Silk from Indian Paper Wasp: Structure Prediction and Secondary Conformational Analysis

Polymer 208 (2020) 122967

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Polymer

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Silk from Indian paper wasp: Structure prediction and secondary conformational analysis

Shikha Chawla , Sinchan Seit , Sumit Murab , Sourabh Ghosh *

Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India

ARTICLE INFO ABSTRACT

Keywords: The pupae of Indian paper wasp, Ropalidia marginata, produce silk that covers the opening of each cells in their Silk hives, which probably helps in thermoregulation and providing mechanical stability. In this study an attempt has Ropalidia marginata been made to investigate the amino acid composition and protein structure of Indian wasp silk using bioinfor Secondary conformation matics and experimental tools. The amino acid analysis of the regenerated solubilized wasp silk revealed high alanine and serine content that increases the propensity of α-helical and random coil and/or β-sheet structure respectively. The Matrix Assisted Laser Desorption/Ionization-Time Of Flight/Time Of Flight based protein fingerprinting analysis of the regenerated solubilized wasp silk revealed its short-range sequences. The protein modeling studies indicated the presence of predominant coiled coil α-helical and random coil structure in the protein isolated from wasp hive caps, similar to previous reports from the Vespoidea family. Fourier transform infrared spectroscopic analysis and X-ray diffraction analysis of the native paper wasp cap revealed the presence of mix of coiled coil α-helix and β-sheet structure in outer regions; whereas inner region contains tannin/ intermolecularly bonded hydroxyl groups of polyphenols. The present study is first report to generate valuable insights about the chemical composition and secondary structure prediction of Indian paper wasp silk and highlight on nature’s novel modus operandi to engineer unique functional fibrous matrix.

1. Introduction Hymenopteran silk from the Vespoidea family like hornets, ants and wasps and Apoidea family like bees. Taken together, it is important to Thousands of creatures are known to produce silk [1], still there are understand how specific secondary conformations and hierarchial many un-explored varieties in nature. Different species, namely Lepi structural assembly impart unique characteristics and functionality to dopterans like the domestic silkworm (Bombyx mori), spiders (Araneae) each variety of silk produced by different species. or aculeates or non-aculeates Hymenopterans [2] produce silk filaments Numerous studies have tried to predict the molecular structure of which exhibit tremendous diversity due to their food habits, utility, Vespoidea superfamily like hornets in order to understand structural metabolic activities. The non-uniformity can be corresponded to dif properties. For example, Japanese hornet Vespa simillima xanthoptera ference in amino acid sequence, secondary structures leading to Cameron reportedly produces fivetypes of silk proteins termed as Vssilk different properties of the silk amongst different species. For instance, 1–5 [5,6]. Matrix-assisted laser desorption/ionization-time of flight hydrophobic domains that contribute to β-sheet crystals are predomi (MALDI-TOF) was used to deduce the partial amino acid sequences of nant in silkworms and dragline spiders contributing to superior me these proteins to predict the secondary structure of Vespa simillima based chanical properties. During silk spinning, poly-Alanine sequences in on MARCOIL bioinformatics server. The analysis deduced that these spider silk and GAGAGS or GAGAGY sequences in silkworm silk undergo proteins are rich in alanine in the middle region which is flanked be self-assembly, due to hydrophobic interaction and salting out effect of tween serine-rich terminal regions [5] leading to coiled coil conforma various metal ions present. Later on once the self-assembly is completed, tion. The X-ray diffraction studies carried out on silk from giant gelation followed by shear induced phase separation, dehydration and Japanese hornet Vespa Mandarinia Japonica suggested the presence of refolding finallydevelop solid, insoluble and uniform fibre[ 3,4]. On the both α-helix and β-sheet secondary structures [7]. other hand, α-helical coiled coils are predominant in Aculeate The current study investigates the structure-property correlation of

* Corresponding author. E-mail address: [email protected] (S. Ghosh). https://doi.org/10.1016/j.polymer.2020.122967 Received 13 June 2020; Received in revised form 14 August 2020; Accepted 19 August 2020 Available online 25 August 2020 0032-3861/© 2020 Elsevier Ltd. All rights reserved. S. Chawla et al. Polymer 208 (2020) 122967 the silk produced by the Indian paper wasp Ropalidia marginata (Hy 2.2. Scanning electron miroscopy (SEM) menoptera, Vespoidea, Polistinae). These wasps make colonies that are non-enveloped nests unlike hornets, on leaves, twigs, elevated rocks and Native as well as washed and degummed Indian paper wasp caps manmade building structures (Fig. 1a–c) [8,9] The wasp larvae spin a were cut into pieces of 4 mm × 4 mm to gain an insight about the cap of silk protein filamentsto cover the openings of each cell of the hive orientation of fibres and distribution of fibre diameter using SEM. The and pupate inside, similar to hornets (Fig. 1b) [10]. This silk cap puta samples for SEM analysis were prepared using ultramicrotome with tively helps in maintaining an optimum temperature inside the cells; diamond blades. Samples were vacuum dried and gold coated using supportive for the larvae to develop and offer protection to the pupa sputter coater (EMITECH K550X) and imaged with SEM (Zeiss EVO 50). [11]. However, composition and properties of the fibrous silk protein The fibre diameter (n = 30, 2 measurements at different positions on produced by Indian paper wasp Ropalidia marginata wasp is not known. each of 15 fibres) was calculated using ImageJ software (NIH) [14]. We have utilized bioinformatics tools, biochemical and material char acterization techniques to interpret its secondary conformations that 2.3. High performance liquid chromatography (HPLC) contribute to the macromolecular structure of Indian paper wasp.

2.3.1. Performic acid oxidation 2. Materials and methods 50 mg of Indian paper wasp silk sample was taken in a conical flask for HPLC analysis for amino acid composition. 5 ml of performic acid 2.1. Wasp silk isolation (Merck) was added to the sample and it was kept in dark for 16 h fol lowed by addition of sodium metabisulphite (Sigma-Aldrich) till the The Indian paper wasp hives were collected from Indian Institute of effervescence ceased. Technology Delhi campus. The cellulosic wall and silk cap portions were carefully separated for analysis. The caps were treated with a 2:1 2.3.2. Acid hydrolysis of silk and drying (chloroform: methanol, Merck) solution to remove the organic impu The hydrolysis of the silk proteins was carried out in 50 × 6 mm rities, if present [12]. The samples were then washed with sodium sample tubes that were then placed in the reaction vials (Agilent). The dodecyl sulfate (SDS) (Merck) and boiled in Sodium carbonate (Merck) reaction was carried out to the above solution by adding 25 ml of 6 N solution for 15 min followed by air drying overnight. The silk caps were hydrochloric acid (Sigma-Aldrich) containing 1% (v/v) phenol vapours then dissolved in 9.3 M lithium bromide (LiBr) (Central Drug House Pvt. ◦ ◦ under oxygen-free conditions at a temperature of 110 C, and it was Ltd.) at 60 C for 4 h, to obtain a 2% w/v solution of silk fibroinprotein attached to refluxair condenser for 24 h. The mixture was then allowed [13]. The silk-LiBr solution was dialyzed in deionized water using to cool at room temperature and neutralized with 50% sodium hy Slide-A-Lyzer cassette (Thermo, molecular weight cut off 3500 kDa). droxide (pH 2.2). The volume of the mixture was made up to 50 ml with This regenerated solubilized Indian paper wasp silk protein was used for HPLC grade water (Agilent). 20 ml of the above mixture was dried using amino acid analysis and Matrix-Assisted Laser rotary evaporator. Desorption/Ionization-Time Of Flight/Time Of Flight (MALDI- TOF/TOF) analysis to predict secondary conformations of the Indian 2.3.3. Derivatization of amino acids paper wasp. The derivatization of the hydrolysed samples was carried out to

Fig. 1. a. Indian paper wasp hive hanging from a leaf. b. Cells in the hive are covered with white silk caps, spun by the larvae. c. Indian paper wasp and a large wasp hive prepared by different generations of wasp.

2 S. Chawla et al. Polymer 208 (2020) 122967 produce phenylthiocarbamyl (PTC) derivatives of amino acids, which Subsequent MS/MS data analysis was carried out using Easy Access were then analysed by HPLC. The derivatization reagent was freshly Wizard™ (AB SCIEX) and QuanTis™ (AB SCIEX) that allowed non- made and consisted of methanol: triethylamine: water: phenyl redundant and automated selection of precursor ions. Protein identifi isothiocyanate (Merck) in the ratio of 7: 1: 1: 1. To this dried mixture 5 cation was done using and MASCOT search engine (Matrix Science) ◦ ml of derivatizing reagent was added and incubated for 20 min at 25 C. against the UniProtKB-SwissProt Database in Arthropoda taxonomy. Then 5 ml of diluent (diluent: 71 mg of sodium phosphate dibasic The protein sequences were then analysed for similarity searches by (Sigma-Aldrich) in 100 ml of water set pH 7.4 with 10% phosphoric acid searching the sequences against the Vespa simillina xanthoptera NCBI (Sigma-Aldrich) was added. The amino acid standard was prepared nonredundant (nr) protein database using the (Basic Local Alignment following manufacturer’s instructions by mixing standard mixture of Search tool) BLAST Tool (BLAST 2.2.30) [18]. amino acid at a concentration of 250 pmol/μL (90 μl, Agilent) and 10 mM norvaline (5 μl, Sigma-Aldrich) [15]. The glass vials to be used for 2.4.4. Protein modeling studies mixing and analyzing the samples were soaked and washed thoroughly The secondary conformational prediction of the Indian paper wasp with detergent solution for 12 h and washed with deionized water to protein sequence obtained from MALDI/TOF/TOF analysis was per eliminate the possible glycine contamination. Both the samples and the formed using MARCOIL, SOPMA [20,21] and GARNIER (GOR4, Institute standards were analysed using HPLC (Column: ZORBAX 300 Extend-C18 of Biology and Protein Chemistry, Lyon, France) [21,22]. MARCOIL HPLC Columns, Agilent Technologies) using the chromatography con predicts existence and location of probable coiled-coil domains in pro ditions according to Agilent analytical protocol [16] with minor modi tein/peptide sequences through a Hidden Markov Model-based program fications as mentioned elsewhere [15]. [21,23]. With respect to MARCOIL, I-TASSER (Iterative Threading AS SEmbly Refinement) server was further used to predict the secondary 2.4. Matrix-assisted laser desorption/ionization time of flight (MALDI- structures of paper wasp and Vssilk 1 (GenBank: BAJ09446) protein TOF) sequence from Vespa simillima xanthoptera. I-TASSER is a hierarchical approach for the prediction of protein structure and function. Multiple 2.4.1. SDS- polyacrylamide gel (SDS-PAGE) run threading approach LOMETS from Protein Data Bank (PDB) is used to Bradford assay was used to determine the concentration of wasp silk identify structural templates; iterative template fragment assembly protein solution using a UV-VIS spectrophotometer (PerkinElmer) at simulations are then used to construct full-length atomic models. Lastly, 595 nm. The required quantity (15 μg) of protein was loaded onto 12% the 3D models of Indian paper wasp and Vssilk1 were threaded through (SDS PAGE) under reducing conditions, for this the protein sample was BioLiP protein function database to gain functional insights of the target mixed with equal quantity of sample buffer containing β-mercaptoe [24]. The quantitative values of the secondary conformations predicted ◦ thanol (Sigma-Aldrich) and denatured at 99 C for 5 min. The gel was were further calculated. stained with silver nitrate after electrophoresis. Molecular weights were determined using a molecular weight marker range 14–97 kDa (Bio- 2.4.5. Attenuated total reflection- Fourier transform infrared (ATR-FTIR) Rad) [17]. spectroscopy FTIR of native paper wasp silk was acquired in transmittance mode 1 2.4.2. In gel tryptic digestion between 4000 and 400 cm using Bruker Alpha P ATR-FTIR having In gel tryptic digestion was performed following the protocol used in deuterated triglycine sulfate IR detector, with spectral resolution of 0.9 1 our previous study [18]. The most prominent band at 37 kDa (Fig. 3a) cm , number of scans 240. The spectra were normalized and the rela was cut using a clean scalpel. The band was further cut into smaller tive area under each peak was used to determine the total crystallization pieces of 1 mm cube. Cut pieces of gel were destained in 50/50, 30 mM in the sample. Fourier self-deconvolution (FSD) of spectra was per potassium ferricyanide (Sigma) and 100 mM sodium thiosulphate formed using PeakFit Version 4.12 (Systat Software). Fitting of FSD FTIR (Sigma) for 30 min. Followed by washing in water for 5 min gel pieces spectra with Gaussian profiles was done in amide I region (1600 and 1 were washed with 50/50, 100 mM ammonium bicarbonate (NH4HCO3, 1700 cm ) and secondary conformation fraction (ratio of these bands Merck)/acetonitrile (ACN, Merck). 100% ACN was added onto the gel to the total amide I bands) was determined by integral of Gaussian pieces for 5 min to dehydrate the pieces followed by air drying. 10 mM profiles [25]. dithiothretol (Merck) in 100 mM NH4HCO3 was added to the sample and ◦ incubated for 30 min at 56 C followed by washing with 50/50, 2.4.6. X-ray diffraction (XRD) ACN/100 mM NH4HCO3. Alkylation process was done using 50 mM Crystalline content in native Indian paper wasp silk was determined iodoacetamide/100 mM NH4HCO3 in dark at room temperature 30 min. using wide-angle XRD (X’Pert PRO PANalytical–DY2022) using the Gel pieces were washed and dehydrated and 10 μl of a 20 ng/μl trypsin protocol mentioned elsewhere [26] using CuKα radiation (1.5405 Å) ◦ ◦ ◦ solution (Sigma) was added to the pieces for 16 h at 37 C. Peptides were from 0 to 50 (2θ) under 40 KV, 30 mA. extracted after incubation with 1% trifluoroacetic acid (TFA, Merck) in distilled water by sonication for 5 min. The extracted peptide solution 2.4.7. Birefringence studies was used further for MALDI-TOF/TOF analysis [19]. To assess the alignment of biomacromolecules of wasp silk Leica DM2500 P microscopy system was used to visualize fibrebirefringence. 2.4.3. MALDI-TOF/TOF analysis Birefringence values were calculated using the classical Michel-Levy Dried droplet method was used to prepare samples for MALDI-TOF/ interference colour chart. TOF analysis. 1 μl of the peptide solution extracted was mixed with 1 μl of a suitable matrix alpha–cyanohydroxycinnamic acid (Bruker) in 1:2 3. Results & discussion v/v of ACN: 0.1% TFA. 1 μl of this mixture was spotted on a MALDI target plate and allowed to air dry at room temperature. MALDI-TOF/ 3.1. Morphological analysis of paper wasp silk cap TOF spectra were acquired in reflection positive ionization mode using AB SCIEX 5800 MALDI-TOF/TOF (AB SCIEX) using 1 kHz Opti The morphological characterization was done using SEM to deduce Beam™ on axis laser. Peptide Mass Standards Kit (AB SCIEX) was used the surface topology as well as fibre distribution and fibre diameter of to calibrate the instrument. 1000 laser shots per MS spectra were ac the silk cap. Fig. 2a shows the native cap part of a wasp hive. The native quired at 400 Hz pulse frequency, while in case of MS/MS 2000 laser wasp cap is chiefly made of randomly oriented round silk fibres, along shots were acquired at 1 kHz pulse frequency. Protein Pilot software V with flat fibres. Similar structure of the cap comprising fibres and flats 3.0 (AB SCIEX) was used to analyse the Mass Spectrometry spectra. have been reported for other vespoidea family like the Oriental hornet,

3 S. Chawla et al. Polymer 208 (2020) 122967

Fig. 2. Scanning electron microscope images of a. Top-view of native unprocessed Indian paper wasp cap, b. Few layers from top were removed and sectional view of native unprocessed wasp cap was captured, c. Top-view of degummed and dewaxed wasp cap, d. Cross-section of degummed and dewaxed cap showing the presence of both round fibrilsand flatfibrillar deposition at higher magnification,e. Some fibresmerged with each other indicating that there was delayed solidificationof the fibres upon spinning from the spinneret. f. Concentric straitions mark covering the fibre.

Vespa orientalis [27]. Thus, we can speculate that the random orientation first level of hierarchical cross-linking as reported by Sutherland et al. of fibresand flatsprovide greater support needed for the internal part of [29]. The N- and C- terminal sequences predominantly consisting of the paper wasp hive cap. The ultramicrotomed cross-sectional surface of serine amino acid residues encompassing the central coiled coil domain wasp cap showed randomly oriented fibres that were covered with determine the second level of cross-linking. This level is mainly wax/organic impurities (Fig. 2b). The top view of dewaxed and responsible for the development hydrogen bonded networks of inter degummed wasp cap shows that the flats are less prominent, depicting molecular β-sheet that imparts wasp silk protein the ability to get the removal of wax and any sericin like protenaceous material present partially dissolved using the concentrated lithium bromide. Further, and clear fibres could be observed (Fig. 2c). The diameter of thicker third level of crosslinking is contributed by the transglutaminase fibreswas measured to be ~13 μm, whereas it was measured to ~5 μm induced iso-peptide bonds between the glutamine and the lysine amino for thinner fibres (Supplementary Fig. 1a). The average fibre diameter acids [30]. Although proteins produced by the hornets and wasps have was found to be 8.4 ± 2.3 μm which is in sync with the earlier reported been reported to contain sparse amount of these covalent crosslinks, but studies on Vespoidea family with fibrediameter varying from 4 to 15 μm they also contribute towards resistance of dissolution [31]. The final [27]. Further, cross-section of fibres at higher magnification revealed level of cross-liking is contributed by the covalent tanning reactions that both round and thick and thinner fibres( Fig. 2d–e). It was observed that provides the brown color to other varieties of silk (such as non-mulberry some fibresare merging with each other (Fig. 2e) indicating that there is silkworm silk and ant silk) [32]. These tanning reaction are carried out indicating that there is delayed solidificationof the fibresupon spinning by phenoloxidases that oxidise phenolic compounds that crosslink the from the spinneret which might be the reason for the observed variation nucleophilic side chains of amino acids; used for hardening of silk in in the fibre diameter of Indian paper wasp cap. An interesting obser some species [31]. Interestingly, while the initial silk was milky white in vation was the presence of ring like structures on the fibre surface color, after dissolution in LiBr, the leftover undissolved silk was brown revealed at higher magnification( Fig. 2f). The reason for the presence of colored probably owing to the presence of the covalent tanning com ring like structures could be originated by the intermittent spinning pounds in the inner core of the paper wasp cap silk fibres (Supplemen behavior of wasp pupa, where the pupa probably applies minimal tary Fig. 1b). stretching shear force, which contributes to the ring like impressions on the silk fibre.In contrast, silkworm silk fibroinpossess smooth texture of fibre due to immense shear stress applied during spinning of the silk 3.2. Amino acid analysis worm silk as well as continuous secretion of fibroinprotein through the spinneret. HPLC analysis was conducted to elucidate chemical composition of The dewaxing protocol resulted in 0.87% w/w loss from hive caps wasp (Supplementary Fig. 1c). There were four major peaks for alanine, suggesting the presence of low hydrocarbon content which may be from serine, glutamic acid and glycine (Table 1). R. marginata silk contains depositions through the wasp saliva that aids in recognition of their alanine as its major component comprising 33.4% of molecular per nests and nest mates [28]. The very low hydrocarbon content indicates centage (Table 1). Other members of Apoidea and Vespoidea families are that they do not play significant structural role in hive cap formation. similarly reported to contain high percentages of alanine, which is hy Further, there was loss of 1.46% w/w from the caps during degumming. drophobic amino acid [33]. Apart from alanine wasp silk also contains Also, during silk dissolution it was observed that only 44–50% w/w of high amounts of serine, glutamic acid, leucine, lysine, glutamine, and the silk got dissolved in LiBr solution while the rest remained undis threonine amino acids. solved even after 24 h. The tendency of wasp silk to not completely Alanine is generally found in the core of long coiled coil domains of dissolve could be contributed to a constellation of cross-linking mech the hymenopteran silk proteins [29] and known to have the highest anisms in the silk proteins produced by the Vespoidea superfamily. helix propensity [34]. Thus highest molecular weight percentage (33% Development of hetero-tetrameric coiled coil structures comprises the of total composition) of alanine residues in wasp silk, as observed by the amino acid analysis, indicates the probability of a predominant coiled

4 S. Chawla et al. Polymer 208 (2020) 122967

Table 1 hydrophilic residues buried in the core of the helices to stabilize the Amino acid composition of wasp silk. structure. This probably suggests a closely packed crystalline structure Name Retention Time (Minutes) Mol % made of coiled coils and β-sheets, which is significantly different from the silkworm silk. The amino acid composition analysis of the Indian Alanine 20.29 33.4 Serine 12.58 20.4 wasp silk putatively indicates towards a convergent evolution with Glutamic acid 6.22 9.3 respect to the presence of both alpha helical coiled coil and β-sheets Glycine 13.21 5.6 structures amongst different insects of Vespoidea family. The silks from Valine 32.53 5 hornets and honeybee have an evolutionary conserved coiled coil Aspartic acid 5.37 4.8 structure due to conservation of four paralogous genes that encode for Arginine 17.88 4.6 Cystine 27.1 3.3 these proteins [38]. Threonine 19.07 3.1 Leucine 37.8 3.1 Proline 20.66 2.1 3.3. MALDI-TOF/TOF analysis Isoleucine 37.25 1.7 Lysine 41.89 1.6 Tyrosine 30.89 0.8 In order to identify the protein sequence of Indian paper wasp; Phenylalanine 40.59 0.7 extracted paper wasp silk proteins was run on SDS PAGE which gave a Histidine 15.74 0.3 major band at 37 kDa (Fig. 3a). The complete probable sequence from 1 Tryptophan 41.23 0.1 to 284 amino acids identified by MASCOT corresponding to unnamed Methionine 34.46 0 protein product of Tetradon species (as represented in Fig. 3b, Supple mentary. Figure 2). Out of the complete sequence, the amino acids in red coil structure, as reported in other members of this family [5,35]. It is represent the exact experimental peptides identified by MALDI-TOF/ interesting to highlight here that in general predominantly large hy TOF that lead to the complete sequence 1 to 284 amino acids once drophobic amino acid residues are known to contribute to the coiled coil searched in MASCOT in Arthropoda category. Moreover, it is important structure of proteins so as to stabilize the core by maximization of the to mention here that MALDI-TOF/TOF and amino acid analysis showed hydrophobic interactions [36,37]. However, Sutherland et al. correlated differences in the amino acid percentage. The MALDI-TOF/TOF analysis high alanine content to coiled coil forming propensity in Vespoidea only provides a partial predicted sequence of the experimental protein, because of metabolic requirement of producing bulk amount of while amino acid analysis provides the exact amino acid quantification. non-recyclable silk by these insects, leading to utilization of Thus, the MALDI-TOF/TOF analysis was only used to predict the prob non-essential amino acids to form coiled coil. Thus, since alanine is the able secondary protein structure. most hydrophobic of all non-essential amino acids and is capable of Similar strategies have been utilized by others where the silks were favoring α-helix, high availability of alanine helps in coiled coil forma solubilized in LiBr to study the compositional analysis of proteins from tion [29]. other hornet species [33]. However, this is the firsttime an attempt has The second highest amount was for serine residue (22% of total been made to study the compositional analysis of silk produced by In composition). Serine has hydrophilic side chain and may slacken the dian paper wasp. The sequence obtained was used to predict the sec inter-plane interactions. Hence high serine content could indicate to ondary structure of Indian paper wasp using I-TASSER. Further, wards the presence of random coil conformations formation. Further similarity searches of resulting protein sequences of unnamed protein more, the presence of serine is also reported to help in stabilization of product in the Tetradon species was searched against the NCBI nonre β-sheet due to the presence of hydroxyl side chains, as demonstrated dundant protein database using the BLAST Tool which showed simi earlier for Japanese hornet, Vespa mandarinia japonica [2,7]. In general, larities with three proteins (Fig. 3c). These proteins are a part of 5 large portions of alanine and serine residues suggest a favorable con membered protein family, in a related hornet species (Vespa simillima), dition for crystallization, although the presence of their bulkier side named Vssilk 1–5 [5] displaying percentage similarities as follows: groups could impede close packing of molecules and thus hamper Vssilk 1 (22% sequence coverage), Vssilk 2 (6% sequence coverage), crystallization [29]. The ratio of bulky/non-bulky amino acid residues in Vssilk 5 (6% sequence coverage) (Fig. 3c). Thus, these three Vssilk wasp silk is 0.53 indicative of closely packed crystalline structure. The proteins were taken up for the prediction of coiled coil domains in their ratio of hydrophilic/hydrophobic groups was found to be 0.84, sug structure as a validation of bioinformatics based modelling approach gesting the possibility of formation of coiled coil structure with the with MARCOIL server (Table 2). The analysis showed highest

Fig. 3. a. SDS PAGE gel picture showing band at 37 KDa, which was taken for MALDI- TOF/TOF analysis. (Samples 1 is B. mori silk while Sample 2 is Indian paper wasp silk. b. Amino acid sequence of Indian paper wasp silk as obtained by MALDI-TOF-TOF experi ment represented as 1 to 284 amino acids identified after MASCOT search in Arthro poda database. The amino acids in red represent the actual identified peptides by MALDI-TOF/TOF corresponding to partial amino acid sequence of Indian paper wasp. c. BLAST search results of Indian wasp silk sequence showing similarities found be tween the present silk and that of Vespa Simillima Xanthoptera. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

5 S. Chawla et al. Polymer 208 (2020) 122967 probability of coiled coil domain in Vssilk 1 while Vssilk 5 showed the predominating, while the structure of former protein is dominated with least probability. X-ray diffraction studies have reported the presence of random coils in conjunction with α-helices in Alanine-rich region. The α-helices and β-sheets in these hornet silk proteins in a related species amino acid compositional analysis allied favorably with this predicted Vespa mandarinia japonica [7], which further supports the possibility of structure of Indian wasp where the core region in the Indian wasp having similar structure of the Indian wasp silk. The sequence similarity protein is alanine-rich which probably helps in the formation of coiled of Indian paper wasp silk protein was highest with Vssilk 1 which coil structure, while serine present in the terminal regions possibly help showed a total of 21% sequence similarity and 22% of query coverage in β-sheet formation as reported in earlier studies related to hornets with E value of 0.11 that counts up to 95.45% of total similarity in the [41]. We also calculated the secondary conformation percentages by sequences of Indian paper wasp silk and Vssilk 1. I-TASSER and compared it with two other bioinformatics structure prediction servers SOPMA, GOR4. The results are presented in Table 3 (Supplementary Fig. 3). All three servers indicated the presence of all 3.4. Protein modeling studies three secondary conformations in a comparable manner. Vssilk1 was taken up for in silico protein structural modeling analysis 3.5. ATR-FTIR analysis to validate the secondary structure model prediction approach used by us for the secondary structure prediction of Indian paper wasp silk. The Infrared bands arising from amino acid side chains are of significance secondary structure prediction by I-TASSER disclosed a predominantly with respect to the extraction of structural information of the protein of α-helical structure of Vssilk 1. The server generated five predicted interest. The different region of infrared spectra could correspond to the structures for the Vssilk1 with different C-scores. The estimated TM- amino acid side chains as reported in the past [42]. Thus the infrared score was 0.52 ± 0.15, while the estimated Root-mean-square devia spectra was utilized by us to deduce the amino acid composition of the tion (RMSD) was 10.4 ± 4.6 Å for the structures. The structures with the wasp silk protein. The spectrum obtained for wasp silk was dominated highest C-scores of 1.60 was taken up as the higher C-score value de by two major peaks at 1619 cm 1 corresponding to Amide I region and picts a model with a higher confidence( Fig. 4a). The structure showed a 1519 cm 1 corresponding to Amide II region [43] (Fig. 5a and b). The coiled coil arrangement of the α-helices in which each coil consisted of amide I region (1600–1700 cm 1) was deconvoluted to analyse the four α-helices. This was in parallel to the silk structures of Hymenopteran secondary structure of the Indian wasp silk (Fig. 5c, Supplementary species, which are known to be predominantly coiled coil in nature [29] Fig. 4a). The deconvoluted spectra of the region disclosed the presence and thus validates our secondary structure model building approach. of three prominent peaks corresponding to different conforms of the silk The study predicted an antiparallel tetrameric configuration for the protein (Fig. 5c). The high intensity peak at 1620 cm 1 observed after coiled coil domains, which fits the present predictions for Indian paper deconvolution corresponds to the presence of aggregate β-strands which wasp silk. Commonly, α-helical proteins have large hydrophobic resi accounts for about ~10% as corroborated from the predicted secondary dues in helix core to stabilize it through their hydrophobic effect [36]. structure from the web-servers (Table 3). But the highest content of As discussed earlier alanine being the most hydrophobic residue among conformation predicted ~50–60% i.e., of random coil, gives a peak at the non-essential amino acids in bees, makes it the best candidate for the 1652 cm 1 overlapping the peaks linked to α-helices with ~35% pre formation of α-helix core [39]. Now, alanine is not hydrophobic enough dicted conformation. Apart from I-TASSER & GOR4, the SOPMA struc to stabilize a classic dimeric or trimeric coiled coil structure and are thus ture prediction server predicts the existence of β-turns with. ~5% stabilized by forming tetrameric coiled coil structures which have a content which has a peak assigned at 1677 cm 1, also somewhat wider hydrophobic interface [40]. Thus, a pro-alanine sequence of In covering the random coil region. Overall conformational analysis per dian paper wasp silk most likely can be stabilized by forming a tetra formed using the FT-IR results corresponded to predominant β-sheet meric coiled coil structure, as also predicted by protein modeling ~45% followed by equal percentage of α-helix and random coil. The studies. peak at 1515 cm 1 can be attributed to Amide II region corresponding to This I-TASSER based validated approach was then used for predic β-sheet conformation [44]. β-sheets are known to crosslink the α-helical tion of secondary structure model of Indian paper wasp silk protein core in order to impart stability to the protein structure in honeybee silk using the sequence predicted by MALDI-TOF/TOF analysis (Fig. 3b). [45]. It is important to mention here that FT-IR results indicates towards Among the fivepredicted structures obtained, the structure with highest a predominant β-sheets conformation alongwith the presence of α-helix, C-score of 3.16 is considered a closest model of the protein. The which is not surprising since similar observations of co-occurrence of modeled structures had 0.36 ± 0.12 expected TM-score with estimated both β-sheets (because of the presence of high serine content) and 13.7 ± 4.0 Å Root-mean-square deviation value. The deduced structure α-helices have been observed earlier in other insects of Vespoidea family, comprised of random coils in high quantity along few β-sheets and corroborating with our observation for Indian paper wasp silk [2,46]. α-helical conformations (Fig. 4b). However, it is important to mention Further, the conformational percentage of α-helix obtained by FT-IR also here that the complete protein sequence predicted by MASCOT (Fig. 3b, corresponds to the conformational predictions of the bioinformatics Supplementary Fig. 2) is only the probable sequence with the red servers (Table 3), corresponding to the occurrence of coiled coil like highlighted peptide sequences being the exact experimental ones. Thus, structures alongwith β-sheets in the silk produced by Indian paper wasp. out of the complete predicted structure (Fig. 4b) we tried to highlight Additionally, as observed from Table 3 there are noteworthy differences only the regions corresponding to experimental sequences (Fig. 4c). The in the random coil percentage between the FT-IR and bioinformatics regions of structure derived from MALDI-TOF/TOF experimental se servers. This may be due to the fact that dewaxed and degummed wasp quences corresponded mainly to predominant coiled coil. Thus, the In hive cap was used for FT-IR analysis, while regenerated solubilized silk dian paper wasp structure is in contrast with the structure of Vssilk1, in protein was used for developing the protein structure of the protein which the latter protein has tetrameric-coiled coil conformation predicted by the MALDI-TOF/TOF analysis. Thus, possibly the some conformational changes from predominant β-sheets to random coil Table 2 might have occurred during the processing of the silk used for protein Coiled-coil domain propensity of the wasp silk protein as predicted by the bioinformatics structure prediction leading to such variations of the MARCOIL server. conformational analysis (Table 3). Protein Threshold Predicted coiled-coil domains Probability With that intention we have performed a detailed analysis of the Vssilk 1 99.0 2 99.9% obtained FT-IR bands represented in Table 4. The band at around 900 Vssilk 2 99.0 1 99.9% cm 1, is attributed to the = C–H out-of-plane bending vibrations of Vssilk 5 99 0 99.9% alanine, which corroborates with the high concentration of this amino

6 S. Chawla et al. Polymer 208 (2020) 122967

Fig. 4. Secondary structure prediction using I-TASSER of a. Vssilk1, b. Indian paper wasp complete sequence predicted by MASCOT, c. Highlighted red region corresponding to experimentally identified sequences by MALDI-TOF/TOF revealing the predominantly coiled coil predicted structure. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

with white native wasp cap (Fig. 5a and b). The most interesting Table 3 observation in the brown remnants of the silk fibres was the highest Secondary conformations as predicted by different conformation prediction intensity peak at 3370 cm 1, which corresponded to the presence of softwares as compared with the experimental FT-IR data. tannin/intermolecularly bonded hydroxyl groups of polyphenols (Gallic Analysis method Conformation (%) acid-based tannins) engaged in tanning reaction. Thus, we can conclude β-sheet α-helix random coil β-turn that inner regions of Indian paper wasp silk fibres contain tanning I-TASSER 8.80 29.23 61.94 – compounds that are involved in the browning of silk core and provide SOPMA 8.80 38.38 48.24 4.58 the final level of crosslinking to the silk [50,51]. The amide I region GOR4 12.63 35.44 51.93 – (1600–1700 cm 1) of the brown wasp cap remnants was further FTIR 45.72 24.64 25.25 4.38 deconvoluted to analyse the secondary structure of the Indian wasp silk (Fig. 5d, Supplementary Fig. 4b). In the amide I region, the prominent 1 1 acid (33.4%) in the HPLC studies. This high percentage of alanine also peak shift from 1619 cm to 1629 cm reveals the transition from β results in the probable coiled coil structure of Indian wasp silk as shown intermolecular to intramolecular -sheets [52] which account for ~84% in the protein modelling studies [47]. of the total conformational composition as determined by peak inte 1 gration followed by peak deconvolution. Along with this, the deconvo The band at 1164 cm could be assigned to Serine, CH2 side chain β 1 wagging vibrations which was the second most prominent amino acid luted curves showed the presence of -turns (1680 cm ) and random 1 (20.4%) demonstrated in HPLC analysis. The band at 1235 cm 1 could coils (1646 cm ) with ~2% and ~14% share respectively. Hence the β be attributed to Aspartic acid and Glutamic acid C–O side chain inside undissolved portion consists more -sheets than outer part of the stretching vibrations again confirming the high presence (9.3%) in the wasp silk. HPLC analysis. Tyrosine (O–H) C–O and CC stretching vibrations also 1 overlap in the same region. Small band at 1390 cm corresponded to 3.6. X-ray diffraction analysis the Aspartatic acid CH3 side chain (in plain bending) while small band at 1 – 1454 cm could be attributed to Proline C N side chain (stretching) To confirmthe predicted molecular model of the protein, wide angle also overlapping with the aromatic amino acid Phenylalanine CH3 side 1 XRD was performed (Fig. 5e, Supplementary Fig. 4c). The D-spacing chain (in plain bending). The band at 1519 cm also corresponds to the pattern of the wasp silk demonstrated prominent peaks at 3.7 Å, 4.6 Å Tyrosine (O–H) CC ring (stretching) and CH (in plane bending) [48]. 1 1 and 4.9 Å. The peak at 4.9 Å suggested the presence of a distorted coiled Very small and low intensity bands at 2852 cm and 2929 cm could coil. The perfect D-spacing of stacked α-coiled coils is suggested by 5.1 Å be because of the hydrocarbon remnants in the silk protein which are spacing that originates from the arrangement of the axial periodicity present in the wasp hive silk caps and are removed by along the length of microfibril(47 nm) through the regular geometry of methanol-chloroform treatment [49]. Thus, the detailed FT-IR band coiled coil secondary structure [2,53]. This spacing indicates the α-helix analysis revealed interesting insights into the secondary conformations pitch along the axis of the coiled coil. The peak at 4.6 Å, confirms the of the wasp silk protein providing additional idea about the amino acid presence of α-helical coiled coil structure [7]. Presence of the second composition of the wasp silk protein. order equatorial peak is evidence of very regular packing and lateral size Additionally, we performed FT-IR analysis of the brown undissolved of the α-helices in the supercoil. The peak at 3.7 Å suggests the presence remnants of the Indian paper wasp silk fibresand compared the spectra of β-sheet structure which is in transition from the turns and α-helices

7 S. Chawla et al. Polymer 208 (2020) 122967

Fig. 5. a. FT-IR spectra of degummed and dewaxed Indian paper wasp silk cap (black line) and remnant brown wasp cap left after dissolution (red line) b. FT-IR spectra comparing the 1600-1700 cm 1 region of degummed and dewaxed Indian paper wasp silk cap (black line) and remnant brown wasp cap left after disso lution (red line), c. Deconvoluted FT-IR spectra of 1600–1700 cm 1 region of degummed and dewaxed Indian paper wasp silk cap to calculate the percentages of different secondary conformations, d. Deconvoluted FT-IR spectra of 1600–1700 cm 1 region of remnant brown wasp cap left after dissolution to calculate the percentages of different secondary conformations, e. XRD spectra of degummed and dewaxed wasp silk fibrous cap. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

spider silk that comprises of 25–35% α-helical secondary structure Table 4 alongwith the presence of 30% β-sheet content [4]. Major peaks identified in the wasp silk FT-IR analysis and their corresponding amino acid or secondary structure determination. 3.7. Birefringence studies S. No Wavenumber Assigned amino acid or secondary structure (cm 1) The paper wasp silk fibresinteracted with polarized light; indicating 900 Ala (=C–H out-of-plane) the presence of birefringent fibrous bars and tactoids, leading to het 1 1058 Pro (CH2) 2 1164 Ser (CH2) erogeneous crystalline structure throughout the fibres (Supplementary 3 1235 Amide III; Asp, Glu (C–O), Tyr (OH) Fig. 5). The wasp silk fibres showed second order birefringence colour 4 1298 Amide III – with the birefringence value of 0.040 0.046 indicating a lesser oriented 5 1390 Asp (COO ) crystalline structure parallel to the fibre axis [56]. Our findings corre 6 1454 Phe (CH3); Pro (C–N) 7 1519 Amide II; β-sheet conformation lates well with the existing concept of structure of silk produced by 8 1619 Amide I; Alanine (α-helical, coiled coil aculeates or non-aculeates Hymenopterans [57], that the coiled coil conformation) structures are roughly arranged with respect to neighbouring coiled 9 2852 alkane CH2 symmetric structures, but poorly aligned parallel to the main fibreaxis. At the same 10 2929 alkane CH symmetric 2 time, this finding supports our conclusion of SEM imaging, that the 11 3070 OH stretching weak band 12 3274 OH stretching strong band paper wasp pupa does not apply much extensive extensional flow and stretching during fibre spinning, which is seen during silk spinning by the silkworm or spider. [54]. Earlier studies have demonstrated the evidence of presence of α β -helical and -sheet structures together in giant hornet Vespa mandar 4. Conclusion inia japonica [7]. The study showed α-helical supercoil formed by β alanine rich sequences associated with -sheet structures formed by The present study highlights a hitherto unexplored fibrous protein, serine rich sequences, which are formed due to transformation of Indian paper wasp silk, for its biochemical and material properties. The random coils due to elongation forces during the process of spinning. high alanine and serine rich primary structure impart unique properties Thus, XRD results also validated the protein modeling studies indicating as compared to the silk produced by silkworm or spider, or aculeates or α β a coiled coil -helical alongwith the presence of -sheet structure of the non-aculeates Hymenopterans. Through protein modeling and physical Indian paper wasp silk thus validating the FT-IR results. Such presence characterization studies it was deduced that wasp silk has a coiled coil α β of -helical secondary structure alongwith the -sheet was reported in α-helical secondary structure along with the presence of β-sheets (in the Vespoidae family as well as some of the spider silks, like Argiope native condition) or predominantly random coils (in regenerated con ’ – α spider s prey-wrapping silks, that comprises of 40 50% -helical dition), which is also unique compared to hornet silk. Inner region of β alongwith the presence of 15% -sheet structure [55] and N. clavipes Indian paper wasp silk contains tannin/intermolecularly bonded

8 S. Chawla et al. Polymer 208 (2020) 122967 hydroxyl groups of polyphenols. We could also detect the trans [12] T. Kameda, T. Akino, K. Kojima, Wax components of larval cocoon silk of the glutaminase induced crosslinking between the glutamine and the lysine hornet Vespa analis Fabricius, Anal. Bioanal. Chem. (2007), https://doi.org/ 10.1007/s00216-007-1137-y. amino acids, which renders this silk partially soluble in chaotropic agent [13] S. Chawla, A. Kumar, P. Admane, A. Bandyopadhyay, S. Ghosh, Elucidating role of such as LiBr. Thus, the hierarchical arrangement of coiled coils and silk-gelatin bioink to recapitulate articular cartilage differentiation in 3D – β-sheets may open up new avenues to use the Indian paper wasp silk for bioprinted constructs, Bioprinting 7 (2017) 1 13, https://doi.org/10.1016/j. bprint.2017.05.001. next generation organic electronics, or advanced biomaterials. This [14] S. Murab, J. Samal, A. Shrivastava, A.R. Ray, A. Pandit, S. Ghosh, Glucosamine study lays the first step in the direction of identification of structural- loaded injectable silk-in-silk integrated system modulate mechanical properties in functional relationship of Indian paper wasp. Furthermore, we agree bovine ex-vivo degenerated intervertebral disc model, Biomaterials 55 (2015) 64–83, https://doi.org/10.1016/j.biomaterials.2015.03.032. that MALDI-TOF/TOF based analysis provides only a partial protein [15] M.P. Bartolomeo, F. Maisano, Validation of a reversed-phase HPLC method for sequence and future studies deducing the complete protein sequence quantitative amino acid analysis, J. Biomol. Tech. 17 (2) (2006) 131–137. identification using protein sequencing are warranted. [16] Y. Mengerink, D. Kutlan,´ F. Toth,´ A. Csampai,´ I. 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