Triple-Helix Conformation of a Polysaccharide Determined with Light

Publication Date (Web): December 6, 2018. Copyright © 2018 American Chemical Society. *(L.Z.) E-mail: [email protected]., *(X.X.) E-mail: ...
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Triple-Helix Conformation of a Polysaccharide Determined with Light Scattering, AFM, and Molecular Dynamics Simulation Yan Meng,† Xiaodan Shi,§ Liqin Cai,† Shihai Zhang,∥ Kan Ding,∥ Shaoping Nie,§ Chuanfu Luo,*,‡ Xiaojuan Xu,*,† and Lina Zhang*,† †

College of Chemistry & Molecule Sciences, Wuhan University, Wuhan, China State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Chang Chun, China § College of Food Science, Nanchang University, Nanchang, China ∥ Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Macromolecules Downloaded from pubs.acs.org by YORK UNIV on 12/07/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: The chain conformation of a β-glucan extracted from black fungus (BFP) was studied by static/dynamic light scattering, viscometry, atomic force microscopy (AFM), and molecular dynamics (MD) simulation. The Mark−Houwink equation and the relationship between Mw and Rg of BFP in water at 25 °C were determined to be [η] = 1.78 × 10−7Mw1.6 and Rg = 5 × 10−4Mw0.9, and the molar mass per unit contour length (ML) and the persistence length (q) were 2724 ± 276 nm−1 and 230 ± 30 nm, respectively, indicating triple-helix conformation. Moreover, the stiff-chain lengths of the BFP fractions were visualized with AFM images, and their ML values were estimated to give a mean of 2212 nm−1, consistent with the above. Importantly, MD simulation confirmed that the triple helix was the most stable conformation of BFP. We identified, for the first time, the triple-helix chain conformation of BFP and also offered an alternative method for the characterization of the rigid macromolecules.

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INTRODUCTION Recently, serious environmental pollution from waste plastics has pushed the research and development of the sustainable polymers from renewable resources such as natural polysaccharides;1−5 thus, there are huge research efforts worldwide to use biomass.6 The theme of the 249th ACS National Meeting in 2015 was chemistry of natural resources. In nature, polysaccharides are widely distributed and can promote healthy development without toxic side effects on the body.7 Polysaccharides, especially those naturally occurring in plants and fungi, are of great importance in human anticancer immunotherapy.8,9 Early studies have shown that polysaccharides are recognized by lectins, which are located on the surface of a cell, stimulating the immune system and inhibiting tumor cells, viruses, or inflammatory factors.10−13 Recent research revealed that (1 → 3)-β-D-glucans having β-D-glucopyranosyl units attached by (1 → 6) linkages can enhance the immune system systemically, resulting in antitumor, antibacterial, antiviral, anticoagulatory, and wound healing activities.14−16 Generally, the biological activities of natural polysaccharides are closely related to their chain conformation; for instance, the polysaccharide from Lentinus edodes (lentinan) with triplehelix conformation shows high antitumor activity, whereas the single chain of lentinan displays almost no biological activity.16 Moreover, Mueller et al. 17 have indicated that rigid © XXXX American Chemical Society

polysaccharides are more likely to bind to cell receptors and exhibit higher bioactivity. Thus, detailed investigation of chain conformation of polysaccharides would be beneficial to better understand the structure−function relationship. However, even though many researchers have contributed to the field of polysaccharides, progress has come to a standstill due to their complicated structure, broad molecular weight distribution, and limited solubility in common organic solvents.18 To date, the main characterization methods of chain conformation of natural polysaccharides are static light scattering and dynamic light scattering (SLS/DLS),19,20 sizeexclusion chromatography (SEC),21,22 viscometry,23 and so forth, based on the polymer solution theory. Atomic force microscopy (AFM), a simple, intuitive and fast method, has also been developed for the observation of the shape, size, and aggregated morphology of macromolecular chains.24 Moreover, molecular dynamics simulation has become a powerful assisting method. For example, the molecular chain parameters, such as chain diameter (d), persistence length (q), and molar mass per contour length (ML), of DNA could be obtained by Monte Carlo simulation based on the bead-model hydroReceived: September 18, 2018 Revised: November 26, 2018

A

DOI: 10.1021/acs.macromol.8b02017 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. Photographs of black fungus (a) and BFP sample (b). SEM image (c) and structure of BFP (d).

dynamic calculation.25 Besides, molecular dynamics can be also used to reveal the formation of a hexaplex by oligomers of artificial nucleic acids bearing bifacial molecules on Dthreoninol.26 However, the dynamic simulation of molecular motion and conformation of polysaccharides have been rarely reported. Black fungus (Auricularia auricula) is an edible fungus and a popular ingredient in many Chinese dishes,27 as shown in Figure 1a. In our laboratory, a white cotton-like polysaccharide was extracted from black fungus,28 which demonstrated significant antitumor activity depending on its molecular weight.29 Moreover, the polysaccharide could self-assemble into hollow nanofibers in water,30 which could be used as carriers for drugs, dyes, and metal nanoparticles.31−33 It was inferred that the polysaccharide is a stiff chain from our primary studies,28 but it is hard to clarify the chain conformation from those limited data. Therefore, a detailed investigation of the chain conformation for this polysaccharide is essential for successful interpretation of their bioactivities and self-assembling behaviors as well as application in the biomedical field. Moreover, new characterization methods for some rigid macromolecules including cellulose and chitin34,35 are greatly needed to be established. We herein reported the chain conformation determination of a polysaccharide extracted from black fungus, coded as BFP, with SLS/DLS, viscometry, AFM, and molecular dynamics simulation. AFM was used to visualize the chain shape and size of the BFP to further identify its conformation, providing a simple alternative method to calculate ML. Importantly, theoretical simulation strongly supported that the BFP polysaccharide exists as a triple-helix conformation in water. This work provides an effective strategy to determine the chain conformations of rigid macromolecules through combination of multiple experimental methods and theories. This is very important for revealing the key roles of polysaccharides in both chemistry and life science.

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then immersed in 70% ethanol/water solution at room temperature for 24 h to remove the supernatant. The residues were dipped into 0.9% aqueous NaCl and stirred at 85 °C for 4 h, and then the resultant cold solution was centrifuged to obtain a supernatant, followed by decolorization (dropping with 50−100 mL of 30% H2O2 at stirring state) and deproteinization according to the reported Sevage method.31 The BFP transparent solution was obtained, and 70% ethanol/water solutions were added for further purification at 25 °C. The precipitates were redissolved in water and dialyzed in ultrapure water, followed by lyophilization to give the final cotton-like sample, denoted as BFP (shown in Figure 1b). The fractions of BFP used in this work were obtained by organic solvent precipitation of the degraded BFP aqueous solutions with ultrasonic treatment (shown in Figure S3). In detail, dissolved aqueous solutions (1 mg/ mL) of BFP sample were treated with an ultrasonic cell disruption system (SCIENTZ, JY 98-IIIDN, 800W) for 5−120 min. Each of the sonicated solutions was precipitated by dropping ethanol (or acetone) until the sediment appeared, after which the solution was centrifuged at 8000 rpm (Beckman Coulter, Avanti J-E, Germany) for 15 min to get a precipitate. To ensure the purity of each BFP fraction, the first sediment was usually discarded. The supernatant was precipitated again, followed by dissolution in water, dialysis, filtration, and lyophilization to get polysaccharide fractions. Moreover, each fraction was screened by SEC-MALLS analysis to ensure the suitable Mw range and narrow Mw dispersion. The final fractions were then coded as BFP-1, BFP-2, ..., BFP-9. Characterizations of Chemical Structure. The total sugar content of BFP was detected by the phenol−sulfate acid method as reported.36 A high performance anion exchange chromatogram (HPAEC) was used to determine the monosaccharide composition of BFP according to the method of Shi et al.37 Monosaccharide standards (glucose, galactose, arabinose, xylose, mannose, rhamnose, fucose, fructose, glucuronic acid, and galacturonic acid), methyl iodide, sodium borodeuteride (NaBD4), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Glucosamine and galactosamine were obtained from USA Acros Organics Co. The glycosidic linkage of BFP was analyzed by methylation analysis38 combined with a gas chromatograph−mass spectrometer (GC-MS, Agilent Technology 7890/7000 QQQ, USA). The GC-MS system was equipped with an SP-2330 (Supelco, Bellefonte, PA) capillary column (30 m × 0.25 mm, 0.2 mm film thickness). The methylated polysaccharides were subsequently hydrolyzed by 4 M trifluoroacetic acid (TFA) at 100 °C for 6 h, followed by reduction with NaBD4 and neutralization with acetic acid. The resultant partial methylated alditol acetates (PMAAs) of BFP were dissolved in CH2Cl2 and subjected to the GC-MS system after

EXPERIMENTAL SECTION

Preparation and Fractionation of Polysaccharides. The polysaccharide was isolated and purified according to the previously reported method.28 Dried fruit bodies of black fungus, purchased from local supermarkets in China, were briefly crushed, defatted with hot ethyl acetate and methanol for 4 h by a Soxhlet extractor, and B

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dissolved in ultrapure water to give concentrations of 1 × 10−8−1 × 10−6 g/mL. The solutions were purified by 0.22 μm membranes, deposited onto freshly cleaved mica, and air-dried overnight prior to imaging. The average chain lengths of BFP fractions were measured by software (Nano Measurer 1.2, Fudan University, China). Molecular Dynamics Simulation: Simulation Software and Force Field. Full atomic molecular dynamics (MD) simulation combined with simulated annealing (SA) algorithm was used to find the lowest energy structure of BFP. The Gromacs-2016.4 software package39−41 was used. The force field model is GLYCAM_06j-1,42 which is based on the AMBER force field.43,44 The optimization of parameters for carbohydrates makes GLYCAM_06j-1 an ideal force field model for studying polysaccharides. The tleap tool in AmberTools16 was used to generate the initial structures of BFP, and the initial structures were then converted into Gromacs’s input file. TIP3P water molecular model was used in the simulation of water solution.45 All 3D structure pictures were generated by VMD.46 The simulation parameters were as follows: the integral method was the frog leaping algorithm (Verlet-LeapFrog), and the integral step length was 2 fs. For simulations in a vacuum, no water molecules were added. Thus, the NVT ensemble and periodic boundary conditions were applied. Because of the periodic boundary conditions, the cutoff distance of 1.2 nm for van der Waals and Coulomb interactions was used. The temperature strategy of simulated annealing was set to drop from 400 to 200 K by 200 ns, and the cooling rate was 1 K/ns. It was found in simulations that the energy barriers between different metastable sets of Φ and Ψ were quite high, so 500 different initial structures with random values of Φ and Ψ were generated. Since the torsion angles of side-branched glycosidic bonds were found to be easily optimized, the initial values of Φ′ and Ψ′ in side chains were randomly set before the structural optimization. In order to obtain a reasonable structure and maintain a moderate amount of calculation, 10 repeating units were selected in the construction of the initial structure of single BFP chain, each of which contained three main-chain sugar rings and two side-chain sugar rings, and then an end sugar ring was added in the tail of the 10 repeating units. A two-step strategy was performed to get the optimized structure of single-chain BFP in a vacuum. At first, steep descent optimization for all the initial 500 randomly generated structures followed by 10 structures with lowest energies was selected to carry out simulated annealing from 400 to 200 K by 200 ns. At the end of simulated annealing, there were three samples converted to the same lowest energy, which was concluded as the final optimized structure. To study the structures of double-helix and triple-helix, we generated five initial structures for both double-helix and triple-helix types of BFP chains by rotating 180° or 120°/240° around the helical axis of a single BFP chain and then combining two or three chains together with slight translations along the helical axis. After simulated annealing at the same conditions above, the stability of double-helix and triple-helix chains was examined. MD simulations in pure water of the triple-helix BFP chain were performed by solving the optimized structures in a vacuum into a water box. The size of the simulation box was 12 × 4 × 4 nm3, and periodic boundary conditions were used. The temperature of simulation was 300 K by using a NVT ensemble, and the water model was TIP3P.47 There were 6097 and 5492 water molecules for single- and triple-helix BFP, respectively. The densities of both systems were about 990 g/L. The cutoff distance for the van der Waals interaction was set as 1.2 nm, and the long-range Coulomb interaction was calculated by the PME method. The systems were first relaxed at 300 K by 20 ns and then followed with a production simulation of 40 ns.

filtration through a 0.22 μm membrane. The FTIR spectrum of BFP was recorded with a Nicolet 170SX FT-IR spectrometer (Spectrum One, PerkinElmer Co., Madison, WI) in the range 4000−400 cm−1. 1 H and 13C NMR measurements of BFP in DMSO-d6 were taken on a Mercury 600 NMR spectrometer (Varian Inc., Palo Alto, CA) at 25 °C. Light Scattering Measurements. The scattering light intensities of the dilute solutions of BFP and its fractions in water at 25 °C were measured on a modified commercial light scattering spectrometer (ALV/SP-125, ALV, Germany) equipped with an ALV-5000/E multiτ digital time correlator and a He−Ne laser (at λ = 632.8 nm). The angular and concentration dependences of the scattered intensities were analyzed using Zimm plots to determine the weight-average molecular weight (Mw), the radius of gyration (Rg), and the second viral coefficient (A2). The basic light scattering equation is as follows: yz KC 1 ijj 16π 2n2 2 jj1 + = ⟨s ⟩ sin 2(θ /2) + ...zzzz + 2A 2 c 2 j Rθ Mw k 3λ 0 { 2 2

(1)

/(NAλ04)

2

and q = (4πn/λ0) sin(θ/2) with c, where K = 4π n (dn/dc) dn/dc, NA, and λ0 being the concentration of polymer solution, the specific refractive index increment, Avogadro’s number, and the wavelength of light in a vacuum, respectively. The specific refractive index increment (dn/dc) of BFP was 0.136 mL/g in water at 633 nm and 25 °C.28 Dynamic light scattering (DLS) measurements were used to characterize the hydrodynamic radii (Rh) of polysaccharides in water at 25 °C by the analysis of the CONTIN program. The tests were carried out on the instrument mentioned above. The average hydrodynamic radius (⟨Rh⟩) can be calculated by using the Stokes− Einstein equation: ⟨R h⟩ =

kBT 6πη0⟨D⟩

(2)

where kB is Boltzmann’s constant and η0 is the solvent viscosity at 25 °C. Size-Exclusion Chromatography with Multiangle Laserlight Scattering (SEC-MALLS) Analysis. The parameters of BFP fractions in water or DMSO were also detected by SEC columns combined with MALS (DAWNDSP, Wyatt Technology Co., USA) equipped with a He−Ne laser at λ = 663.4 nm, differential refractive index detector RI (Opitilab T-rEX, λ = 658.0 nm), and Vis (ViscoStar-II). The measurements were carried out on the above instruments equipped with a combination of two Shodex-OHpak columns (SB-806 M HQ, SB-803M HQ) at a flow rate of 0.5 mL/ min. The injection volumes were 500 μL, and the eluent was 0.9% aqueous NaNO3 solution or DMSO, which was filtered by 0.22 μm membranes and degassed before use. The temperature of columns was kept at 25 °C in the water phase and 50 °C in the organic phase, and the dn/dc of BFP in DMSO was 0.068 mL/g.30 Viscometry. Intrinsic viscosities ([η]) of the BFP solutions in water with different molecular weights were measured at 25 °C by an Ubbelohde capillary viscometer. The kinetic energy correction was always negligible. Huggins and Kraemer plots were used to calculate the [η] values.

ηsp /c = [η] + k[η]2 c

(3)

(ln ηr )/c = [η] − β[η]2 c

(4)

where k and β are constants for a given polymer under the desired conditions, ηsp/c is the reduced viscosity, and (ln ηr)/c is the inherent viscosity. AFM Measurements. AFM images of BFP fractions were measured with an atomic force microscope (AFM, Cypher ES, Asylum Research, USA) in ac mode at 35 °C. Silicon probes with a spring constant of 2 N/m and resonance frequency of 70 kHz (OLTESPA-R3, Bruker) were employed. All data from the images were analyzed by using AFM accessory software, and the images presented were flattened only when necessary. All fractions were

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RESULTS AND DISCUSSION

Chemical Structure of the Black Fungus Polysaccharide. The BFP polysaccharide appeared as a white cotton-like sample (Figure 1b), which exhibited parallel fibril microstructure, supported by SEM observation (Figure 1c). It was not hard to imagine that the formation of such submicrofibers C

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Table 1. Experimental Data of BFP Fractions in Water at 25 °C Obtained from SLS, DLS, SEC-LLS, and Viscometry samples BFP BFP-1 BFP-2 BFP-3 BFP-4 BFP-5 BFP-6 BFP-7 BFP-8 BFP-9

Mw × 10−4

sonication time 0 5 10 12 15 18 20 40 60 120

a

216 173a 142a 122a 100a 85a 81a 76a 58a 46a

240b 161b 128b 116b 93b 88b 84b 82b 56b 41b

PDI (Mw/Mn)

Rg (nm)

Rh (nm)

ρ

[η] (mL/g)

nk

1.2b 1.4b 1.5b 1.4b 1.2b 1.1b 1.4b 1.2b 1.4b 1.5b

215a 167a 131a 118a 100a 96a 90a 81a 60a 50a

88a 69a 53a 46a 46a 45a 42a 39a 29a 25a

2.5 2.2 2.3 2.4 2.2 2.1 2.1 2.1 2.0 2.0

2148 1446 836 644 561 526 414 318 230 155

1.70 1.36 1.11 0.96 0.78 0.67 0.64 0.60 0.46 0.36

105c 48c 37c 30c 20c 17c 24c 22c

a Parameters of BFP fractions measured with SLS/DLS in water. bMolecular weight of BFP fractions measured with SEC-LLS in water. cMolecular weight of BFP fractions measured with SEC-LLS in DMSO.

at least 2−3 times. Each fraction was screened by size exclusion chromatography combined with laser light scattering (SECLLS) analysis (Figure S4), and the results are listed in Table 1. As shown in Figure 2a, with an increase of ultrasonication time,

is related to their rigid chain conformation. We further clarified the chemical structure of BFP with FT-IR, high performance anion exchange chromatography (HPAEC), GC-MS, 13C NMR, and so on. The total sugar content of BFP was calculated as 91.3 ± 1.3% by the phenol−sulfate acid method.36 The HPAEC data (Figure S1a) exhibited that only one peak corresponding to glucose appeared, indicating that BFP was a glucan. A characteristic absorption peak of the BFP sample at 890 cm−1 appeared in the FT-IR spectrum (Figure S1b), suggesting the β-glucan structure of BFP.48 Moreover, GC-MS analysis of methylated BFP (Table S1) revealed that the molar ratio of terminal glucose, 1,3-linked glucose, and 1,3,6-linked glucose was around 2:1:2, revealing a β-(1 → 3)-D-glucan with two β-(1 → 6)-D-glucosyl residues for every three main-chain glucosyl residues. The NMR data (Figure S2 and Table S2) further confirmed the chemical structure. The typical signal belonging to uronic acid (176.5 ppm) was not observed in the 13C NMR spectrum, demonstrating BFP was a neutral polysaccharide. Moreover, the ratio of the integral value of C2t (side chain) at 74.3 ppm of glucan BFP to that of C2 (main chain) at 73.0 ppm, corresponding to the ratio of the terminal units on side residue to the backbone units to be 1:1.4, further supported the conclusion obtained by GC-MS. Therefore, BFP was identified as a β-(1 → 3)-D-glucan with two β-(1 → 6)-D-glucosyl residues for every three main-chain glucose residues, as shown in Figure 1d, which is consistent with our previous report.28 It was demonstrated that the preparation of the polysaccharide BFP from black fungus is repeatable, providing the base of scale production of BFP for its applications. Moreover, the structure was similar to other well-characterized triple-helix polysaccharides, such as schizophyllan,49 scleroglucan,50 and lentinan,51 which have the same backbone of β-(1 → 3)-Dglucan but different branching degrees at C6. Chain Conformation of BFP Determined on the Basis of Solution Theory. To study the chain conformation of polymers, it is necessary to prepare several fractions with different molecular weights and a narrow distribution. As reported, ultrasonic treatment is considered as one of the most promising, effective, and environmentally friendly physical treatments in preparing fractions with different molecular weights as it allows the degraded products to be more easily recovered and purified with no change in structure.52 Herein, ultrasonication in combination of precipitation by acetone (or alcohol) was used to prepare BFP fractions (Figure S3). Notably, to ensure a relatively narrow distribution of molecular weights, the degraded polysaccharide solution was precipitated

Figure 2. Mw dependence on sonication time of BFP fractions (a) and the Rh distributions of different BFP fractions (b). The doublelogarithm relationship between Mw and [η] (c) and Rg (d). The fitted curves from experimental results via viscosity (e) and Rg (f) according to the HW model.

the Mw of BFP fractions decreased dramatically at first 15 min and then kept stable. The results provided important information for controlling Mw of BFP. Generally, the measured Mw values of polysaccharides in aqueous solution might be too much higher due to the aggregation of their chains; hence, extremely diluted solution was usually used to keep chains dispersed without aggregation.53,54 Take the hydrodynamic radius (Rh) distribution of BFP-1 as an example (Figure S5a); a shoulder peak or even new peak corresponding to aggregation appeared once the concentration beyond 0.1 mg/mL but exhibited as a single peak while the concentrations D

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Macromolecules were 2.0 on the whole, indicative of a stiffchain conformation in water. Moreover, the Mark−Houwink equation ([η] = KMwα) was calculated from the doublelogarithmic plot of [η] against Mw (Figure 2c) as follows: [η] = 1.78 × 10−7M w1.6 (mL/g)

(11)

log(dr 2/A 0) = 0.173 + 2.158 log d r

(12)

where Φ0 is the Flory constant (2.86 × 1023 mol−1) when dr ≤ 0.1 (for stiff chains), v̅ is the partial specific volume (the value is 0.68 cm3g−1 for polysaccharides), and NA is the Avogadro constant. The chain diameter (d) can be obtained from the formula d = dr 2q

(13) 2

1/3

Mw1/2

From the plot of (Mw /[η]) against of the BFP fractions (Figure S7a), the parameters of ML, q, and d were calculated to be 3362 nm−1, 192 nm, and 4.6 nm (the black line in Figure 2e), respectively. Moreover, according to the Yamakawa−Fujii−Yoshizakai theory mentioned above,60 the theoretical line (ML = 3000 nm−1, q = 200 nm, and d = 2.2 nm) was obtained (the red line in Figure 2e), which was more fitted with the experimental data than the black line. Therefore, the parameters of BFP approached ML = 3000 nm−1, q = 200 nm, and d = 2.2 nm. Furthermore, the Benoit−Doty equation (BD theory)61 has also been used to calculate ML for the wormlike chain model; Zhang et al.62 had simplified the Benoit−Doty equation as follows:

(5)

Generally, the exponent α is related to the shape of polymer, which is also used to describe the stiffness of polymer chains. The α value is in the range 0.5−0.8 for flexible polymers in good solvent, whereas those above 0.8 and even beyond 1.0 represent stiff chains.55 Herein, the α value of 1.6 was much higher than those of some stiff polysaccharides, such as lentinan (1.31)56 and xanthan (1.14),57 suggesting the super rigid chain conformation of BFP. Similarly, the relationship between Rg and Mw of BFP fractions (Rg = K′Mwα′) was established as follows: R g = 5 × 10−4M w 0.9 (nm)

dr 2/A 0 = (4Φ0 /1.215πNA )(υ ̅ /A η)Bη4

(M w 2 /12R g 2)2/3 = ML 4/3 + (2/15)(ML1/3/q)M w

(14)

(M w /R g 2)1/2 = (3ML / q)1/2 + 3ML(3qML)1/2 /2M w

(15)

For stiff chains, namely, where the nk value (nk = Mw/2qML) was