Letter Cite This: ACS Macro Lett. 2018, 7, 672−676
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Effects of Water Content of the Mixed Solvent on the SingleMolecule Mechanics of Amylose Lu Qian, Yu Bao, Weili Duan, and Shuxun Cui* Key Laboratory of Advanced Technologies of Materials (Ministry of Education), Southwest Jiaotong University, Chengdu 610031, China S Supporting Information *
ABSTRACT: It is generally recognized that water is deeply involved in the structures and functions of DNA and proteins. For polysaccharides, however, the role of water remains unclear. Due to the force-induced conformational transition of the sugar rings, a fingerprint plateau can be observed in the single-chain force− extension (F−E) curves of amylose and some other polysaccharides in aqueous solutions. In this study, the effects of water content of the mixed solvents on the fingerprint plateau of amylose are explored by single-molecule AFM. The experimental results obtained in a series of water/alcohol mixed solvents clearly show that both the appearance and the fingerprint plateau height in the F−E curves of amylose are dependent on the water content. Since water is a good solvent for amylose but alcohols are not, the higher water content of a mixed solvent corresponds to a better solvent quality. Thus, the observed results can be associated with the solvent quality to amylose. The present study implies that water is not only a solvent but also an active constituent in the amylose solution.
T
he pyranose ring-based polysaccharides play crucial roles in many vital biological activities such as energy storage, structural support, and cellular recognition.1,2 Meanwhile, polysaccharides are important raw materials in the renewable energy industry, textile industry, food industry, etc.3 It has been recognized that life is stressful, in a mechanical sense.4,5 By using the atomic force microscopy (AFM)-based singlemolecule force spectroscopy (SMFS),6−22 the single-chain mechanics of polysaccharides can be better understood.6,23−25 The chair conformation is the most stable one for the glucopyranose ring.26,27 When a single polysaccharide chain is stretched, the chair conformation of sugar rings will switch to a boat or an inverted chair conformation, which will result in the fingerprint plateau in the single-chain force−extension (F−E) curve.28−30 As an atomic lever, the glycosidic bond produces the necessary torque to trigger the conformational transition in the sugar rings.27−29 Marszalek and his co-workers found that the single-chain mechanics of polysaccharides can be strongly affected by the solvent.25,31 Water, which is deeply involved in the structures and functions of biomacromolecules (e.g., DNA and proteins), plays a fundamental role in biological systems.32−34 Yet, the role of water in the structures and functions of polysaccharides remains unclear. Here in this study, we attempt to elucidate the effects of water content of the mixed solvents on the singlechain mechanics of amylose. The single-chain F−E curve of amylose obtained in deionized (DI) water displays a fingerprint plateau (Figure 1a and Figure S1). When the first derivative of force reaches the minimum, the corresponding force is 320 pN (Figure 1), which is defined as the height of the fingerprint plateau. However, the © XXXX American Chemical Society
Figure 1. (a) Normalized single-chain F−E curve of amylose obtained in DI water. The red dotted line is the polynomial fitting curve. (b) The first derivative of the polynomial fitting curve shown in (a).
fingerprint plateau disappeared when the force measurements are carried out in a typical nonpolar solvent, octane (Figure Received: May 14, 2018 Accepted: May 21, 2018
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DOI: 10.1021/acsmacrolett.8b00375 ACS Macro Lett. 2018, 7, 672−676
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ACS Macro Letters S2). It is interesting to find out whether the single-chain mechanics of amylose can be influenced by the water content of the liquid environment.35 Alcohol, which is miscible with water, can be a suitable system to investigate the effects of water content of the mixed solvents on the single-chain mechanics of polysaccharides. SMFS experiments of amylose are carried out in a series of water/1-propanol mixed solvents, in which the water content (χwater) is increased gradually from 0 to 100% (W/W). When the water content is CWC, the proportion is increased to 100%; i.e., all of the F−E curves show a plateau in this case. These results indicate that the amylose chains have two different structures at CWC, while the population of each structure (“all” and “none” modes) is dependent on the water content of the mixed solvents. The two-state modified freely jointed chain (M-FJC) model has been used to describe the conformational transition of the modules in a polymer chain upon stretching (see the Supporting Information for details).45 Here, the two-state MFJC model is used to fit the F−E curve of amylose with a plateau obtained in water/1-propanol mixed solvent at CWC (see Figure 3). The lengths of the structural units of the two states used in the model before and after the conformational
Figure 2. Normalized single-chain F−E curves of amylose obtained in water/1-propanol mixed solvents. The water contents are χwater = 15% (blue), χwater = 4%−14% (CWC, red and black), and χwater < 4% (green), respectively.
the water content is 15%, only one type of F−E curve can be observed (Figure 2), each presenting a plateau at 253 pN (Figure S3). Interestingly, when the water content is gradually increased from 4% to 14%, we find that two kinds of F−E curves can be observed: one with a plateau (similar to those obtained when water content is 15%) and the other without (similar to those obtained when the water content CWC and < CWC, respectively. Therefore, the higher the water content of the environment, the higher the plateau height in the F−E curves (Figure 4). A similar phenomenon can be found in water/ethanol mixed solvents (see Figure S6 and Table S2). Surprisingly, for the case of water content of 98%, the plateau height (285 pN) is still lower than that in neat water (320 pN) (see Figure 4). The H-bonding network of water will be disturbed upon the addition of alcohol.38,39,41 Alcohol molecules can influence the distribution of bound water around the sugar rings through the H-bonds. Therefore, the hydration number in mixed solvent is less than that in neat water, even if the alcohol content is as low as 2%. This may explain the sudden increase in the plateau heights at very high water contents (98%−100%) in Figure 4b. Similar results can be observed at high water content in water/ethanol mixed solvents (see Figure S6 and Table S2). The present study implies that the water content not only determines the appearance of a fingerprint plateau but also affects the height of it. In a word,
transition are 0.45 and 0.54 nm, respectively, which consist of the conformational transition from chair to boat.28 The fitting curve is superposed well with the experimental curve, confirming that the conformation of sugar rings will be changed from chair to boat upon force stretching (when water content is ≥ CWC). Interestingly, the F−E curve without plateau (water content < CWC) can be superposed well with the fitting curve when the free energy difference (ΔG0) between these two states is 0.5 kBT/unit (Figure 3). This means that the ΔG0 may be very small in mixed solvents when water content < CWC. With this small ΔG0, the chair−boat conformational transition may be finished in the low force region by the thermal disturbance (∼kBT). Therefore, no apparent plateau can be observed in this case. However, the ΔG0 is 5.1 kBT/unit for the F−E curve with a fingerprint plateau obtained at CWC. The only difference between the two conditions is the water content. These results indicate that the ΔG0 will be significantly increased when the amylose chain is turned from the “none” mode to the “all” mode. In other words, the ΔG0 will be increased by the bound water around the sugar rings. The proportion of chair conformation prior to extension will be increased from 62.2% to 99.4% when the ΔG0 is increased from 0.5 to 5.1 kBT/unit. That is, the chair conformation can be stabilized by bound water when the water content ≥ CWC. It can be speculated that the hydration number of the chair conformation is larger than that of the boat conformation. Similar experiments are carried out in water/ethanol mixed solvents (see Figure S4 for details). Interestingly, CWC can also be found in these mixed solvents. The only difference between the water/ethanol and water/1-propanol mixed solvents is the CWC values (see Table 1). The CWC ranges Table 1. CWC in Different Alcohol/Water Mixed Solvents mixed solvent
water/ethanol
water/1-propanol
CWC
8%−15%
4%−14%
in these two water/alcohol mixed solvents are very close, implying that these mixed solvents may share a similar fine structure at CWC. MD simulations and neutron diffraction are powerful tools to study the alcohol/water mixed solvents. We plan to study the fine structures of the mixed solvents at CWC by these methods in the future.
Figure 4. (a) Normalized single-chain F−E curves of amylose obtained in water/1-propanol mixed solvents. The water contents are χwater = 4%−14% (CWC, black and red), χwater = 50% (green), χwater = 98% (purple), and χwater = 100% (blue), respectively. To show the differences clearly, the F−E curves have been filtered. (b) The plateau height of amylose as a function of water content (from 0 to 100%). Error bars represent standard deviation. The height of the plateau is defined as zero if no fingerprint plateau can be observed in the F−E curve. Therefore, some of the data points do not have error bars. 674
DOI: 10.1021/acsmacrolett.8b00375 ACS Macro Lett. 2018, 7, 672−676
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ACS Macro Letters water is the key to the fingerprint of single-chain mechanics of amylose. In addition, more neat solvents (with various solvent polarities, see Table S3) are used as the environment in the force measurements. No fingerprint plateau can be observed from the F−E curves obtained in each of the neat organic solvents (Figure S7), indicating that the solvent polarity is not a key factor for the fingerprint plateau of amylose. Water is a good solvent for amylose, but alcohols are not. It has been reported that the solvent quality to amylose will be improved with the increase of water content of water/alcohol mixed solvents.47 This behavior is closely related to the Hbonds between water and the amylose chain. More H-bonds correspond to a better solvent quality. Our results show that the conformations of sugar rings are dependent on the H-bonds with water. The proportion of the “all” mode chains (sugar rings in one chain are fixed to the chair conformation) will be increased with the increase of water content. In other words, the solubility of amylose is positively correlated with the number of H-bonds between water and amylose. When water content < CWC, mixed solvent is a poor solvent for amylose. No plateau can be observed in this case. At CWC, the solvent quality reaches a threshold value, where a plateau can be observed in some F−E curves. When water content reaches 100% (neat water), the solvent quality is maximized, where a highest plateau can be observed. Thus, both the appearance and the plateau height in the F−E curves of amylose are dependent on the solvent quality. In summary, AFM-based SMFS is used to study the effects of water contents of the water/alcohol mixed solvents on the single-chain mechanics of amylose. Extensive results show that both the appearance and the fingerprint plateau height in the F−E curves of amylose are dependent on the water content. It is speculated that the chair conformation of the sugar rings can be stabilized by bound water, when the water content > CWC. The amount of bound water around the sugar rings will be increased with the increase of water contents beyond CWC. Since water is a good solvent for amylose but alcohols are not, the higher water content of a mixed solvent corresponds to a better solvent quality. Thus, the observed results can be associated with the solvent quality to amylose. The present study implies that water is not only a solvent but also an active constituent in the amylose solutions. These findings provide new insights into the role of water in the structures and functions of polysaccharides.
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(Bruker Corp., CA) and the sample surface. Alcohols and some other solvents can absorb water from air, which may affect the results of force measurements (Figure S8). To keep the water content of the mixed solvents, the sample is settled in a closed liquid cell of AFM. The spring constant of each AFM cantilever, which is around 45 pN/ nm, is obtained by the thermal excitation method. The instrumentation details of SMFS can be found elsewhere.7,46
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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/acsmacrolett.8b00375. Details of the two-state M-FJC model. Single-chain F−E curves of amylose obtained in various liquid environments, such as DI water, octane, water/ethanol, etc (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel./Fax: +86-28-87600998. ORCID
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 Natural Science Foundation of China (21774102) and the Sichuan Province Youth Science and Technology Innovation Team (2016TD0026, 2017JQ0009). The authors would like to thank Prof. Dr. Piotr Marszalek for helpful discussions.
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REFERENCES
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EXPERIMENTAL METHODS
Amylose is purchased from Sigma (Type III from potato, SigmaAldrich). Other chemicals are analytically pure and used without further treatment, unless specified otherwise. Deionized (DI) water (>18 MΩ·cm) is used when water is involved. Amylose is dissolved in DI water (by heating to 90 °C) to a concentration of 2 mg/L. In the force measurements, glass slides are used as the substrates. 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 by air flow. (Warning: Piranha solution is extremely oxidizing and should be handled with care!) To prepare the sample for SMFS, a few drops of the polysaccharide solution are deposited onto the clean substrate for 30 min. Then, the substrate is rinsed with abundant DI water to remove the loosely adsorbed polymers. Finally, the sample is immediately used in the force measurements. All the force measurements are performed on a commercial MFP3D AFM (Asylum Research, CA). Prior to the measurements, a drop of liquid is introduced between the V-shaped Si3N4 AFM cantilever 675
DOI: 10.1021/acsmacrolett.8b00375 ACS Macro Lett. 2018, 7, 672−676
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