Montmorillonite Nanocomposites: Effect of pH on the

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Silk Fibroin/Montmorillonite Nanocomposites: Effect of pH on the Conformational Transition and Clay Dispersion Qinqin Dang, Shoudong Lu, Shen Yu, Pingchuan Sun,* and Zhi Yuan* Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry and College of Chemistry, Nankai University, Tianjin, 300071, People’s Republic of China Received March 5, 2010; Revised Manuscript Received May 4, 2010

By adjusting the solution pH value below the isoelectric point (pI) of silk fibroin (SF) protein, the SF was in the cation state and it could interact strongly with unmodified anionic montmorillonite (MMT) surface. In this way, novel SF-MMT nanocomposites with good clay dispersion were successfully obtained, which were confirmed by X-ray diffraction and transmission electron microscopy. Further 1H CRAMPS and 13C CP/MAS NMR experimental results revealed that β-sheet content of SF was remarkably enhanced for nanocomposite prepared below the pI of SF (SF-MMTA) due to the strong interaction between MMT and SF. In SF-MMTA nanocomposite, clay layers acting as an efficient nucleator could efficiently enhance the β-sheet crystallization. On the contrary, SF preserved the native random coil conformation in SF-MMTN nanocomposites due to the weak interaction between MMT and SF. A tentative model was suggested and used to explain the mechanism of clay dispersion and conformational transition of silk protein.

Introduction The combination of natural polymers (proteins) and inorganic solids (e.g., clays) has become an emerging group of hybrid materials, namely, bionanocomposites.1-5 Because of their excellent performance, low cost, and eco-friendly characteristic, protein-based “green” materials have attracted great interest in the past decades.6-8 Bombyx mori silkworm silk, one of the most intensely studied structural proteins, has generated significant interest because of its remarkable mechanical properties, which has been successfully used in biotechnological and biomedical areas.9-11 Silk contains a fibrous protein known as fibroin, which forms a thread core, and glue-like proteins called sericin that surround the fibroin fibers to cement them together. Natural silk is produced as fiber; this, in turn, has limited the use of silk to applications that are amenable to this significant conformational constraint. To expand its application, it is necessary to dissolve silk fiber and regenerate it. After efforts of many researchers, however, the regenerated silk protein materials exhibit poorer mechanical properties and more brittleness compared with their origin form.12 Therefore, plasticizers are usually used to overcome the brittleness of the SF films, which unavoidably leads to the significant decrease of its tensile strength.13 To obtain the flexible materials with high strength, some suitable reinforcing fillers have been introduced in SF materials.14,15 Clay has been successfully used as a nanoscale reinforcing phase for a wide range of commodity and natural polymers.16,17 Some natural polymer/clay materials with high-performance such as improved mechanical, thermal, and barrier properties have been achieved by several research groups.18-21 One of the obstacles in obtaining an excellent clay nanocomposite is the dispersion of the clay sheets in polymer matrix. Proteins are natural copolymers containing both hydrophobic and hydrophilic domains, which could interact with clay surface. Therefore, proteins could be used as excellent dispersants to * To whom correspondence should be addressed. Tel.: +86-2223508171. Fax: +86-22-23494422. E-mail: [email protected] (P.S.); [email protected] (Z.Y.).

clay. Because silk fibroin has potential use in the biomedical and composite industries, it is envisioned that incorporating clay platelets into silk fibroin would impart high tensile modulus and strength to the nanocomposites. This may open the gateway to produce multifunctional silk materials that may be used as tissue scaffold, artificial tissue, and biodegradable structural materials. The conformation of protein in the nanocomposite and the relevant interaction between clay and protein are the key to understand the particular properties of bionanocomposites. Solidstate NMR is one of the most powerful and versatile techniques to elucidate the molecular structure and dynamics of species at a molecular level.22 It has been successfully applied to the study of organic-inorganic interfaces. Under certain circumstances, this technique can provide a direct three-dimensional picture of polypeptides in the bulk or in the adsorbed state.23,24 Many solid state NMR studies have been performed on silks by Asakura and co-workers.25-28 They concerned on structural studies of model peptides and isotopically enriched silks, especially conformational analyses of 13C and 15N chemical shifts; determinations of torsion angles and atomic distances by various kinds of NMR techniques. Recently, solid-state NMR was also applied to study the structural evolution of silk fibroin by some exogenous factors like pH, metal ion, temperature, and so on.29-32 As far as we know, little solid state NMR studies have been reported on silk fibroin protein/clay systems. In our previous work, we have demonstrated that polymer-clay interaction and end group of polymers are key factors in controlling the clay dispersion.33-35 In this work, by adjusting the protein-clay interaction through changing pH value below or above pI of SF, we reported novel biomaterial silk fibroinmontmorillonite (MMT) nanocomposites. Wide-angle X-ray diffraction (WAXD) and transmission electron microscopy (TEM) were applied to examine the behaviors of clay dispersion in the nanocomposites. Moreover, the influence of the clay on the conformational feature of silk fibroin protein was investigated by solid-state NMR techniques. We used these methods

10.1021/bm1002398  2010 American Chemical Society Published on Web 05/28/2010

Silk Fibroin/Montmorillonite Nanocomposites

Biomacromolecules, Vol. 11, No. 7, 2010

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to elucidate the mechanism of clay dispersion and conformation features of SF in SF-MMT nanocomposites.

Experimental Section +

Materials. Na -montmorillonite was purchased from Tianjin Clay Chemical Corporation in China. The cation exchange capacity of this Na+-MMT was 100 mequiv/100 g. The chemical formula for Na+MMT was Na0.67[Si8O20][Al3.33Mg0.67(OH)4], with an octahedrally coordinated alumino-hydroxide layer bound by two tetrahedrally coordinated silica layers.36 Preparation of Regenerated Silk Fibroin. Degummed Bombyx mori silk was prepared following Jin et al.’s procedure.37 Bombyx mori silk cocoon was degummed in 0.5 wt % (w/w) Na2CO3 aqueous solution for about 40 min and then rinsed thoroughly with distilled water to extract the glue-like sericin proteins. Each step was repeated twice and, finally, the degummed silk was dried at 50 °C. The degummed fiber was shortened as SF-D. The degummed silk was dissolved in 9.3 mol/L LiBr aqueous solution at room temperature to obtain SF aqueous solution with a concentration of 10 wt %, then dialyzed against distilled water for 3 days with Slide-a-Lyzer dialysis cassettes (Pierce, MWCO 3500) to remove the salt. The dialyzed solution was then centrifuged at 9000 rpm for about 10 min. The supernatant was collected and then lyophiled. The corresponding sample was shortened as SF-L. The lyophiled silk fibroin powder was redissolved in pH ) 2.8 HCl solution, and then centrifugated. The supernatant was collected and diluted to 0.05 wt % and then lyophiled. The sample was shortened as SF-2.8. Preparation of Nanocomposites. All nanocomposites were prepared using the solution-intercalation technique. Na+-MMT (100 mequiv/ 100 g, 15 mg) powder was added into 7.5 mL of deionized water to form 0.2 wt % suspensions. The water/clay suspension was stirred for 30 min and then ultrasonicated for 30 min to improve dispersion. The lyophiled silk fibroin powder was dissolved in deionized water and centrifugated. The supernatant was collected and diluted to 2 wt %. Then 7.5 g 2 wt % silk fibroin solution was added to the clay suspension and diluted to 40 mL. The mixtures were maintained at 4 °C for 24 h and then lyophiled. The final clay/protein ratio was 1/10. The nanocomposite powder obtained was shortened as SF-MMTN. The nanocomposite prepared under pH ) 2.8 HCl conditions was same as SF-MMTN. It was shortened as SF-MMTA. X-ray Diffraction (XRD) Measurements. X-ray diffraction (XRD) experiments were performed in reflection on a Rigaku D/max-2500 X-ray powder diffractometer with Cu KR (λ ) 0.154 nm) radiation at a generator voltage of 40 kV and a current of 100 mA. The diffraction data was collected from 2θ ) 1-70° in a fixed time mode. Transmission Electron Microscope (TEM) Measurements. The microstructure and morphology of clay in SF-MMT nanocomposites were visualized by a HITACHI-800 transmission electron microscope at an accelerating voltage of 200 kV. SF-MMT powder was embedded in epoxy and cured at room temperature overnight. The specimens for TEM observation were prepared by microsectioning on a RMC PowerTome with a 35° diamond knife at room temperature and were mounted on a copper grid. Solid-State NMR Measurements. All solid-state NMR experiments were performed on a Varian Infinityplus-400 wide-bore (89 mm) NMR spectrometer operating at a proton frequency of 399.7 MHz. All experiments were carried out at room temperature (25 °C). A conventional 4 mm double-resonance HX CP/MAS NMR probe was used for all CRAMPS experiments, and samples were placed in a 4 mm zirconia PENCIL rotor with 52 µL sample volume. (1) 1H CRAMPS (combined rotation and multiple pulse spectroscopy) experiments: The DUMBO-1 pulse sequence was used for homonuclear decoupling of 1H. The DUMBO-1 decoupling cycle time was set to 32 µs divided in 64 discrete phase steps of 500 ns duration each, and 1H decoupling RF field strength of DUMBO-1 was set to 100 kHz.38 The magic angle spinning (MAS) was automatically

Figure 1. XRD patterns of pristine clay and SF-MMT nanocomposites prepared in nearly neutral (SF-MMTN) and acid media (SF-MMTA). The inset displays the in-plane 060 clay reflection of SF-MMTA.

controlled at 9.8 kHz within (2 Hz with a MAS speed controller. The 1 H chemical shift was calculated using an experimentally determined scaling factor of 0.58 using the continuous phase modulated 1H CRAMPS spectrum of alanine and was internally referenced using silicone rubber (δ ) 0.12 ppm) relative to tetramethylsilane (TMS; CH3)4Si (δ ) 0 ppm). (2) 13C CP/MAS spectra were acquired with a ramp CP contact time of 0.8 ms at MAS of 12 kHz. A total of 5000-8000 scans were collected over a spectral width of 50 kHz. SPINAL-64 1H decoupling with a field strength of 80 kHz was applied during acquisition. The 13 C chemical shifts were referenced to external HMB (hexamethylbenzene) (δ ) 17.3 ppm for CH3). In previous work on regenerated Bombyx mori silk fibroin, Zhou et al.30 confirmed that Ala Cβ of SF with different conformation has the same cross-polarization properties, and they used 13C cross-polarization spectrum of Ala Cβ to quantitatively analyze the relevant conformational (random coil and β-sheet) contents of silk fibroin. Following their approach, the NMR peak of Cβ (chemical shifts between 5 and 30 ppm) for alanine residue was deconvoluted using Guassian functions to analyze quantitatively the secondary structure components in our samples.

Results and Discussion Structure of SF-MMT Nanocomposites. From the theoretical prediction, nanoclay would have a good dispersion in polymer matrix if a relatively strong attractive interaction between polymer and clay sheets is introduced.39 This has been confirmed experimentally by our previous work.33-35 Silk fibroins are composed of relatively large hydrophilic chain end blocks (N and C-termini) with smaller hydrophilic internal blocks and large internal hydrophobic blocks.37 Therefore, the amphiphilic block copolymer-like properties of silk fibroin may make it a potential dispersing agent for clay. It is known that the isoelectric point (pI) of amphoteric polyelectrolyte silk fibroin is 3.8-3.9.40,41 Below the pI, the SF chain is with positive charges. Owing to the presence of negative charges on the MMT surface (tSiO-Na+ for Na+-MMT), the approach of SF to the clay surface may be predominant by the balance of surface charges of the clay and the charges on the protein. So the pH of media is crucial to control the interaction of SF and MMT and then the final structure of the SF-MMT nanocomposites. Therefore, we prepared SF-MMT nanocomposites just below and above pI to compare the interaction effect. The degree of clay dispersion is usually characterized by XRD. Figure 1 shows the XRD patterns for original clay and SF-MMT nanocomposites. From Figure 1, the d001 spacing of the pristine MMT clay layers is 1.32 nm. This represents the spacing between the silica layers that constitute the clay structure. XRD patterns of SF-MMT nanocomposites change dramatically in comparison with pristine clay. In the XRD

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Figure 2. TEM micrographs of (a) SF-MMTA and (b) SF-MMTN nanocomposites.

pattern of SF-MMTN, a weak and broad hump at lower angle corresponding to d001 ) 1.84 nm can be observed, indicating the existence of intercalated clay (some swelling tactoids).42 In contrast, the XRD pattern of SF-MMTA nanocomposite prepared in the acid condition displays no obvious (001) basal reflection peak in the 2θ range from 1.3° (corresponding to gallery distance of about 7 nm) to 10°, indicating possible exfoliation of clay.43 The absence of the (001) peak does not result from the low quantity of clay in the nanocomposite, as the (060) in-plane reflection peak of the single clay sheet can still be detected (the inset of Figure 1).44 XRD results indicate that the control of pH value has a significant influence on clay dispersion. In general, the absence of diffraction peaks in the XRD pattern can be attributed to two possible reasons: (a) exfoliated structure and (b) intercalated structure, with an average d-spacing larger than about 7 nm, which exceeds the detection range of XRD.34 To further investigate the dispersion and morphology of clay layers in SF-MMT nanocomposites, TEM measurement was applied. The TEM image of SF-MMTA shown in Figure 2a indicates that most of the clay layers are disorderly dispersed in the silk fibroin matrix (the dark lines are the silicate layers), and only a small amount of clays are intercalated with a large gallery distance that exceeds the detection range of XRD. Because the clay content reaches as high as 9 wt % in our system (SF/MMT ) 10/1), absolute and complete exfoliation is very difficult. From the TEM image of SF-MMTN shown in Figure 2b, we can see a large amount of swelling tactoids, together with some exfoliated single clay platelets. Overall, the TEM results show much better clay dispersion in SF-MMTA than in SF-MMTN. The TEM observations agree well with the above XRD results. On the basis of XRD patterns and TEM images, SF-MMT nanocomposites with good clay dispersion were successfully prepared via a solution intercalation process. The effect of pH value on clay dispersion is subtle and can be directly visualized by comparing TEM images of SF-MMTA and SFMMTN nanocomposites. The above phenomena would be ascribed to the different protein-clay interactions, which can be tuned by adjusting the pH value. Conformational Features of SF-MMT Nanocomposites. Silk fibroin protein can assume three different forms, namely, random coil, R-helix, and β-sheet. Generally, high crystallinity and stable β-sheet structure are critical for silk fibroin to achieve high breaking strength and stiffness. The mechanical, thermal, optical, and degradation properties in silk fibroin materials are related to the formation of β-sheet crystals.45-47 So it is very important to control silk fibroin transformations from random coil to antiparallel β-sheet in vitro to produce high performance materials. It has been known that the chemical shifts of 1H and 13 C nuclei are strongly influenced by the local conformation of protein. Kimura had already studied the correlation between the

Figure 3. 1H CRAMPS NMR spectra of (a) SF-L, (b) SF-2.8, (c) SFMMTN, (d) SF-MMTA, and (e) SF-D.

1

H chemical shifts and conformation of silk fibroin by 1H CRAMPS experiments.48 They found that the HR chemical shifts of the random coil structure were 3.9 ppm (singlet-like), whereas those of β-sheet structure exhibited peaks at 5.0 and 3.9 ppm. Asakura reported that the 13C chemical shift of Ala Cβ showed a strong dependence on the silk fibroin secondary structure.49 The 13C chemical shifts of Ala Cβ in random coil were 16.6 ppm, whereas those of the β-sheet structure exhibited peaks at 20.4 ppm. Therefore, it is sufficient to identify the secondary structure of silk proteins by 1H and 13C solid-state NMR spectroscopy. 1 H CRAMPS is a unique technique to obtain high-resolution solid-state 1H NMR spectrum. Figure 3 showed the 1H CRAMPS spectra of silk fibroin and SF-MMT nanocomposites. The spectra of these samples are separated into four regions (amide proton NH, side chain phenyl, R-proton HR, and β-proton Hβ). It is noteworthy that the HR signals of SF-L (random coil) and SF-D (β-sheet) show one (δ 3.9) and two resonances (δ 5.0 Ala CR and 3.9 Gly CR), respectively. In addition, the 1H chemical shifts of Ala Hβ protons of SF-L were slightly different from those of SF-D. For SF-2.8 and SF-MMTN, the location of the peaks was almost same as SF-L, indicating that silk fibroin in both SF-2.8 and SF-MMTN materials is a random coil structure. But for SF-MMTA, the HR exhibits a typical asymmetric broad peak at 4.1 ppm, with a shoulder peak at about 5.0 ppm. Besides, the Hβ shows the same peak positions as SF-D, suggesting that silk fibroin in SF-MMTA is the mixture of random coil and β-sheet. Moreover, we found that the asymmetric line shape of NH peaks is different among the silk materials. The signal intensity of NH peak centered at 8.6 ppm increases in the order SF-L ≈ SF-2.8 ≈ SF-MMTN < SF-MMTA < SF-D. According to Kimura’s previous work,50 the amide proton chemical shifts and signal intensity were sensitive to the conformation of solid peptides. The 1H chemical shift of the NH group in the β-sheet is 0.6 ppm downfield from that of the R-helix form (R-helix, δ ) 8.0 ppm; β-sheet, δ ) 8.6 ppm). Thus, it is reasonable to conclude that the β-sheet content increases in the order SF-L ≈ SF-2.8 ≈ SF-MMTN < SF-MMTA < SF-D. This is in accordance with the above analysis about HR and Hβ. To further confirm the above 1H CRAMPS ascription, we performed 13C CP/MAS experiments, which give much better spectra resolution compared with 1H CRAMPS experiments. 13 C CP/MAS has high spectral resolution for macromolecules due to its wide chemical shift range of 13C. It is sensitive to the rigid materials where strong dipolar interactions in the system allow polarization transfer from protons to nearby carbons, thus, efficiently enhancing the carbon intensities. The 13C CP/MAS spectra of silk fibroin and SF-MMT nanocomposites prepared



shoulder peak appears at 170.0 ppm, which again suggests that silk fibroin in SF-MMTA is the mixture of random coil and β-sheet. On the basis of the above 1H CRAMPS and 13C CP/MAS analyses, it is suggested that SF is intercalated within the NaMMT clay gallery without the loss of the secondary structure in the SF-MMTN nanocomposite. However, there are significant changes in the secondary structure of constrained SF in the SFMMTA nanocomposite, where the content of the β-sheet increased. It is noteworthy that the increase of β-sheet content is not due to the pH value below the pI of SF or the presence of sodium ion, because the silk fibroin in pH 2.8 adopts in random coil form and the presence of equivalent sodium ion to Na+-montmorillonite alone does not cause the structural change of silk fibroin (the results not shown here). Zhou et al. reported that pH and certain amounts of sodium ion favored β-sheet formation in regenerated silk fibroin.30,32 However, low pH and equivalent sodium ion to Na+-montmorillonite does not convert the random coil to β-sheet in our prepared samples. We assume that the silk fibroin solution is too dilute in our system, 0.05 wt %, so the molecular chains of SF are far away and can not get close to each other even if at low pH and in the presence of sodium ion. When solvent was quickly extracted in the process of lyophilization, the silk fibroin still kept their random coil structure. Thus, we infer that the increase of β-sheet content should be predominately caused by the clay layers. To explain why clay has different effects on the secondary structure of silk fibroin in SF-MMT nanocomposites, we ascribe it to the interactions between clay and silk fibroin and the existence of clay layers acting as efficient nucleators. Mechanism of Clay Dispersion and Conformational Transition of Silk Protein. Clay clusters are known to break down into nanoscale thick clay layers in dilute solution by mechanical stirring and sonication.53 In acid solution below the isoelectric point of SF, the silk fibroin with positively charges forms a coating around each negatively charged clay layer. Such a strong interaction between clay and SF prevents clustering of the disordered clay layer. Meanwhile, due to large molecular size of the SF chain with a hydrodynamic radii, RH, of about 22 nm,54 the ordered restacking of disordered clay layers was inhibited when solvent was quickly extracted in the process of lyophilization. Therefore, the nanocomposites with good clay dispersion were formed. While in solution above the isoelectric point of SF, the silk fibroin protein becomes negatively charged. So, it is reasonable to infer that the anionic residues of SF generate repulsive interactions with clay sheets and the protein can not climb into the clay galleries. However, the presence of both swelling tactoids and some exfoliated clay platelets in SF-MMTN implies that there must be some kind of interaction between SF and MMT layers. Beall et al.55 suggested that organic molecules containing partial negative charged groups can ion-dipole bond to the interlayer exchangeable cations. Ion-dipole interactions

Figure 4. 13C CP/MAS spectra of (a) SF-L, (b) SF-2.8, (c) SF-MMTN, (d) SF-MMTA, and (e) SF-D.

under different conditions are shown in Figure 4. To quantitatively analyze the components of the conformations of silk fibroin and SF-MMT nanocomposites, spectral deconvolution for NMR peaks of Cβ for alanine residues was performed. The extracted chemical shifts arising from the major amino acid residues and contents of the corresponding conformation were summarized in Table 1. It should be mentioned that MMT is paramagnetic and the NMR signals of 1H and 13C nuclei in close proximity to the paramagnetic centers within 0.5 nm will experience a serious line broadening and are very difficult to be detected. Because the content of silk fibroin in our samples reaches as high as 91 wt % (SF/MMT ) 10/1(w/w)), thus, the obtained relative narrow NMR signals should be mainly attributed to proteins that are 0.5 nm far away from fixed paramagnetic centers within the lattice of the aluminosilicate. As shown in Figure 4 and Table 1, the peaks for Ala Cβ in SF-L, SF-2.8, and SF-MMTN are similar, which dominate at 17.1 ppm, with a weak shoulder at 20.8 ppm, indicating that the Ala residues are mainly in random coil structure. For the degummed fibers (SF-D), the Ala Cβ 13C chemical shift changes to 20.7 ppm, which can be assigned to a β-sheet structure. The Ala Cβ peak of SF-MMTA, however, shows a double pattern at 20.9 and 16.8 ppm, indicating that the SF-MMTA adopts two kinds of secondary structures, namely, mixtures of random coil and β-sheets. The analysis of the conformation contents from the spectral deconvolution shows that the β-sheet content increases in the order SF-L ≈ SF-2.8 ≈ SF-MMTN < SFMMTA < SF-D. Also, the 13C chemical shift of the carbonyl carbons reflects the state of secondary structure in silk materials.51,52 As shown in Figure 4, the widths of Gly and Ala carbonyl carbons in SF-D are very sharp and the two peaks are completely separated. This proves the existence of the β-sheet structure and the presence of a well ordered state of hydrogen bond. On the other hand, one broad peak is observed in SF-L, SF-2.8, and SF-MMTN. Such unresolved carbonyl peaks arise from the superposition of 13C NMR signals from segments in the random coil form, whose respective (Φ, Ψ) torsion angles were distorted to some extent. For SF-MMTA nanocomposites, the carbonyl peak of the Gly residue is not resolved, but a Table 1. Silk Fibroin Sample, Conformational Characteristic, and their sample chemical shifts

SF-D CdO CR Ala Cβ Ser CR Ser Cβ

main conformation β-sheet content

Ala 173.2 Gly 170.0 Gly 43.3 Ala 50.0 20.7 55.5 64.8 β-sheet 86.0%

SF-L

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C Chemical Shifts (ppm) SF-2.8

SF-MMTN

172.8

172.4

172.6

Gly 43.7 Ala 51.2 17.1 56.2 62.1 random coil 28.4%



SF-MMTA Ala 173.2 Gly170.0 Gly 43.7 Ala 50.0 16.8 20.9 55.5 56.2 63.6 β-sheet + random coil 58.3%

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Figure 5. Schematic illustration of fabricating SF-MMT nanocomposites in acid and nearly neutral media.

between the exchangeable cations of smectite and the polar functional groups are the dominant bonding for a series of organic compounds such as amide, alcohols, and ethers.56 In our system, silk fibroin has many polar functional groups that possess partial negative charge, so the SF chain can directly ion-dipole bond to the interlayer exchangeable sodium cations above the isoelectric point of SF. Therefore, ion-dipole interaction could be the main interaction between pure SF and clay. On the other hand, according to Matsumoto’s report,57 the predicted pI of heavy chain N-terminus and the C-terminus of SF are 4.59 and 10.53, respectively, and the predicted pI of the light chain linked at the C-terminus of heavy chain is 5.06. Thus, in a solution of pH 6.4, the heavy chain C-terminus is always positively charged and could replace Na+ and anchor silk fibroin segments to the negatively charged clay layers. Overall, above the isoelectric point, silk fibroin interacts with clay mainly through ion-dipole bonding and below isoelectric point via electrostatic attraction. As the ion-dipole interactions are relatively weak compared to electrostatic attractive interactions, the clay dispersion in SF-MMTN nanocomposite was poorer than that of SF-MMTA nanocomposite. This is in accordance with the theoretical prediction by Balaz at al. that nanoclay would have a good dispersion in polymer matrix if a relatively strong attractive interaction between polymer and clay sheets is introduced.39 Figure 5 presents a schematic illustration of the formation of SF-MMT nanocomposites and suggested interaction between SF and MMT, as well as conformation features of SF in SFMMT nanocomposites. Beall et al.58,59 reported that there are three regions around the clay plates: the surface modifier region, the constrained polymer region, and the unconstrained polymer region. The surface modifier region is about 1-2 nm. The constrained polymer region may extend 50-100 nm from the surface of the clay and the restriction of polymer motion is a function of the interaction of the polymer with the surface of the nanoparticle. In our system, below the pI of SF, the silk fibroin with positively charges acting as the surface modifier can form strong electrostatic attractive interactions with the negatively charged clay layers. While above the pI of SF, the ion-dipole interaction between the exchangeable sodium cations of smectite and the polar functional groups of SF could be the main interaction between pure SF and clay. As electrostatic attractive interactions are stronger compared to ion-dipole interactions, the silk fibroin in SF-MMTA nanocomposites is more tightly constrained than that in SF-MMTN nanocomposites. According to molecular dynamic simulations, the clay surface can have a denaturing effect on compactly absorbed proteins because they are dehydrating agents.60 Brovchenko reported that when peptide-surface attraction is strong enough the two-dimensional surface should provide orientational ordering of the peptides as well as a preferential formation of twodimensional aggregate.61 In the SF-MMTA nanocomposites

system, the possible mechanism of β-sheet transition was supposed as below: First, the positive residues of SF molecules are expected to be located in the close vicinity of the negatively charges of clay due to overall electrostatic attraction. Such interactions between SF molecules and clay surface would decrease their hydration, making SF molecules embedded in the protein-clay contact region and shielded from the bulk aqueous medium. Then the SF chains close each other, creating a hydrophobic environment. Subsequently, SF chains readjust and reorganize, forcing SF chains into an antiparallel β-sheet structure near the clay surface. Surface-induced β-sheet of protein has been reported by many authors.62 Previous work using ATR/FTIR63 and CD64 reported that the β-sheet-rich structure for globular proteins could form at interfaces. Theng et al. suggested that when protein was absorbed compactly to clay minerals, they may collapse mainly to an extended β-structure.65 Sethuraman et al. also reported that hen egg white lysozyme forms macroscopic aggregates enriched in intermolecular β-sheets at a surface.66 On the contrary, the native random coil conformation of silk fibroin preserves in SF-MMTN nanocomposite. This is due to the weak ion-dipole interaction. The protein-clay surface interaction is too weak to cause the conformation change of SF. Above all, positively charged silk fibroin proteins result in good clay dispersion with the formation of a β-sheet structure below the pI of SF. While the ion-dipole interactions could be the main bonding force between pure SF and clay above the pI of SF, the ion-dipole interactions are relatively weak, leading to a poor dispersion of the clay layer in the protein matrix.

Conclusion By adjusting the solution pH value below the isoelectric point of silk fibroin protein, we have successfully prepared SF-MMTA nanocomposites with good clay dispersion. The structures of SF-MMT nanocomposites were characterized by XRD, TEM, and NMR techniques. It was found that the clay dispersion of SF-MMT nanocomposites strongly depends on the protein-clay interaction. The SF-MMT prepared in acidic conditions below the isoelectric point of SF showed better clay dispersion than SF-MMT prepared above the isoelectric point. 1H CRAMPS and 13C CP/MAS showed that the conformation of silk fibroin protein in SF-MMT nanocomposites was pH-dependent. In SFMMTN nanocomposite, the SF preserves random coil conformation both before and after addition of clay layers, while in SF-MMTA nanocomposite the secondary structure of SF changed significantly. Clay layers were shown to enhance the β-sheet crystallization of SF significantly, acting as an efficient nucleator. Finally, a schematic illustration was proposed to describe the clay dispersion mechanism, interaction between SF and MMT as well as the conformation features of silk protein in SF-MMT nanocomposites. The dispersion degree of the clay


layers in the polymer matrix is critical for material performance in many applications. The opportunity to combine silk fibroin and clay could lead to environmentally friendly materials that are suitable for widely usage. Acknowledgment. We are grateful to Dr. Xiaoliang Wang for his helpful discussions. This work was financially supported by the National Science Fund for Distinguished Young Scholars (No. 20825416), and the National Natural Science Foundation of China (NSFC) through the Key and General Programs (Nos. 20634030 and 20774054).

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