NC can form more intrachain H-bonds than amylose. This finding, which is supported by MD simulations, indicates that NC is more hydrophobic than amylose at the single-molecule level. These results reveal that water is a special solvent for these two polysaccharides: Both mechanics and affinity for water of them can be effectively affected by water through regulation of the Hbonds. The present study provides new insight into the role of water (the key environment of organisms) in the structures and functions of polysaccharides.

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INTRODUCTION Water, which is deeply involved in the structures and functions of biomacromolecules (e.g., DNA and proteins), plays a fundamental role in biological systems.1,2 In general, the effects of water on the properties of biomacromolecules are closely associated with the hydrogen bonds (H-bonds).1,2 Having abundant hydroxyl groups and oxygen atoms, polysaccharides tend to form extensive intrachain and/or interchain Hbonds.3−5 Amylose and natural cellulose (NC) have very similar molecular structures, but they present different statuses of H-bonding in the crystals. NC can form both interchain and intrachain H-bonds in the crystals. These H-bonds contribute to the stability of the flat ribbon structure of NC molecules and promote the fibrous aggregation of NC.3,6 In a starch granule, however, only intermolecule (interchain and polysaccharide− water) H-bonds are formed.7,8 The solubility of a polysaccharide in water is closely related to the status of H-bonds. For example, NC cannot be dissolved in water due to the extensive H-bonds among sugar rings. However, amylose shows a better water solubility than NC, possibly due to the different statuses of H-bonds. In a polysaccharide/solvent mixture, polysaccharides can form three types of H-bonds, namely, interchain, intrachain, and © 2019 American Chemical Society

polysaccharide−solvent H-bonds. In a very dilute solution of polysaccharide (i.e., at the single-molecule level), the interchain H-bonds between polysaccharide chains can be ignored due to the relatively long distance between them. Only the intrachain and polysaccharide−solvent H-bonds need to be considered under this condition. The interactions between the nonpolar organic solvent molecules and solute molecules are van der Waals forces in general; no H-bonds can be formed between them. If a nonpolar organic solvent is used as the experimental environment, only intrachain H-bonds need to be considered in this case. If a polar solvent (which can form Hbonds with the polysaccharide) is used as the experimental environment, the effects of the polysaccharide−solvent Hbonds can be studied under this condition. Thus, the effects of these three kinds of H-bonds on the properties of polysaccharides can be studied by changing the experimental conditions. It is known that the interchain H-bonds of NC will be broken during the dissolution process. Yet, there are sparse Received: January 24, 2019 Revised: April 29, 2019 Published: June 20, 2019 5006

DOI: 10.1021/acs.macromol.9b00179 Macromolecules 2019, 52, 5006−5013

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Figure 1. (a) Normalized single-chain F−E curves of NC obtained in nonane (red) and DMSO (blue). (b) Force difference (ΔF) between the two F−E curves shown in (a) vs the normalized extension. The F−E curves have been smoothed before calculation. The full-range F−E curves of NC obtained in nonane and DMSO are shown in Figure S2. by air flow. (Warning: piranha solution is extremely oxidizing and should be handled with care!) To prepare the sample for single-molecule AFM, a few drops of the polysaccharide solution are deposited onto the clean glass substrate for 30 min. Then, the substrate is rinsed with abundant DI water to remove the loosely adsorbed polysaccharides. Force Measurements. All the force measurements are performed on a commercial MFP-3D AFM (Asylum Research, Goleta, CA). Prior to the measurements, a drop of liquid is introduced between the V-shaped Si3N4 AFM cantilever (Bruker Corp., San Jose, CA) and the sample surface. Dimethyl sulfoxide (DMSO) can absorb water molecules from air (Figure S1), which may affect the results of force measurements.30 To avoid water absorption in DMSO, the sample is settled in a closed liquid cell of AFM. For the force measurements at elevated temperatures, the sample is also settled in a closed liquid cell. Then the sample is heated to the target temperature (nominal specifications of the heating accessory: precision, 0.02 °C; accuracy, 0.1 °C).33,34 Force measurements are performed when the temperature is stable for at least 5 min. The spring constant of each AFM cantilever, which is ∼45 pN/nm, is obtained by the traditional thermal excitation method.35 The instrumentation details of singlemolecule AFM can be found elsewhere.11,34,36 Molecular Dynamics (MD) Simulations. MD simulations are performed with GROMACS 5.0.2, using the force field parameter set GROMOS 53A6carbo.37 In the simulations, both the NC and amylose molecules contain nine sugar units. The details of the MD simulations are described in the Supporting Information.

studies on the effects of solvent properties (such as dielectric constant or polarity) on the intrachain H-bonding of NC. It has been reported that the status of H-bonding in a single amylose chain is closely related to the dielectric constant of solvent.9 Intrachain H-bonds of amylose can be formed in a solvent with a low dielectric constant. However, the intrachain H-bonds are significantly weakened in a solvent with a high dielectric constant.9,10 The single-molecule mechanics of amylose is dependent on the dielectric constant of solvent, which is an important factor to regulate the strength of the intrachain H-bonds. Yet, the role of the intrachain H-bonds in the properties and functions of NC and amylose remains unclear. Single-molecule atomic force microscopy (AFM)11−27 is a powerful tool to investigate the single-molecule mechanics of polysaccharides,28−31 which in turn can be used as a measure for the strength of the intrachain and polysaccharide−solvent H-bonds under various solvent conditions. Here in this study, single-molecule AFM and molecular dynamics (MD) simulations are used to study the status of H-bonding of NC and amylose in different experimental environments. The experimental results show that NC and amylose share the same single-molecule mechanics and status of H-bonds in a given organic solvent, no matter whether the solvent is polar or nonpolar. In water, however, the single-molecule mechanics and statuses of H-bonds of these two polysaccharides differ largely. This study is an important update to the understanding of the physical and biological properties of these important polysaccharides at the single-molecule level.

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RESULTS AND DISCUSSION Nonane and DMSO are used to study the effects of the solvent polarity on the status of H-bonding in a single NC chain. These two solvents differ largely in polarity (Table S2). As shown in Figure 1a, the normalized force−extension (F−E) curves obtained in nonane and DMSO can be superposed well in the low or high force region (F < 50 pN or F >2500 pN). However, the F−E curve obtained in nonane is higher than that in DMSO in the middle force region (50−2500 pN). Because DMSO is a strong H-bonds breaker, it can be speculated that the deviation between these F−E curves is caused by the difference of the intrachain H-bonds. In a nonpolar organic solvent, the interactions between solvent molecules and solute molecules are van der Waals forces, which can be ignored in single-molecule AFM studies.38,39 NC, with abundant hydroxyl groups and oxygen atoms, tends to form extensive intrachain H-bonds in nonane (a nonpolar solvent) due to the very weak disturbance from the solvent molecules. However, the intrachain H-bonds of NC may be broken by DMSO, which is a highly polar solvent. Compared to the case in DMSO, more energy will be consumed to

MATERIALS AND METHODS

Materials and Chemicals. The ionic liquid 1-allyl-3-methylimidazolium chloride (AMIMCl) is synthesized following the literature.32 Four polysaccharides are used in this study, i.e., amylose (Type III from potato, Sigma-Aldrich), amylose triacetate (ATA, a generous gift from Prof. Takahashi of Osaka University), NC (Product No. C6288, Sigma-Aldrich), and cellulose triacetate (CTA, Acros-Organics). Deionized (DI) water (>18 MΩ·cm) is used when water is involved. Other chemicals (analytically pure) are used without further treatment. Sample Preparation. Amylose is dissolved in DI water (by heating to 90 °C) to a concentration of 2 mg/L. NC is dissolved in AMIMCl by stirring the mixture at 60 °C to a concentration of 2 mg/ L. ATA or CTA is dissolved in DI water to a concentration of 2 mg/L. The low concentrations mentioned above are helpful to realize the single-chain measurements later. Before use, the glass slides are treated by a hot piranha solution (98% H2SO4 and 35% H2O2, 7:3, v/ v) for 30 min, followed by rinsing with abundant DI water and dried 5007

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Figure 2. (a) Normalized single-chain F−E curves of CTA obtained in nonane (red) and DMSO (blue). The inset shows the structure of the CTA. (b) Normalized single-chain F−E curves of NC in DMSO (blue) and CTA in nonane (red).

Figure 3. (a) Normalized single-chain F−E curves of amylose obtained in nonane (red) and DMSO (blue). (b) Normalized single-chain F−E curves of amylose in DMSO (blue) and ATA in nonane (red). The inset shows the structure of ATA. The full-range F−E curves of amylose obtained in nonane and DMSO are shown in Figure S5.

rupture the intrachain H-bonds during the single NC chain elongation in a nonpolar solvent (nonane). Thus, the singlechain F−E curve obtained in nonane is remarkably higher than that in DMSO (Figure 1a). As shown in Figure 1b, the force difference (ΔF) between the two F−E curves shown in Figure 1a increases slowly when the normalized extension is increased from 0.7 to 0.85. Beyond 0.85, ΔF increases rapidly until it reaches the maximum at ∼0.97 (F ∼ 2000 pN). With further elongation, ΔF decreases rapidly to zero, indicating that almost all the intrachain H-bonds have been broken during the chain elongation, and the statuses of NC in nonane and DMSO are virtually the same at high forces (F > 2500 pN). The derivatives (dF/dx) of the normalized F−E curves of NC obtained in nonane and DMSO are shown in Figure S3, where the differences between the two F−E curves in Figure 1a are displayed in another way.40,41 The dF/dx in nonane increases first and then decreases (i.e., there is a turning point where the dF/dx reaches the maximum; see Figure S3b), while the dF/dx in DMSO increases monotonically in the whole range (Figure S3d). The turning point, which can be used as an indicator for the structural transition during the single chain elongation,30,42 supports that the rupture of the intrachain Hbonds occurs in the single chain elongation of NC in nonane. As described above, we speculate that the single-molecule mechanics of NC can be affected by the solvent polarity through regulation of the intrachain H-bonds. To confirm this hypothesis, similar force measurements of CTA (Figure 2a, inset) are performed in nonane and DMSO. Because all the hydroxyl groups are substituted by acetate groups, no intrachain H-bond can be formed in CTA. The size of side

chain of CTA (acetate group) is larger than that of NC (hydroxyl group). However, the previous study showed that for the vast majority of macromolecules (including the case of CTA) the size of side chain do not remarkably affect the single-chain elasticity of the macromolecule in an organic solvent.43 Therefore, the results observed after group substitution should be associated with the H-bonding capability of the group. The F−E curves of CTA obtained in nonane and DMSO can be superposed well (Figure 2a), indicating that the single-molecule mechanics of CTA is independent of the solvent polarity. In addition, we find that both dF/dx of CTA obtained in the two organic solvents increase monotonically in the whole range (see Figure S4). These results validate that the appearance of the turning point in dF/dx of NC in nonane (Figure S3b) is closely related to the intrachain H-bonds. Interestingly, the F−E curve of NC obtained in DMSO can be superposed well with that of CTA obtained in nonane in the entire force region (see Figure 2b). NC and CTA present the same single-molecule mechanics, implying that the statuses of intrachain H-bonds for the two systems (NC in DMSO and CTA in nonane) are identical. Note that no intrachain Hbonds can be formed in CTA. Thus, it can be confirmed that the intrachain H-bonds of NC are completely eliminated in DMSO. Furthermore, similar force measurements of amylose and ATA are performed in both nonane and DMSO. As shown in Figure 3a, the normalized F−E curve of amylose obtained in nonane is remarkably higher than that in DMSO. This result implies that amylose can form intrachain H-bonds in nonane, 5008

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Figure 4. Normalized single-chain F−E curves of NC (blue) and amylose (red) obtained in DMSO (a) and nonane (b).

Figure 5. (a) Normalized single-chain F−E curves of NC obtained in DI water (blue), nonane (red), and DMSO (green thin curve). (b) Normalized single-chain F−E curves of amylose obtained in DI water (blue), nonane (red thin curve), and DMSO (green).

like NC? It is interesting to find out the dominant factor for the biological properties of NC and amylose. Water (the most abundant molecule in a living cell), which is deeply involved in the structures and functions of DNA and proteins, plays a fundamental role in biological systems.1,2,45 However, the effects of water on the biological properties of polysaccharides remain poorly understood. It is interesting to investigate the role of water in the structures and properties of NC and amylose. When water is used as the environment in the force measurements, a remarkable difference between the F−E curves of NC and amylose can be observed (see Figure 5 and Figure S7). It has been reported that the long flat plateau (hereafter referred as the flat plateau) observed in the F−E curve of NC in DI water is closely related to the hydrophobic hydration of NC chain upon pulling (see Figure S7a).33 The fingerprint shoulder-like plateau (hereafter referred as the shoulder plateau) in the F−E curve of amylose in DI water is attributed to the force induced conformational transitions of sugar rings (see Figure S7b).30,46,47 In addition to the difference in plateaus, there is a remarkable deviation in the high force region of the F−E curves obtained in DI water (see Figure 5). The F−E curves of NC obtained in DI water and nonane can be superposed well when F > 200 pN (Figure 5a). However, the F−E curve of amylose obtained in nonane is higher than that in DI water when F > 700 pN; the F−E curve in DI water can be superposed with that obtained in DMSO when F > 1100 pN (see Figure 5b). These results imply that the intrachain H-bonds of NC can be formed in both nonane and DI water. The intrachain H-bonds of amylose can be formed in nonane. However, the intrachain H-bonds of amylose can be largely eliminated in DI water. Thus, we

while these intrachain H-bonds will be eliminated completely in DMSO. Similar to CTA, no intrachain H-bond can be formed in ATA due to the absence of the H-bond donor (Figure 3b, inset).9,44 The F−E curves of ATA obtained in nonane and DMSO can be superposed well in the entire force region (Figure S6), indicating that the single-molecule mechanics of ATA is independent of the solvent polarity. The F−E curve of amylose obtained in DMSO can be superposed well with that of ATA obtained in nonane in the entire force region (Figure 3b), implying that the intrachain Hbonds of amylose can be completely eliminated in DMSO. Amylose and ATA show the same single-molecule mechanics when the formation of the intrachain H-bonds is prohibited due to the effect of solvent (amylose in DMSO) or the absence of hydroxyl group (ATA in nonane). As shown in Figure 2b, NC and CTA also show the same single-molecule mechanics when the formation of the intrachain H-bonds is prohibited due to the effect of solvent (NC in DMSO) or the absence of hydroxyl group (CTA in nonane). These experimental results demonstrate that the single-molecule mechanics of both NC and amylose can be effectively affected by the solvent polarity through regulation of the intrachain H-bonds. Figure 4a shows that in DMSO the normalized single-chain F−E curves of NC, and amylose can be superposed well in the entire force region. Surprisingly, the F−E curves of NC and amylose obtained in nonane can also be superposed well in the entire force region (see Figure 4b). These results show that NC and amylose share the same status of H-bonds in a given organic solvent (DMSO or nonane). In a given organic solvent amylose and NC present the same single-molecule mechanics even if the linkages between the sugar rings are different (α or β). Thus, why amylose cannot be used as structural material 5009

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cooperativity of intrachain H-bonds but amylose cannot due to the much fewer intrachain H-bonds. Therefore, the F−E curve of NC in water is remarkably higher than that of amylose in the force region of 700−2500 pN. The impressive mechanical strength is necessary for NC which serves as the structural material. In one word, the extensive intrachain H-bonds is a key factor for the mechanical strength and relative hydrophobicity of NC in water at both the single-molecule and aggregate state. In general, starch is fragile and not suitable for structural material due to the relatively low crystallinity. In a starch granule, only intermolecule (i.e., interchain and amylose− water) H-bonds are used to promote the aggregation of amylose molecules.7 These H-bonds contribute to the solubility and degradability of amylose.50,52 The force measurements and MD simulations results indicate that at the single-molecule level amylose can form extensive H-bonds with water molecules, while the intrachain H-bonds are largely eliminated in water. These results suggest that the extensive Hbonds with water contribute to the relative hydrophilicity of amylose at the single-molecule level. In a given organic solvent (nonane or DMSO), NC and amylose present the same status of H-bonding and singlemolecule mechanics, which, however, differ largely in water. The mechanics and affinity for water of NC and amylose can be effectively affected by water through regulation of the Hbonds (intrachain and polysaccharide−water). These results demonstrate that water is deeply involved in the structures and properties of both NC and amylose. In addition, the effects of ionic strength and temperature on the single-chain mechanics of NC and amylose are studied. Recently, Walker and Li studied the effects of salt concentration on the plateau height in the F−E curve of a hydrophobic polymer (polystyrene).13 They found that the plateau height is proportional to the polymer−solvent interfacial free energy, which will be increased with the increasing of salt concentration. As shown in Figure 7a, the height of the flat plateau of NC is increased with the increasing of ionic strength, indicating that the flat plateau is related to the hydrophobic hydration of NC chain upon pulling.13,33

speculate that in DI water NC can form more intrachain Hbonds than amylose. This speculation is supported by MD simulations, where the H-bonds of NC and amylose oligomers in water can be analyzed. The number of H-bonds of NC (or amylose) in water can be described by the equation NT = Nintra + Nw

(1)

where Nintra and Nw are the number of H-bonds of the oligomer that can form with the neighboring repeating units and water molecules, respectively, and NT is the total number of H-bonds of the oligomer. It is well-known that glucose is the structure unit for both NC and amylose.48 This means that in water NT of NC is the same as that of amylose, which is supported by MD simulations (Figure S8). Interestingly, the MD simulations results (Figure 6) show that Nintra of NC is

Figure 6. Number of intrachain H-bonds of NC and amylose oligomers in the MD simulations. The time-averaged number of intrachain H-bonds is 4.18 for NC and 1.80 for amylose.

remarkably larger than that of amylose, which supports the speculation from the force measurements. According to eq 1, one can conclude that Nw of NC in water is less than that of amylose. This result indicates that NC is more hydrophobic than amylose at the single-molecule level. The force measurements and MD simulations results suggest that the status of Hbonds (intrachain and polysaccharide−water) is a key factor for the affinity for water of these two polysaccharides at the single-molecule level. It is well-known that the biological properties of NC and amylose are closely related to their affinity for water: the hydrophobicity is very important for NC that serves as the structural material, while the hydrophilicity is necessary for amylose that serves as the energy storage compound.49−52 Extensive intrachain and interchain H-bonds can be formed in the NC crystals. These H-bonds contribute to the stability of the flat ribbon structure of NC molecules and promote the fibrous aggregation.3,6 NC exploits these H-bonds to impart impressive mechanical strength and water insolubility to the cell wall of plants.51,53 The force measurements results demonstrate that NC forms fewer H-bonds with water than amylose. Thus, NC is more hydrophobic than amylose. This conclusion is supported by MD simulations (Figure 6). It has been reported that the stability of both DNA double helices and protein folds can be strengthened by the cooperativity of H-bonds.54−56 It can be speculated that the single-molecule mechanics of NC can be effectively strengthened by the

Figure 7. Height of the flat plateau of NC obtained in KCl solutions with different concentrations (a) and in DI water with different temperatures (b). Height of the shoulder plateau of amylose obtained in KCl solutions with different concentrations (c) and in DI water with different temperatures (d). For each condition, n ≥ 15. 5010

DOI: 10.1021/acs.macromol.9b00179 Macromolecules 2019, 52, 5006−5013

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Macromolecules The height of the flat plateau of NC is increased with the increasing of temperature (see Figure 7b). It has been reported that the plateau height in the F−E curve of a hydrophobic polymer is closely associated with the hydration free energy per monomer in the chain (ΔGHyd), which is temperaturedependent. For the size of sugar ring, ΔGHyd is a monotonic increasing function of temperature in the accessible range up to 100 °C.33 The result shown in Figure 7b further confirms that the flat plateau of NC is closely related to the hydrophobic hydration. Cui and co-workers found that the height of the shoulder plateau of amylose is positively associated with the amount of bound water around amylose.30 As shown in Figure 7c, the heights of the shoulder plateau of amylose are almost constant, even if the concentrations of KCl vary largely (0.01−1.0 M). This result means that the amount of bound water around amylose is virtually independent of the ionic strength. As shown in Figure 7d, the height of the shoulder plateau of amylose decreases with an increase in temperature. This result is reasonable since the amount of bound water around amylose will decrease with increasing temperature.34,57 Beyond the plateau (i.e., F > 400 pN), the F−E curves of NC (or amylose) obtained in KCl solutions with different concentrations (or in DI water with different temperatures) can be superposed well (Figures S9 and S10), indicating that the ionic strength and temperature range in this work do not significantly affect the statuses of intrachain H-bonds of the polysaccharides.

environment of organisms) in the properties and functions of polysaccharides.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.9b00179. Details of MD simulations, details of interfacial free energy, supporting data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel/Fax +86-28-87600998. ORCID

Zhong-yuan Lu: 0000-0001-7884-0091 Shuxun Cui: 0000-0002-7713-7377 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21774102 and 21604013), the Sichuan Youth Science & Technology Foundation (2017JQ0009), and the Fundamental Research Funds for the Central Universities (2682019JQ01 and 2682018CX08). Helpful suggestions from the anonymous reviewers are greatly appreciated.

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CONCLUSIONS In this study, single-molecule AFM and MD simulations are used to investigate the effects of H-bonds on the singlemolecule mechanics and affinity for water of NC and amylose. The experimental results show that both NC and amylose can form intrachain H-bonds in a nonpolar solvent (nonane). These intrachain H-bonds can be completely eliminated by a highly polar solvent (DMSO). Thus, the single-molecule mechanics of both NC and amylose can be effectively affected by the solvent polarity through regulation of the intrachain Hbonds. This conclusion is supported by the results from control samples (CTA and ATA) as well as salt- and temperature-dependent experiments. The F−E curves of NC and amylose obtained in a given organic solvent can be superposed well, implying that they share the same status of H-bonds in a given organic solvent (nonane or DMSO). However, the statuses of H-bonds (intrachain and polysaccharide−water) of NC and amylose differ largely in water. NC forms fewer H-bonds with water than amylose. Thus, NC is more hydrophobic than amylose. During single-chain elongation, NC consumes more energy than that of amylose in the force region of 700−2500 pN due to the cooperativity of intrachain H-bonds. The properties and functions of NC and amylose differ largely, even though they share the same structure unit (glucose). The relative hydrophobicity and impressive mechanical strength are very important for NC which serves as the structural material, while the relative hydrophilicity is necessary for amylose which serves as the energy storage compound. These results reveal that water is a special solvent for amylose and NC: both mechanics and affinity for water of these polysaccharides can be effectively affected by water through regulation of the Hbonds. This study casts new light on the roles of water (the key

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DOI: 10.1021/acs.macromol.9b00179 Macromolecules 2019, 52, 5006−5013

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DOI: 10.1021/acs.macromol.9b00179 Macromolecules 2019, 52, 5006−5013