Modulation of Self-Assembly Process of Fibroin: An Insight for

Nov 17, 2015 - ATR-FTIR data of fibroin solution incubated with CaCl2 (100 μM) revealed the formation of intermediate α-helical structure during its...
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Modulation of Self-Assembly Process of Fibroin: An Insight for Regulating the Conformation of Silk Biomaterials Priyanka Dubey,† Sumit Murab,† Sandip Karmakar,‡ Pramit K. Chowdhury,‡ and Sourabh Ghosh*,† †

Department of Textile Technology and ‡Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India ABSTRACT: Controlling the mechanism of self-assembly in proteins has emerged as a potent tool for various biomedical applications. Silk fibroin self-assembly consists of gradual conformational transition from random coil to β-sheet structure. In this work we elucidated the intermediate secondary conformation in the presence of Ca2+ ions during fibroin self-assembly. The interaction of fibroin and calcium ions resulted in a predominantly α-helical intermediate conformation, which was maintained to certain extent even in the final conformation as illustrated by circular dichroism and attenuated total reflectance-Fourier transform infrared spectroscopy. Further, to elucidate the mechanism behind this interaction molecular modeling of the N-terminal region of fibroin with Ca2+ ions was performed. Negatively charged glutamate and aspartate amino acids play a key role in the electrostatic interaction with positively charged calcium ions. Therefore, insights about modulation of self-assembly mechanism of fibroin could potentially be utilized to develop silk-based biomaterials consisting of the desired secondary conformation.

1. INTRODUCTION Tuning the self-assembly of silk protein has emerged as an important and advanced strategy for the fabrication of silkbased biomaterials. Silk fiber consists of two component proteins namely: fibroin (70−80%) and sericin (20−30%).1 Fibroin is a self-assembling protein with a high molecular weight block copolymer composed of heavy (∼350 kDa) and light (∼26 kDa) chains linked by a single disulfide bond.2 The fibroin heavy chain consists of four different segments: the Nterminus region, the C-terminus part, 11 spacer regions, and 12 large repeat domains. The N- and C-termini and 11 spacer regions contribute toward the hydrophilicity of fibroin, while the 12 large bulk domains consisting of the repeating hexapeptides GAGAGS and GAGAGY sequences render the protein its hydrophobic character. This repeated amino acid sequence with small side chains, allows tight packing of the individual strands of molecular chains leading to the formation of β-sheet structure,2 which is accountable for the observed thermal stability and high mechanical strength of silk fiber.3 Fibroin exists in three distinct structural conformations, that is, silk I, silk II, and silk III. Even after decades of research, there are some uncertainties about secondary conformations in fibroin; for example, silk I is reported to have ordered secondary structure.4,5 However, some investigations state that silk I is a S-shaped zigzag/crankshaft6 or repeated β-turn type II structure.7,8 Silk II consists of highly ordered β-sheet crystalline regions4 and silk III structure is defined as a 3-fold helical conformation present at the interfaces (air−water interface4 and aqueous fibroin−organic solvent interface).9 During silk fiber spinning by silkworm, assembly process of fibroin protein is initiated by water extraction, gradual changes © XXXX American Chemical Society

in salt concentration, pH and ultimately triggered by alignment of polymer chain due to mechanical stretching force. During the self-assembly process, from solution state to solid state, fibroin undergoes a structural transition from random coil to stacked β-sheet crystal rich (silk II) structure due to hydrogen bonding and hydrophobic interactions. Different metallic ions such as Na+, K+, Ca2+, Cu2+, and Mg2+ are known to play a crucial role in self-assembly, during the spinning of silk fibers within the glands of silkworm,10−13 due to the conformational transitions induced by binding of these mono and bivalent cations to the fibroin chains. Various studies have been carried out to investigate the effect of individual ion on self-assembly process of fibroin solution in vitro.10 Among these ions, calcium has been reported to promote the formation of a stable silk gel, whereas the sodium and potassium ions were reported to destabilize the gel network.10 Calcium ions may stimulate aggregation of fibroin by interacting with negatively charged amino acids present predominantly in the vicinity of fibroin heavy chain ends using electrostatic interactions.14,15 It has been demonstrated that physiological concentrations of calcium ions promotes the formation of β-sheet and distorted β-sheet characteristic of silk II. On the contrary, higher concentrations of calcium ion sustain random coil conformation.16 Hence, it can be concluded that optimum concentration of calcium ions play a crucial role in the self-assembly process of silk fibroin; however, none of the studies elucidates clearly the presence of Received: September 18, 2015 Revised: November 15, 2015

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DOI: 10.1021/acs.biomac.5b01258 Biomacromolecules XXXX, XXX, XXX−XXX

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in an air incubator shaker. Control experiments were also carried out to determine the effect of temperature and shaking independently. In the first control, fibroin solution (10 mg/mL) was incubated at 37 °C and examined for its self-assembly kinetics. Next, fibroin solution was incubated at room temperature (RT) with shaking at 150 rpm. Another study was performed in the presence of CaCl2 at 37 °C with 150 rpm shaking. Fibroin solution with 80% methanol at 37 °C with 150 rpm shaking was used as positive control to monitor the kinetics of fibroin. During the incubation period, aliquots were collected at regular intervals in all the cases. 2.5. Fluorescence Spectroscopy. All ThT fluorescence spectra were monitored at 25 °C using Edinburg Spectrofluorimeter (FLS920, Edinburgh Instruments, U.K.). All emission spectra were recorded in the range of 440 to 700 nm with excitation at 430 nm. The slit widths for excitation and emission were kept at 2 and 5 nm, respectively. The final concentration of ThT used was 20 μM. 2.6. Turbidity Measurements. Turbidity of fibroin solution, fibroin solution with CaCl2, and fibroin solution with 80% methanol was studied using UV visible spectrophotometer (UV-2450, Shimadzu, Japan). Samples were incubated at 37 °C with shaking at 150 rpm from 0 to 200 min. Aliquots were collected at regular intervals. Absorbance was recorded at 600 nm and baseline was corrected using the respective solutions without the protein. 2.7. Dynamic Light Scattering (DLS) Measurements. DLS measurements were carried out with a Malvern Zetasizer Nano ZS instrument (Worcestershire, U.K.) for characterizing the size of fibroin molecules with optimized calcium ions concentration along with negative (fibroin without calcium ions) and positive (fibroin with 80% methanol) controls. Samples were collected at different time point during the incubation at 37 °C with shaking at 150 rpm. A total of 2 mL of sample was introduced manually in DLS cuvette with a path length of 10 mm and analyzed at 25 °C. 2.8. Circular Dichroism Measurements. Far-ultraviolet (UV) CD spectra were recorded using AVIV 420 SF (AVIV- Biomedical Inc. Lakewood, NJ, U.S.A.) in the range of 190−260 nm with a path length of 0.1 cm. Fibroin solution of 10 mg/mL concentration was incubated at 37 °C with shaking for 0 to 200 min. Aliquots were collected at 20 min interval until 200 min. All CD spectra were corrected for deionized water contributions. 2.9. Attenuated Total Reflectance-Fourier Transforms Infrared Spectroscopy. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was recorded using PerkinElmer instrument with DTGS (deuterated triglycine sulfate) IR detector in transmission mode (Spectrum BX Series, MA, U.S.A.). A total of 50 scans were recorded per sample at a resolution of 4 cm−1 with the wavenumber ranging from 1700 to 1600 cm−1 in the amide I region. Origin Pro 8.5 (Origin Lab Corporation, Northampton, MA, U.S.A.) was used for deconvolution of spectra by following the protocol described elsewhere.33 2.10. Molecular Dynamics Simulations. The calcium ion binding sites on the N-terminal region of Bombyx mori fibroin (PDB ID: 3UA0) were determined using BION (binding ions nonspecifically) server. It is a web server for the identification of ions that bind to the protein surfaces nonspecifically because the electrostatic attraction is strong enough to immobilize them. BION server utilizes a method that uses a DelPhi-calculated potential map in conjunction with a clustering algorithm to predict nonspecific ion-binding sites. The predicted sites generated by the BION server as a PDB file were visualized using PyMOL.34

intermediates during self-assembly process of silk in the presence of calcium. Various methods for inducing conformational transition in regenerated fibroin protein have been reported earlier such as exposure to organic solvents,17,18 mechanical treatments19 such as shear,20 sonication,21 vortexing,22 increased temperature,23 pH,24 ions,10 high pressure carbon dioxide,25 and laser exposure.26 Electrogelation,27 incorporation of ionic liquids,28 and blending with other copolymers29,30 are some other reported methodologies for inducing transition in secondary conformations in fibroin. Addition of organic solvents having low dielectric constants such as methanol or ethanol is the most common and fastest method for inducing conformational transitions in fibroin.31 The present study relates to the understanding of kinetics and sequences of formation of intermediate secondary structural motifs during silk fibroin self-assembly process induced by calcium ions toward utility as silk-based biomaterials. Three different conditions were chosen in order to study the kinetics of conformational transition of silk fibroin. First, we studied the self-assembly process of fibroin solution at 37 °C as a function of time. Second, we explored the selfassembly process of fibroin solution with optimized concentration of calcium ions with time. In third condition, methanol was used as a positive control to investigate the self-assembly process with respect to time. The self-assembly process of fibroin solution under all three different conditions was monitored by using UV−visible spectrophotometry, thioflavin T (ThT) fluorescence, dynamic light scattering (DLS), circular dichroism spectroscopy, and attenuated total reflectanceFourier transform infrared spectroscopy (ATR-FTIR). The data suggested that presence of calcium ions leads to the formation of α-helical secondary structural intermediate during fibroin self-assembly process which was not present in other two conditions. Further, the interaction of calcium ions with specific amino acids on N-terminal region of fibroin was predicted through molecular modeling.

2. MATERIALS AND METHODS 2.1. Materials. Sodium carbonate (Na2CO3), lithium bromide (LiBr), and calcium chloride (CaCl2) were purchased from Merck (Mumbai, India). Thioflavin T (ThT) dye was purchased from SigmaAldrich (St. Louis, MO, U.S.A.). Rests of the chemicals used were of analytical grade. Bombyx mori cocoons were procured from Central Silk Technological Research Institute (Central Silk Board), Bangalore, Ministry of Textiles, Government of India. 2.2. Isolation of Regenerated Aqueous Solution of Fibroin from Cocoons. Fibroin solution was prepared as described earlier.32 Bombyx mori cocoons were degummed twice with 0.02 M Na2CO3 for 20 min each in boiling water to take out sericin. The degummed silk fibers were washed thoroughly with deionized water and dried overnight at 50 °C in an oven. Silk fibers (degummed) were then dissolved in 9.3 M solution of LiBr. After complete dissolution of silk fibers, the solution was dialyzed against the ultrapure deionized water for 48 h. The final obtained concentration of regenerated fibroin solution was 5 wt %. 2.3. Preparation of Calcium Chloride Solution. Stock solution of CaCl2 with a concentration of 2 mM was prepared and diluted using ultrapure deionized water to get the desired final concentrations of CaCl2 (0, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, and 1000 μM). 2.4. Kinetics of Fibroin Self-Assembly Process. Stock solution of fibroin with 50 mg/mL (∼5% w/v) concentration was diluted with deionized water to obtain a final concentration of 10 mg/mL. Fibroin self-assembly kinetics was monitored at 37 °C with shaking at 150 rpm

3. RESULTS AND DISCUSSION Fibroin is known as a versatile building block for fabricating biomaterials. Protein self-assembly, which may involve various intermediate protein conformations, is one of the best strategies to fabricate unique supramolecular architectures. However, such diverse protein conformations may offer different immunogenicity and lead to different immune responses when exposed to cells in vitro or in vivo. We have previously demonstrated that human monocytes, cultured on fibroin-based B

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accepted tool to investigate the changes occurring in β-sheet content during the structural transition process and fibril formation.41 3.1.1. Fibril Formation of Fibroin Solution. ThT fluorescence of fibroin solution showed increased intensity with time due to progressive fibril formation during its selfassembly process. Fibroin incubated at 37 °C with shaking at 150 rpm showed highest intensity with maximum fibril formation at 180 min. After that it reached to saturation, without showing any further increase. However, fibroin incubated at 37 °C without shaking or fibroin at 150 rpm shaking at RT (25 °C) as controls, showed slower kinetics, as illustrated in Figure 1. Therefore, fibroin aggregation kinetics was observed to follow the conventional nucleation-dependent or seeded polymerization process.42

3D biomaterials with silk II conformation, showed immune activation at initial time point of culture as evidenced by enhanced IL-1β and IL-6 gene expression and protein production. However, in contrast, fibroin-based 2D films with silk I conformation were unable to stimulate monocyte responsiveness.35 Prion protein is another example wherein protein conformation significantly affects the immune responses. It has been observed that mice immunized with either α-prion protein or β-prion protein elicits different cytokine milieu and antibody isotype profile.36 Another study showed that precise antibacterial activity of LL-37 protein needs oligomeric α-helical conformation prior to interaction with bacterial membrane.37 Increase in the helical conformation of LL-37 is correlated to enhancement of antibacterial activity.37 Hence, it is crucial to study about the precise control of fibroin protein conformations, which can then be exploited to optimize the immunological outcome of silk biomaterials. Several studies have been reported about the interaction of fibroin with various metallic ions (Na+, K+, Cu2+ Mg2+, Zn2+, and Fe3+),10−13,16,38,39 which facilitates the conformational transition from silk I to silk II structure in fibroin solution. For example, Ruan et al.12 demonstrated that sodium ions at higher concentration than that present in vivo promoted conformational transition from random coils to β-sheet.12 A similar study was carried out on the effect of potassium ions on fibroin conformation by NMR and Raman spectroscopy.11 Mg2+, Fe3+, and Zn2+ ions are also known to induce subtle conformational transitions from disordered random coils to ordered β-sheet structures in fibroin.10,13,38 Cu2+ ions induce β-sheet formation in fibroin by forming coordination complexes with histidine residues and induce the formation of β-sheet structures.39 Apart from the aforementioned studies, only a few studies have indicated the crucial role of calcium ions in facilitating the selfassembly process by encouraging conformational transition from random coil to β-sheet.10,16 But none of these studies focused on the intermediates that are formed during the conformational transition during self-assembly process. Also, Ca2+ ion concentration plays a key role in silk spinning process in vivo. The anterior part of silk glands contain a relatively low concentration of Ca2+ ions, which helps to keep the silk in solution to ensure smooth flow through the duct.40 In the posterior and middle parts of the silk glands of silk worm, calcium ions at high concentrations are employed to keep the fibroin in predominantly α-helical structural conformation16 that prevents the solidification and subsequent choking of the silk glands. Apart from this, a gradient of calcium concentrations is also utilized in the silk glands to maintain the rheology of silk solution for efficient spinning process14 as well as control sol−gel transition regime. Studies have also shown that divalent cations like Ca2+ and Mg2+ have a significant effect on the silk fibroin self-assembly as compared to monovalent cations like K+ and Na+.15 This effect was attributed to the fact that divalent cations lower the pH of the silk fibroin solution thus decreasing the repulsion between silk fibroin chains resulting in easy interactions between the chains to selfassemble.15 Based on the above stated physiological relevance of Ca2+ ions, it was preferred to evaluate its effects on intermediate structures during the self-assembly process by using different biophysical techniques. 3.1. Fibril Formation. It has been illustrated previously that aggregation kinetics of protein follows two pathways, that is, isodesmic (exponential) aggregation kinetics and nucleation dependent aggregation kinetics. ThT fluorescence is a widely

Figure 1. ThT fluorescence intensity of fibroin was measured at 485 nm with the excitation wavelength at 430 nm. ThT fluorescence of fibroin was recorded from 0 to 200 min under three different incubation conditions: (black circle) at 37 °C with shaking at 150 rpm, (red square) at 37 °C without shaking, and (blue triangle) at 150 rpm shaking at room temperature.

3.1.2. Fibril Formation of Fibroin Solution with Different Concentrations of CaCl2. After studying the aggregation kinetics of fibroin solution with respect to time (Figure 1), fibroin solution (10 mg/mL) was incubated with different concentrations (0 to 1000 μM) of CaCl2 at 37 °C with shaking at 150 rpm for 100 min to investigate the specific concentration of CaCl2 which induces the maximum β-sheet formation during self-assembly process. Figure 2a demonstrated that fibroin solution incubated with 100 μM CaCl2 showed maximum fibril formation. However, fibroin incubated with more than 100 μM concentration of CaCl2 resulted in a sharp decrease in the fibril formation, which attains saturation eventually at 400 μM CaCl2. This inhibition in fibril formation after further addition of calcium ions could be due to mutual repulsion of similarly charged calcium ions bound in excess to the fibroin chains.43 3.1.3. Fibril Formation of Fibroin Solution with Optimized Concentration of Calcium Ions with Time. Based on aforementioned results (Figure 2a), we further investigated the kinetics of fibroin solution with optimized concentration (100 μM) of CaCl2 with respect to time using ThT fluorescence. Fibroin solution incubated with optimized concentration of CaCl2 showed an increase in fibril formation with maximum fibrils at 80 min. Thereafter, it reached stationary phase and no further change was observed (Figure 2b), whereas fibroin incubated without CaCl2 showed maximum fibril formation at 180 min (Figure 1). Methanol is reported as one of the fastest stimulating agents to induce the secondary conformational transition in fibroin.44 Therefore, it was used as a positive control to study fibroin self-assembly C

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Figure 2. Illustrating the ThT fluorescence intensity of (a) Fibroin incubated for 100 min at 37 °C with shaking at 150 rpm with different concentration of CaCl2. (b) Fibroin Incubated with optimized concentration of CaCl2 at 37 °C with shaking at 150 rpm. (c) Fibroin incubated with 80% methanol at 37 °C with shaking at 150 rpm from 0 to 100 min.

3.3. Particle Size Measurements. To investigate the kinetics of self-assembly and to identify the average size and size distribution of species generated during conformational transition study of fibroin, DLS was performed. The DLS data (Figure 4) of fibroin with optimized concentration of CaCl2 at different incubation time revealed a similar trend in accordance to ThT fluorescence data (Figure 2). The data revealed that fibroin with and without calcium ions at initial time point (0 min) had an average diameter of ∼10 nm, which corresponds to low molecular weight species of fibroin. With increased incubation time, fibroin with (40 min) and without calcium ions (90 min) undergoes transition toward higher molecular weight species of fibroin with the formation of 60−160 and 40−150 nm, respectively. Fibroin with calcium ions further increased in size due to self-assembled fibroin chain or aggregated chains (∼2100 nm) at around 80 min and no further increase in average size was observed. However, fibroin without calcium showed maximum particle size (∼2000 nm) at around 180 min. Fibroin with methanol as control showed higher particle size (∼2500 nm) instantly after methanol addition. This transition of lower molecular weight species to larger aggregates contributes toward our understanding of fibroin protein self-assembly process (Figure 4). 3.4. Conformational Transition of Fibroin Solution Monitored by Circular Dichroism. The conformational transition during the aggregation kinetics of fibroin solution with calcium ions, without calcium ions and with methanol was monitored by CD. Fibroin solution without calcium ions incubated at 37 °C with shaking at 150 rpm showed a random coil structure at 0 min whereas with prolonged incubation, the conformational transition from random coil to β-sheet was observed at around 120 min. No further structural change was observed later, as illustrated in Figure 5a. However, in contrast fibroin solution incubated with 100 μM CaCl2 showed the transition from random coil to α-helix in 20 min which lasted until 40 min of incubation. Thereafter, it showed transition from α-helical to βsheet structure and eventually showed β-sheet conformation at about 80 min as illustrated in Figure 5b. Fibroin solution treated with methanol at 37 °C with 150 rpm shaking attained the stationary phase of β-sheet conformation immediately after methanol exposure (Figure 5c). Since methanol is a hydrophilic agent with high dehydrating property, leading to the formation of β-sheet due to removal of water molecules from the fibroin chains as a result of the lower dielectric constant.31 Taken together the data suggested that at initial lag phase fibroin macromolecules, with and without calcium ions, existed in random coil conformation whereas fibroin with methanol

kinetics. Fibroin solution with 10 mg/mL concentration was incubated with 80% methanol at 37 °C with shaking at 150 rpm. It showed formation of fibrils immediately after methanol exposure, as shown in Figure 2c. It has been demonstrated previously that aggregation kinetics of fibroin protein follows the nucleation dependent aggregation pathway that comprises of three crucial steps:45 (a) first, lag phase nucleation, (b) second, log/exponential phase with protofibril formation, and (c) final third phase consists of maturation of protofibrils to full length fibrils.46 Similarly, in our study ThT fluorescence data demonstrated that fibroin with or without calcium ions during the initial (lag) phase of incubation represented lack of β-sheet structure. However, fibroin with methanol (positive control) showed abundance of β-sheet structure even at initial phase of incubation. Prolonged incubation of fibroin with calcium ions showed increased ThT fluorescence intensity during exponential (log) phase as compared to fibroin without calcium ions, which further attained saturation with progression of time, that is, the stationary phase (Figure 2). 3.2. Turbidity Measurements. Turbidity analysis was performed to investigate the extent of fibril formation as a function of time. Absorbance of fibroin solution incubated at 37 °C with shaking at 150 rpm was increased with respect to time due to enhanced fibril formation during self-assembly process, as illustrated in Figure 3. Fibroin solution incubated with CaCl2 showed more fibril formation in contrast to fibroin solution incubated without CaCl2. However, fibroin solution with 80% methanol showed abundant fibrils immediately after methanol exposure. Hence, turbidity experiment evidently validated the results obtained through ThT fluorescence (Figure 1 and 2).

Figure 3. Turbidity analysis: (black square) fibroin, (red circle) fibroin with optimized concentration of CaCl2, and (blue triangle) fibroin with 80% methanol. Absorbance recorded at 600 nm with respect to time. All samples were incubated at 37 °C with shaking at 150 rpm. D

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Figure 4. Dynamic light scattering: (a−c) Fibroin at 0 min (initial), 90 (intermediate), and 180 min (final) incubation; (d−f) Fibroin with 100 μM CaCl2 at 0 (initial), 40 (intermediate), and 80 min (final) incubation; (g−h) Fibroin with 80% methanol at 0 (initial) and final 100 min. All samples were incubated at 37 °C with shaking at 150 rpm.

Figure 5. CD spectra were recorded at 25 °C from 0 to 200 min at 190 to 250 nm wavelength. All samples were incubated at 37 °C with shaking at 150 rpm. (a) Fibroin incubated without adding calcium and methanol. (b) Fibroin incubated with 100 μM CaCl2 from 0 to 200 min. (c) Fibroin incubated with 80% methanol from 0 to 100 min.

and final (80 min) aggregation phase, respectively. However, fibroin incubated without calcium ion showed β-turn as intermediate structure which further transformed into native β-sheet conformation. Fibroin solution with 80% methanol showed β-sheet conformation without forming any intermediate structure, as illustrated in Figure 6. Therefore, data obtained through ATR-FTIR showed the similar trend in accordance to CD spectroscopy, as depicted in Table 1. Therefore, the present study showed a clear transition of fibroin conformation from random coil to more ordered helical structure as an intermediate step and then to the hydrophobic β-sheet structure in the presence of calcium ions. Interestingly, fibroin without calcium ions showed β-turns as intermediate phase before going into native β-sheet conformation. The formation of β-turns as intermediate structures could be due to the tightening or close packing of loose structures, which is known as hairpin loops.46 Positive control, that is, fibroin with methanol showed no such intermediates. Methanol instantly

showed β-sheet conformation even at initial time point. Prolonged incubation revealed that in the presence of calcium ions fibroin assumed α-helical conformation before transforming to β-sheet conformation. However, other two controls, namely, fibroin without calcium ions and fibroin with methanol did not show α-helical intermediate structure by CD. 3.5. ATR-FTIR Analysis. Amide I region is a widely accepted region to evaluate the secondary conformational changes of proteins by using ATR-FTIR. The bands in the range of 1616 to 1637 cm−1 are assigned to the β-sheet conformation. However, 1638 to 1655 cm−1 are characteristic band of random coil structure. Band from 1656 to 1662 cm−1 are attributed to the α-helical conformation.47 ATR-FTIR data of fibroin solution incubated with CaCl2 (100 μM) revealed the formation of intermediate α-helical structure during its conformational transition from random coil to β-sheet; presence of 34.8 and 25.4% of α-helical conformation were estimated in the intermediate (40 min) E

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Figure 6. ATR-FTIR of (a−c) fibroin at 0 (initial), 90 (intermediate), and 180 min (final) incubation; (d−f) fibroin with 100 μM CaCl2 at 0 (initial), 40 (intermediate), and 80 min (final) incubation; (g) Fibroin with 80% methanol at 0 min. All samples were incubated at 37 °C with shaking at 150 rpm.

intermediate. Whereas, higher calcium concentrations (20 mg per gram of fibroin) could hinder the formation of silk II conformation and lead to distinctive silk I or silk I-related intermediates.16 In contrast to their findings our study demonstrated that a significantly lower concentration of calcium ions/fibroin (1.1 mg/g) can modulate the self-assembly of fibroin. The conformational transitions taking place at this calcium ion concentration were found to be drastically different from those happening at higher concentrations. The hindrance with higher concentrations of calcium ions can be due to the electrostatic repulsions between calcium ions bound to silk chains that come closer during the folding of fibroin chains, thus resulting in a total change in the hydrophobic interaction profile of the protein thus hindering the self-assembly. It was observed that addition of 2 mg/g calcium to fibroin solution favored the formation of an intermediate structure that was able to stabilize the fibroin chains but the nature of intermediate structure was not clearly described.10 The authors used Raman spectroscopy to look at the conformational transition and reported that a peak at 1680 cm−1 corresponding to the formation of an intermediate conformation whose nature they did not comment on. But the results of this paper indicated that the optimized concentration of calcium ions (1.1 mg/g) that we found is in the range of the in vivo calcium concentration (0.5− 2.5 mg/g) that is present in the middle region of silk gland to keep the silk solution in a gel form for spinning.10

Table 1. ATR-FTIR Deconvolution Data of the Amide I Region (1600−1700 cm−1) of (i) Fibroin, (ii) Fibroin with 100 μM CaCl2, (iii) Fibroin with 80% Methanola S. No. i.

ii.

iii.

a

time (min) fibroin (a) at 0 min incubation (b) at 90 min incubation (c) at 180 min incubation fibroin incubated with 100 μM concentration of CaCl2 (d) at 0 min incubation (e) at 40 min incubation (f) at 80 min incubation fibroin incubated with 80% methanol (g) at 0 min incubation

random coil (%)

αhelix (%)

βsheet (%)

βturns (%)

72.2 34.3 4.1

0.0 0.0 0.0

0.0 34.0 50.0

27.8 31.7 45.9

64.6 20.1 10.3

0.0 34.8 25.4

0.0 31.2 38.6

35.4 14.9 25.7

26.4

0.0

50.6

23.0

All samples were incubated at 37 °C with shaking at 150 rpm.

induced β-sheet crystalline structure in fibroin by dehydration of polypeptide chains resulting into closely packed structures as reported earlier.48 Earlier it has been reported that calcium ions present at a concentration of 10 mg per gram of fibroin promoted the formation of β-sheet with distorted β-sheet silk II related F

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Biomacromolecules 3.6. Molecular Dynamics Simulation of Fibroin Protein with CaCl2. In order to understand the mechanism involved in the conformational transition of fibroin induced by calcium ions, molecular modeling was performed. BION server was used to predict the binding sites engaged in the interaction of N-terminal fibroin with calcium ions (Figure 7). BION

Table 2. Amino Acids Involved in Interaction between NTerminal of Fibroin Protein and Calcium Ions Using Molecular Modeling by BION Servera interaction ionic

Figure 7. Molecular modeling of N−terminal sequence of fibroin (PDB ID: 3UA0) with CaCl2 to determine its binding sites using BION server. A total of 36 residues were identified on N-terminal region of fibroin protein as illustrated in Table 2. The BION server showed 8 glutamate and 14 aspartate residues along with 5 threonine, 2 phenylalanine and 1 each of alanine, aspargine, arginine, serine, glycine, isoleucine, and valine. Red color balls are depicting the calcium ions interacting with yellow and green β-sheets and β-turns, respectively, present in the N-terminal of fibroin.

nonspecific

server also predicted the coordination distance of calcium ions and fibroin residues. The BION server predicted role of specific amino acids in fibroin protein involved in binding with calcium ions (Figure 7). A total of 36 residues were identified on Nterminal region of fibroin protein (Table 2). The BION server showed 8 glutamate and 14 aspartate residues along with 5 threonine, 2 phenylalanine, and 1 each of alanine, aspargine, arginine, serine, glycine, isoleucine, and valine. The aspartate and glutamate residues carry a negative charge, which facilitate the electrostatic interaction with positive charged calcium ions. The server also predicted the distance between bound calcium ion and amino acid residues that generated an insight about the nature of interaction occurring between the calcium ions and these residues. The residues with a bond length in the range of 2−3 Å were predicted to electrostatically interact with calcium ions while those with higher bond lengths were predicted to interact nonspecifically with calcium ions. The calcium binding sites present in the N-terminus of fibroin consisted of highly hydrophilic amino acids like glutamate and aspartate, which have a −COO− side chain that can electrostatically interact with positively charged calcium ions. Similar mechanism was reported earlier in calcium binding proteins like cytolytic bacterial toxins, where calcium ion binding sites consist of negatively charged aspartic acid residues.49 These negatively charged amino acid residues bridge two calcium ions with the fibroin protein chain. Glutamate residues are known to increase the solubility and bioavailability of calcium ions by means of chelation helping in their absorption in organisms.50,51 It has been reported previously that both glutamate and aspartate play a key role in binding and coordinating with calcium ions in sarcoplasmic reticulum ATPase.52 Another study illustrated that glutamate

residue

chain

position

bound to

bond distance (Å)

Glu Ala Asp Glu Phe Asp Asp Asp Glu Asn Arg Asp Glu Asp Arg Glu Asp Asp Asp Asp Glu Thr Thr Thr Phe Glu Ser Asp Asp Asp Val Thr Asp Glu Ile Gly

B A A A A A A A B A B B A A A A B A A B B A A B B A B B A B A B B B B A

99 50 44 45 31 108 91 27 78 93 48 89 28 108 48 57 108 29 49 49 57 43 60 87 84 28 90 27 100 44 85 43 25 56 23 92

Ca[5] Ca[28] Ca[7] Ca[13] Ca[27] Ca[21] Ca[11] Ca[19] Ca[9] Ca[34] Ca[30] Ca[14] Ca[10] Ca[33] Ca[35] Ca[4] Ca[18] Ca[22] Ca[20] Ca[17] Ca[8] Ca[31] Ca[26] Ca[36] Ca[6] Ca[32] Ca[29] Ca[15] Ca[2] Ca[23] Ca[16] Ca[12] Ca[3] Ca[1] Ca[25] Ca[24]

2.426 2.458 2.458 2.465 2.494 2.495 2.501 2.510 2.518 2.524 2.546 2.551 2.593 2.614 2.616 2.671 2.750 2.780 2.819 2.850 2.905 3.048 3.074 3.202 3.266 3.375 3.471 3.623 3.635 3.708 3.767 3.958 3.995 4.281 4.398 4.897

a

Server predicted 36 amino acids which includes 14 aspartate, 8 glutamate with 5 threonine, 2 phenylalanine, and 1 each of alanine, aspargine, arginine, serine, glycine, isoleucine, and valine residues.

and aspartate are responsible for bidentate ligation of calcium to parvalbumin protein.53 Furthermore, the glutamate and aspartate residues also seem to be involved in the stabilization of the α-helical intermediate conformation through manipulation of charge densities in the fibroin chain. The presence of aspartate in the N-terminus of a neutral alanine based peptide was found to stabilize the αhelical conformation.54 Another study showed that aspartate is the most potent residue in stabilizing helical conformations in short peptides at the N-terminus followed by asparagines, serine, glutamate, glutamine, and alanine.55 Studies have also indicated that polyglutamate peptides tend to have a helical conformation.56,57 During sol to gel transition of fibroin (toward a β-sheet conformation rich structure) conformational transition is hindered by the interaction of calcium ions with fibroin residues that drives the conformation toward α-helical intermediate. Finally, fibroin forms a predominantly β-sheet structure but due to the interaction of calcium ions a part of G

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Figure 8. Proposed mechanism involved during conformational transition of fibroin incubated with CaCl2 at 37 °C with shaking at 150 rpm.

helical intermediates remain intact even in the final conformation that fails to go into β-sheet form. Thus, a comprehensive prediction of calcium ion binding sites helped to explain the underlying mechanism of the role of calcium ions in modulating the conformational transition during fibroin selfassembly. Based on aforesaid observations we propose the following mechanism of self-assembly of fibroin in the presence of calcium ions (Figure 8). The regenerated solution of fibroin consists of polypeptide chains in random coil conformation. As calcium ions are added into this solution, they start interacting with the polypeptide chains disrupting the existing hydrophobic interactions. Calcium ions have chaotropic property, which helps in disrupting intramolecular forces like hydrogen bonding prevailing among the residues of fibroin by charge shielding and thus preventing the stabilization of salt bridges. The modified hydrophobic interactions lead to the formation of α-helical intermediate conformation before adopting its native structure. This intermediate stage of predominantly α-helical structure is then driven toward its native β-sheet conformation, which also retains some percentage of the intermediate conformation due to the changes brought by calcium ions. We propose that the transition from α-helix to β-sheet involves unraveling and subsequent reordering. This process of reordering is caused due to the charge shielding done by the calcium ions. Thus, the mechanism of calcium ion induced conformational transition of silk fibroin described in the current study could have broad utilization toward development of fibroin based biomaterials. Secondary conformations in silk matrix are known to modulate degradation kinetics, matrix stiffness.58 Mesenchymal progenitor cells embedded in 3D printed constructs having higher β-sheet content of sonicated silk fibroin (25.4%)

demonstrated higher propensity toward osteogenic differentiation, compared to tyrosinase-induced cross-linked constructs (having 14% β-sheet content).59 This finding highlighted a strong significance of controlling the secondary conformation and stiffness of scaffold to govern a lineagespecific of progenitor cells. Silk fibroin protein as a biomaterial do not elicit long-term adaptive immune response,35 but presence of different secondary conformations can impart immunostimulatory response to silk biomaterials. Taken together, fibroin protein assembly can be well optimized with calcium dependent chemistry in order to achieve tunable immune response to fabricate biomaterials for tailor-made tissue engineering applications. The limitations of the present study include the use of a specific concentration of silk fibroin protein. The effect of calcium ions may vary with change in concentration of fibroin which needs to be studied further. Second, all the studies reported so far have looked into the effect of various metallic ions separately but not in combination. While in vivo various ions participate together to modulate the silk fibroin conformation so as to help in silk spinning process. Thus, the combinatorial study of multiple ions together needs to be done to gain further insight about the self-assembly process of silk fibroin. This understanding can have a significant implication on the development of tailor-made silk biomaterials in tissue engineering.

4. CONCLUSIONS Calcium ions bind to fibroin at negatively charged amino acids like glutamate and aspartate. This binding hinders the hydrophobic interactions prevailing in the fibroin chains and modulates the self-assembly process. A predominantly α-helical H

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intermediate is formed as a result of this interaction which is also retained to some extent in the final conformation attained by fibroin. This calcium ion-fibroin interaction can thus be utilized for modulating the self-assembly process of fibroin solution to get tunable secondary conformations.

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AUTHOR INFORMATION

Corresponding Author

*Fax: 91-11-2659-1103. Tel.: 91-11-2659-1440. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.biomac.5b01258 Biomacromolecules XXXX, XXX, XXX−XXX