X-ray diffraction pattern from locally available Tassar fibers was recorded using imaging plate system (Dip-100S). The pattern was used along with fiber processing software to identify the reflections and to compute X-ray intensities. A molecular model was first constructed with standard bond lengths and angles using helical symmetry and layer-line spacing observed in the X-ray pattern. The model was then refined against observed X-ray data using linked-atom least-squares (LALS) method. The crystal and molecular structure of Tassar fibers is reported. It is concluded that the sheet structures formed by hydrogen bonds assume the antipolar-antiparallel arrangement.

1. Introduction

Silk is a composite material and generally defined as protein polymers that are spun into fibers by some Lepidoptera larvae such as silkworms, spiders, mites, and flies. Silk fibers are paracrystalline in nature and they have found extensive applications in textile industry. Compared to cellulose and other synthetic polymers, silk is peculiar for its durability, high strength, luster, and other unique features [1]. Among the Lepidopterans, the silk moths, especially Bombyx mori, Antheraea pernyi, Antheraea mylitta, and Antheraea assama play an important role in rural economy of many developing nations [2]. The importance of silk protein increased because of its potential use as a natural biopolymer for tissue engineering and biomedical applications. There are two classes of silk, namely, mulberry (Bombyx mori) and nonmulberry (Tassar, Eri and Muga). Antheraea mylitta (A. mylitta) is one of the wild varieties of non-mulberry silkworm, which produces Tassar silk. Few other wild silks are Mopani silk from South Africa, Saturniidae silk from Thailand, Assam silks (Muga, Eri, and Pat) from India, Tussah Silk from China, and Tassar Silk from India. Six million people in India alone are involved in sericulture, and one of the most economically important species for sericulture in India is the wild type nonmulberry silkworm, A. mylitta [2]. Tassar is a multivoltine type of silk and is mostly produced by tribal by rearing silkworms on forest plants (Terminalia arjuna, Terminalia tomentosas and Shorea robusta) [3]. Tasar silk fiber has its own distinctive color, is coarse to feel, but has higher tensile strength, elongation, and stress-relaxation values than the mulberry silk fiber secreted by Bombyx mori.

Different techniques were used by many researchers to understand the crystal and molecular structure of domestic and wild silk fiber varieties. Crystal structure of silk fibroin was reported by Marsh et al. who revealed that silk is made up of regular arrangements of antiparallel sheets [4]. Amino acid sequence, basic building blocks, of silk fibers, has been reported by number of researchers [5]. Refined molecular and crystal structure of Silk I base on Ala-Gly peptide sequence was carried by Okuyama et al. [6]. Crystal structure of pure Mysore silk (PMS) was carried out by Sangappa et al. [7]. Crystal and molecular structures of raw Bivoltine silk fibers were carried out by Mahesh and Somashekar who reported the comparison of parameters with pure Mysore silk fiber [8]. A rough computation of crystal structure of Tassar fiber in wet and dry condition was carried out by Reddy and others to find reason for two-load build-up regions in tensile graph [9].

In the present study, an attempt has been made to emphasis on modifications in crystal and molecular structure of Tassar silk (Indian) fibers employing X-ray diffraction data and linked-atom least-squares (LALS) refinement modeling, so that one can have a better perspective of structure property relation in these fibers.

2. Experimental

2.1. Sample Preparation

Locally available raw Tassar silk fibers belonging to Antheraea mylitta family, which comes under the classification multivoltine on the basis of shape, color, denier and life cycle of the fibers/cocoons. Cocoons were collected from the germ plasma stock of the Department of Sericulture, University of Mysore, which were then cooked in boiling water (100ยฐC) for 2โ€‰min and transferred to water bath at 65ยฐC for 2โ€‰min. Then the cocoons were reeled in warm water with the help of mono cocoon reeling equipment Epprouvite. These fibers were mounted on rectangular frame in just taut condition. The whole process, starting from reeling to mounting of fibers, does not involve any type of mechanical deformation.

2.2. X-Ray Diffraction Measurement

XRD patterns from raw Tassar silk fibers were recorded on an imaging plate. A rotating anode X-ray generator (ULTRA-X, RIGAKU) was operated in a normal focus mode to provide a monochromatic beam of wavelength (ฮป = 7107โ€‰ร…), at 50โ€‰kV and 250โ€‰mA. Diffraction data was recorded on a disk-shaped imaging plate with the sample to plate distance of 150โ€‰mm. The measurement of X-ray diffraction data was implemented by the hardware system DIP100S (MACSCIENCE). The intensity values were thereby converted into pixel data in a rectangular coordinate system. A whole area of the imaging plate (diameter ~200โ€‰ฮผm) was divided into 1600 ร— 1600 pixels each of a size of 125โ€‰mm2. For this purpose, the program available in CCP13 suite was used (CCP13, 2004). For computation, a workstation, OCTANE, version 6.5 with an operating system IRIS 64, was used. Ivanova and Makowshiโ€™s (1998) method for background estimation, which is more reliable and consistent, was adopted [10]. It makes use of the fact that the background of a fiber diffraction pattern is typically composed of lower spatial frequencies than the diffraction maxima. Image plate X-ray pattern for Tassar fiber is given in Figure 1.

Each diffraction spot was picked by positioning the mouse on its centre, and coordinates were measured using SUN SP/2, a SUN microsystem Computer Corporation Business. After determining the centre and inclination angle of the X-ray pattern, the interplanar spacing was obtained by averaging the distances between the centre of the diffraction pattern and the positions of two or four equivalent reflections. The dimensions of unit cells were determined by least-squares method with the preliminary cell dimensions being obtained by a trial and error method on the computer display. We found no significant variation in the cell parameters of different silk fibers. The averaged cell parameters are ๐‘Ž=9.44(5)โ€‰ร…, ๐‘=10.66(3)โ€‰ร…, and ๐‘=6.95(7)โ€‰ร…. The space group of the structure being P212121 and has four asymmetric units in a cell each comprising two residues. The whole pattern fitting is carried out in two stages. In the first stage, X-ray diffraction pattern taken on a flat imaging plate system was transformed to reciprocal space using the specimen to film distance, rotation of image, wavelength, and tilt of the system and employing a program FTOREC, available in the software suite CCP13. In the second step, for the whole pattern fitting, we have used a program LSQINT available in CCP13 suite for which the input file is the output of FTOREC program. By inputting unit cell parameters, the space group symmetry, and the profile parameters, further processing of the pattern was carried out. Using CCP13 package โ€œXCONVโ€ with appropriate file options suitable for DIP image system, we can simulate cylindrical image and also calculate the integrated intensities with standard deviations [11]. The output of this routine give simulated cylindrical pattern and computed integrated intensities. These intensities were corrected for polarization and the Lorentz factors [12]. The output file which contains the integrated intensities corresponding to various (hkl) reflections with standard deviations are further used to determine the molecular and crystal structures of Tassar silk fibers.

3. Structure Determination

3.1. Molecular Model

Silk is a composite material with amino acid sequence as its primary structure. Amino acid analyses of Antheraea fibroins [5, 12] show a uniformly high content of alanine ([Ala]n). There is an option in linked-atom least-squares (LALS) program to indicate two molecular chains by defining the constrains for only one of them. Here, the geometry for Ala is for the D-Ala and not L-Ala. The molecular conformation must satisfy both the sterical and mathematical requirements. Hence, we have chosen initial ฯ†โ€‰โ€‰and ฯˆโ€‰โ€‰for Ala and Gly residues to be the same as that of Marsh et al. (1955) results [4], and the main chain was constructed with appropriate helical parameters together with bond lengths and angles.

Takahashi et al. (1999) have shown that there are four models for the sheet structure formed by hydrogen bonds. They are (i) polar-antiparallel [PA (1)]; (ii) polar-parallel (PP); (iii) antipolar-antiparallel (AA); (iv) antipolar-parallel (AP). In the polar model, the methyl groups of alanine residues are on one side of the sheet only. While in the antipolar model, the methyl groups alternately point to both sides of the sheet along the hydrogen bonding direction [13]. For the positioning of the molecular model in the unit cell, two additional parameters were used to define the relative axial rotation (ฮผ) and translation (w) along the ๐‘-axis. At each stage in the modeling and refinement of the structure, we minimized the quantity ฮฉ in the following least-squares fashion [14]. The first summation in ฮฉ ensures the optimum agreement between the observed (๐น๐‘œ) and the calculated (๐น๐‘) X-ray structure amplitudes. The w is the weight of each reflection. The second๎“๐‘ค๎€ท||๐นฮฉ=๐‘œ||โˆ’||๐น๐‘||๎€ธ2๎“+๐‘†๐‘—โˆˆ๐‘—๎“+๐‘†โ„Ž๐œ†โ„Ž๐บโ„Ž(1) ensures the optimization of noncovalent interatomic (โˆˆ๐‘—). S is the scale factor used to adjust the overall weight of the second term with respect to the first. Third imposes, by the method of Lagrange undetermined multipliers (lh), the exact constraints (Gh) we have chosen the exact constraints (Gh). Atomic scattering factors for calculating structure factors were obtained using the method and values given in International Tables for X-ray Crystallography (1974). Computations were carried out and compiled the LALS program using LINUX operating system based PC.

3.2. Molecular and Crystal Structure for Repeating Unit

The refinement was carried out for the crystal structure in which two antiparallel molecules related by a 2-fold rotation axis parallel to ๐‘-axis. In order to get appropriate packing parameters, the discrepancy factors ๐‘…๐‘ (conventional R factor) and ๐‘…๐‘ค (weighted ๐‘…๐‘ factor) and shortest contact between nonbonded atoms were calculated. Here, ๐‘…๐‘ and ๐‘…๐‘ค were defined by๎“||||๐น๐‘…=๐‘œ||โˆ’||๐น๐‘||||โˆ‘||๐น๐‘œ||,๐‘…๐‘ค=๎“๐‘ค๎€ท||๐น๐‘œ||โˆ’||๐น๐‘||๎€ธ2โˆ‘๐น2๐‘œ.(2) A dipeptide Ala-Gly was used as chemical repeating unit for refinement. The weight of each reflection โ€œwโ€ was fixed to 1.

4. Results and Discussion

The refined parameters and the final values are summarized in Table 1. Here, the azimuthal angles of the two molecules forming a sheet were also refined independently. The refinement gave good agreement between the observed and calculated structure factors (Table 2). Fractional atomic coordinates for (Ala-Gly) residues are given in Table 3. The crystal structure of Tassar silk in raw form is shown in Figure 2. The internal rotation angles (ฯ‰) of glycine and alanine residues about Nโ€“C(=O) bonds are 179.06 and 177.87, respectively, which are nearly trans conformation. The ฯ†โ€‰โ€‰and ฯˆโ€‰โ€‰for the glycine residue are โˆ’146.16 and 138.52, respectively, which are also between skew and trans conformations. The ฯ†โ€‰โ€‰and ฯˆโ€‰โ€‰for the alanine residue are โˆ’141.76 and 148.44, which respectively are in agreement with the values (ฯ† = โˆ’146.67ยฐ and ฯˆโ€‰โ€‰= 143.00ยฐ) given by Pauling and Corey for antiparallel-chain model [15, 16]. The torsional angles and Eulerian angles are the same for both chain-a and chain-b as they are symmetric. The steriochemical energy which is represented by ฯƒ is found to be 2.37E + 04. Here the ฯƒ is given by the sum of second term of (1) [6]. We observe that the molecular modification is essentially same as ฮฒ-pleated structure with antipolar-antiparallel arrangements formed by hydrogen bonds.

5. Conclusions

Crystal and molecule structure of locally available Tassar fiber, belonging to A. mylitta family, was carried out by X-ray diffraction data and employing linked-atom least-squares method. Four molecular chains are contained in the rectangular unit cell with parameters ๐‘Ž=9.44โ€‰ร…, ๐‘=10.64โ€‰ร…, and ๐‘=6.95โ€‰ร… and the space group being P212121. From the obtained values of torsional angles, Eulerian angles and other parameters, we conclude that, in raw Tassar silk fiber, the sheet structures formed by hydrogen bonds assume the antipolar-antiparallel arrangement, which is in conformity with the results of Takahashi et al. (1999) [13].