Emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers

1 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 www.nature.com/scientificreports emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers José pérez-Rigueiro 1,2,3, Rodrigo Madurga 1,2, Alfonso M. Gañán-Calvo 4, Manuel elices1,2, Gustavo V. Guinea1,2,3, Yugo tasei5, Akio Nishimura5, Hironori Matsuda5 & tetsuo Asakura5 the conditions required for the emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers are assessed through an experimental approach that combines the spinning of regenerated fibers with controlled properties and their characterization by 13C solid-state nuclear magnetic resonance (NMR). Both supercontracting and non-supercontracting regenerated fibers are produced using the straining flow spinning (SFS) technique from 13C labeled cocoons. The shortrange microstructure of the fibers is assessed through 13C CP/MAS in air and 13C DD/MAS in water, and the main microstructural features are identified and quantified. The mechanical properties of the regenerated fibers and their microstructures are compared with those of natural silkworm silk. The combined analysis highlights two possible key elements as responsible for the emergence of supercontraction: (1) the existence of an upper and a lower limit of the amorphous phase compatible with supercontraction, and (2) the existence of two ordered phases, β-sheet A and B, which correspond to different packing arrangements of the protein chains. Silks1 are remarkable materials when considered in terms of their processing route, microstructure and properties. Silk fibers are produced by some groups of arthropods, most conspicuously spiders and moths (Lepidoptera), for such critical biological functions as protecting the offspring or building the web2. In spite of having appeared as the result of two independent evolutionary events3, silks from spiders and worms share an extensive number of features in terms of sequence4,5, spinning mechanism6,7, mechanical properties8,9 and biocompatibility10. Probably the most salient feature in which silks from spiders and silkworms differ is the existence of the supercontraction phenomenon in the former. Supercontraction is a characteristic feature first identified in the major ampullate gland (MA) silk of Orbicularian spiders. Initially, the term supercontraction was used to refer to the significant reduction of its length when the fiber is immersed in water with at least one of the ends unrestrained11. It was later realized that the shrinkage of the fiber is simply the most evident effect of a much more profound property of the material: the existence of a ground state to which the fiber can revert independently from its loading history by immersion in water12,13. The existence of such a ground state implies that the fiber can be stretched following an arbitrary sequence of loads, but it will recover the tensile behaviour of this ground state by being simply allowed to contract in water. Later studies showed that spider silks different from MA, such as flagelliform silk, also exhibited a ground state14 and that supercontraction extended outside the Orbicularian group15,16. In contrast to this wide distribution of the supercontraction phenomenon among spider silk fibers, silkworm (Bombyx mori) silk presents a negligible shrinkage when immersed in water which, in addition, is not related to the existence of a ground state in the material17,18. Consequently, the possibility of producing regenerated silkworm silk fibers with the composition of silkworm silk, but endowed with the property of supercontraction19 was considered as a nice opportunity to understand the principles that underlie the emergence of this phenomenon. 1Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223, Pozuelo de Alarcón, (Madrid), Spain. 2Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, 28040, Madrid, Spain. 3Biomedical Research networking center in Bioengineering, Biomaterials and nanomedicine (CIBER-BBN), Madrid, Spain. 4Escuela Técnica Superior de Ingenieros, Universidad de Sevilla, 41092, Sevilla, Spain. 5Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo, 184-8588, Japan. Correspondence and requests for materials should be addressed to J.P.-R. (email: jose.perez@ctb. upm.es) or T.A. (email: [email protected]) Received: 30 July 2018 Accepted: 17 December 2018 Published: xx xx xxxx opeN www.nature.com/scientificreports/ 2 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 Prior to the production of supercontracting regenerated silkworm silk fibers, the basic strategy for analysing the origin of this phenomenon relied on the comparison of the sequences and microstructures of natural fibers, either endowed or not with this property20–22. Silks such as those produced by the major and minor ampullate glands of the spiders and that produced by silkworms (B. mori) were included in the analysis, but no clear correlation could be established to relate the microstructures of the different fibers and the emergence of supercontraction23–25. In particular, solid-state nuclear magnetic resonance (NMR) was a preferred technique for the microstructural characterization of silk fibers, since it allows unveiling some singular microstructural features in the short-range organization of the fibers26–32. Following these ideas, this work exploits the versatility of the straining flow spinning (SFS) technique33,34 to (1) tune the appearance of supercontraction in regenerated silk fibers by modifying the processing parameters, and (2) produce the relatively large amount of material required for solid-state NMR analysis. Thus, supercontracting and non-supercontracting regenerated silkworm silk fibers were produced by simply applying (or not) a post-spinning drawing step in water to regenerated SFS fibers. The mechanical properties of the fibers were characterized both in air and immersed in water in the as spun and supercontracted states, and their short range organization was explored with the unique insight offered by 13C solid-state NMR32. Both the mechanical properties and NMR characterization were compared with the data obtained from the natural fibers23. As shown below, this combined experimental approach allows identifying the microstructural features that may underlie the emergence of supercontraction. The identification of these features might have far reaching consequences, since it would offer the possibility of incorporating this property to a whole range of new artificial fibers and lead to the development of novel applications. Results Tensile behaviour of natural (N) and SFS regenerated (R-) silkworm silk fibers. In spite of sharing a similar composition, natural (N) and regenerated (R-) silkworm (Bombyx mori) silk fibers may exhibit very different tensile properties as illustrated in Fig.1. The true stress-true strain curves of N, R-NoPS and R-3.6PS regenerated silk fibers tested in air are compared in Fig.1a. It is apparent that N fibers outperforms their artificial counterparts in terms of elastic modulus and tensile strength as found in a number of previous studies17,18. It was found, however, that regenerated silk fibers may reach higher values of strain at breaking under adequate processing conditions19. These higher values of strain at breaking, in turn, lead to values of work to fracture in some regenerated fibers19,33 that are comparable to those of the natural material. Differences between the tensile behaviour of natural and regenerated silks are even more apparent when the fibers are tested in water. Figure1b compares the true stress-true strain curves of N, R-NoPS and R-3.6PS fibers tested in water. As illustrated in Fig.1b, immersion in water of N fibers induces a moderate reduction in the elastic modulus and tensile strength, while the strain at breaking is not affected. The effect of water on N fibers is explained by the collapse of the hydrogen bonds established between the fibroins in the dry fiber. In the wet fiber the protein-protein hydrogen bonds initially present in the dry fiber are substituted by water-protein hydrogen bonds, which do not contribute to the tensile behaviour of the fiber. In this regard, the mechanical behaviour of the wet fiber is controlled by the stiff β-nanocrystals and by the protein chains that interact among them and with the β-nanocrystals through van der Waals forces35. In comparison, water exerts more extreme changes in regenerated silkworm silks, although the details depend on the concrete processing parameters23. In this case, both R-NoPS and R-3.6PS samples tested in water show a significant reduction in the elastic modulus (over two orders of magnitude for R-NoPS samples), and an increase in the strain at breaking. Besides, the regenerated fibers tested in water show tensile properties characteristic of an elastomer as occurs with MA spider silk tested in water36. The effect of water on the regenerated silk fibers was explained as a result of the collapse of the hydrogen bond network found in the dry fiber, so that protein chains with an elastomeric behaviour control the mechanical behaviour of the wet material36–38. In this regard, the true stress-true strain curves of the regenerated fibers tested in water are more similar to those of major ampullate spider silk (MAS)11 than to the natural silkworm silk from which the regenerated fibers are produced. The similarity between spider silk and regenerated silkworm silk fibers suggests exploring the possible existence of supercontraction in the latter. Figure1c compares the true stress-true strain curves of the fibers after being immersed in water, allowed to contract and dried. No contraction is measured in the R-NoPS samples and their tensile properties concur with those of the as spun fibers. Natural silk shows a small contraction (~1–2%)17, but immersion in water and subsequent drying modifies the true stress-true strain curve. This modification concentrates around the yield point and results from changes in the hydrogen bond network, mainly in the amorphous regions of the material, and subsequent conformational changes of the fibroin proteins9. As observed in Fig.1a,b, the most evident change in the tensile properties of N fibers upon immersion in water and subsequent drying is a significant reduction in the yield stress of the material. In contrast to both N and R-NoPS samples, R-3.6PS regenerated silk fibers show a contraction of 17% from the original length. In addition, a significant increase in the strain at breaking is observed after the R-3.6PS samples are allowed to contract, resulting in a work to fracture of Wf = 34 MJ/m3 for these samples. As indicated above, the contraction of a silk fiber in water is just an initial hint of the supercontraction ability of the material. In order to substantiate this property, it is necessary to prove the existence of a true ground state by using recovery tests39. Four recovery tests were performed on R-3.6PS-SC samples and a representative recovery test is shown in Fig.2a. In an initial step, the fiber is stretched in air up to a strain of 0.18. Then, the fiber is unloaded and allowed to contract in water (this step is indicated in the Figure by the light blue rectangle) and, finally, tensile tested until breaking. The concurrence of the true stress-true strain curves of both tensile tests observed in Fig.2a is considered as the definitive evidence of a ground state in R-3.6PS samples. From Fig.2a it is also apparent that the properties of the as-spun R-3.6PS sample are recovered upon stretching the R-3.6PS-SC sample in air. www.nature.com/scientificreports/ 3 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 In spite of the differences in the true stress-true strain curves of the R-3.6PS and R-NoPS samples when tested in air, R-3.6PS are obtained from R-NoPS samples upon adding a post-spinning drawing step during processing. Thus, the similarity of the mechanical behaviour of R-3.6PS and R-NoPS samples when tested in water hints to the existence of some common underlying microstructural features between both types of fibers. The existence of supercontraction in R-3.6PS fibers, which reflects the significant structural flexibility of the material, supports the possibility of finding a procedure to interconvert the microstructure and properties of both types of regenerated silks. In this regard, it was found that the tensile properties of regenerated silk fibers prepared from an N-methyl morpholine oxide (NMMO) solution can be modified through a wet stretching process17, in parallel with the behaviour of MA spider silk40. Following this rationale, R-NoPS samples were subjected to wet stretching and allowed to contract. The true stress-true strain curve of a wet-stretched R-NoPS (R-NoPS + WS) sample is compared with that of a R-3.6PS-SC sample in Fig.2b. The concurrence of both true stress-true strain curves indicates that R-NoPS fibers are converted into R-3.6PS-SC fibers through a wet-stretching process. The comparison of the distinct behaviour of the N, R-NoPS, R-3.6PS and R-3.6PS-SC silk fibers offers an intriguing scenario. Firstly, both natural and regenerated fibers share essentially a common composition, so that the observed differences must be related with their different processing routes and microstructures. Secondly, in spite of their diverging mechanical properties, R-NoPS, R-3.6PS and R-3.6PS-SC samples share a common composition and processing conditions, except for the absence of a post-spinning drawing step in water during the spinning of the R-NoPS samples. In addition, it is also found that the behaviour of the R-NoPS samples can be 0 80 160 240 320 400 480 560 640 00.1 0.2 True stress (MPa) True strain aAs spun - Air N R-3.6PS R-NoPS 0 80 160 240 320 400 480 560 640 00.2 0.40.6 0.811.21.4 True stress (MPa) True strain bAs spun - Water N R-3.6PSR-NoPS 0 80 160 240 320 400 480 560 640 00.1 0.20.3 0.4 True stress (MPa) True strain c Supercontracted AirN R-3.6PS-SC R-NoPS Figure 1. True stress-true strain curves of N (red), R-NoPS (black) and R-3.6PS (blue) fibers. (a) As spun fibers tested in air, (b) as spun fibers tested in water, and (c) fibers immersed in water, allowed to contract, dried and tested in air. A representative curve for each material and testing condition is presented, on which the average values of tensile strength and strain at breaking of at least three tests are indicated by a square symbol. Error bars correspond to the standard errors of the tensile strength and strain at breaking for each material and testing condition. Data of the N fibers in (a,c) are reproduced from the reference9. Data of the N in (b) are reproduced from the reference35. www.nature.com/scientificreports/ 4 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 modified to concur with that of the R-3.6PS-SC fibers through a simple wet-stretching process. Finally, R-3.6PS and R-3.6PS-SC fibers can be interconverted into one another by stretching in air and maximum supercontraction, respectively. In this context, the connections among these natural and regenerated fibers offers a unique opportunity to explore the relationships between microstructure and properties in silk fibers, and NMR arises as a powerful microstructural characterization technique especially adequate for this task. In particular, usage of 13C labelled samples allows (1) isolating the structural information obtained from different amino acids and, consequently, distinguishing between crystalline and amorphous regions, (2) obtaining microstructural information on the effects related with the interactions between silk proteins and water molecules, and (3) obtaining information on the dynamics of the different microstructural features. 13C CP/MAS NMR spectra of natural (N) and regenerated (R-) SF fibers in the dry state. Figure3 shows representative 13C CP/MAS NMR spectra of [3-13C] Ser-, [3-13C] Tyr- and [3-13C] Ala-labeled (a) R-NoPS, (b) R-3.6PS, (c) R-3.6PS-SC and (d) N fibers, in the dry state together with the assignment of the main peaks. The selective 13C labeling of the Tyr Cβ and Ser Cβ carbons makes possible to study the local conformation of these residues in detail41,42. The assignment of Tyr Cβ, Ser Cβ and Ala Cβ peaks is shown in Fig.3(a), although the naturally abundant Gly Cα peak overlaps at the lower field with the Tyr Cβ peak. Structural and dynamical information on the crystalline domain can be obtained from the Ser peak and that of the non-crystalline domain from the Tyr peak independently. Figure4 shows the expanded Cβ peaks of Ser, Tyr and Ala residues in 13C CP/MAS NMR spectra of the R-NoPS, R-3.6PS, R-3.6PS-SC and N fibers, together with the deconvolution of the peaks. 0 50 100 150 200 250 00.050.1 0.15 0.20.250.3 True stress (MPa) True strain riAa R-3.6PS-SC 0 50 100 150 200 250 00.050.1 0.15 0.20.2 50.3 True stress (MPa) True strain R-3.6PS-SC R-NoPS + WS b Air Figure 2. (a) A representative recovery test of a R-3.6PS-SC regenerated silk fiber. The maximum supercontraction step between the first and the second tensile tests is indicated by the light blue rectangle. (b) Comparison of the true stress-true strain of a R-3.6PS regenerated silk fiber after maximum supercontraction (R-3.6PS-SC) and an R-NoPS regenerated silk fiber subjected to wet stretching. Figure 3. 13C CP/MAS NMR spectra of (a) R-NoPS, (b) R-3.6PS, (c) R-3.6PS-SC and (d) N fibers in the dry state. www.nature.com/scientificreports/ 5 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 Two main contributions can be identified in all the spectra, which correspond to β-sheet and random coil (r.c.) conformation. β-sheet, in turn, comprises two kinds of packing arrangements, which are labelled as β-sheet A and B. The interpretation of the latter β-sheet A and B is given in Fig.S1 (Supporting Information) and corresponds to differences in the packing arrangement of the β-sheet secondary structure. The deconvolution of the spectra to determine the fraction of the various conformations for each residue followed the method presented in previous papers23,42. In particular, the chemical shifts of the 13C NMR spectra were used to identify each elementary contribution28–32,41. The fractions of the different conformations obtained from the deconvolution are shown in Fig.5, and the values are summarized in TableS1 (Supporting Information). The fraction of β-sheet increases in the order of R-NoPS, R-3.6PS, R-3.6PS-SC and N fiber for Ser and Ala residues. The Tyr residue also shows a similar tendency, but changes are comparatively smaller. 13C DD/MAS NMR spectra of natural (N) and regenerated (R-) SF fibers in the hydrated state. Hydration leads to a more heterogenous structure that reflects the different effect of the presence of water molecules on the individual domains of the silk fibers. In order to analyze the spectra of the hydrated samples quantitatively, 13C DD/MAS NMR was used instead of 13C CP/MAS NMR, although there is no significant difference between 13C DD/MAS NMR and 13C CP/MAS NMR spectra of B. mori silk fibroin fiber in the dry state42. Figure6 shows 13C DD/MAS NMR spectra of [3-13C] Ser-, [3-13C] Tyr- and [3-13C] Ala-labeled (a) R-NoPS, (b) R-3.6PS, (c) R-3.6PS-SC and (d) N fibers in the hydrated state. A remarkable difference between the spectra of the dry and hydrated SF samples is the appearance of sharp peaks in the spectra of the latter. These peaks can be assigned to the hydrated random coil peaks23,42, and result from the increased mobility of some protein regions upon interaction with water molecules. The appearance of the sharp peaks was observed for all residues characterized in Fig.6. Detailed analyses were performed from the deconvoluted spectra as shown in Fig.7. Ser Tyr Ala (b) (c) (d) (a )R- NoPS Dry R-3.6PS Dry R-3.6PS-SC Dry N Dry Figure 4. The expanded Cβ peaks in 13C CP/MAS spectra of Ser, Tyr and Ala residues of (a) R-NoPS, (b) R-3.6PS, (c) R-3.6PS-SC and (d) N fiber samples in dry state together with the deconvolution of the peaks. www.nature.com/scientificreports/ 6 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 Ser Tyr Ala (c ) (b) (a ) (d) R- NoPS Dry R-3.6PS Dry R-3.6PS-SC Dry N Dry Figure 5. Pie charts of the fractions corresponding to the different conformations of Ser, Tyr and Ala Cβ peaks in the 13C CP/MAS NMR spectra of (a) R-NoPS, (b) R-3.6PS, (c) R-3.6PS-SC and (d) N fiber samples in the dry state obtained from the deconvolution of the NMR peaks. The color code is: yellowrandom coil; redβ-sheet or β-sheet A; pinkβ-sheet B. Figure 6. 13C DD/MAS NMR spectra of (a) R-NoPS, (b) R-3.6PS, (c) R-3.6PS-SC and (d) N samples in the hydrated state. www.nature.com/scientificreports/ 7 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 The fractions of the various conformations obtained from the deconvolution are shown in Fig.8, and their values are summarized in TableS2 (Supporting Information). Relatively small differences are found between the fractions of the four components in the regenerated silk fibers. In contrast, significant differences are observed when the regenerated fibers are compared with N samples. In particular, N fibers show a smaller proportion of hydrated random coil, and a higher proportion of β-sheet B. Discussion As indicated previously, the characterization of the system formed by R-NoPS, R-3.6PS, R-3.6PS-SC and N fibers offers a privileged opportunity for finding correlations between the singular properties of these materials and some of their microstructural features. This opportunity is especially clear with regard to the emergence of supercontraction in silk fibers, due to the possibility of spinning both supercontracting and non-supercontracting regenerated fibers with the composition of (non-supercontracting) natural silkworm silk by simply switching on and off a post-spinning drawing step. Ser Tyr Ala (c) (b) (a) (d) R-NoPS Hydrated R-3.6PS Hydrated R-3.6PS-S C Hydrated N Hydrated Figure 7. The expanded Cβ peaks of Ser, Tyr and Ala residues of (a) R-NoPS, (b) R-3.6PS, (c) R-3.6PS-SC and (d) N fiber samples in the hydrated state together with the deconvolution of the peaks. In addition to the contributions previously identified from the 13C CP/MAS NMR spectra, a new hydrated random coil contribution is identified in the wet samples using 13C DD/MAS NMR. www.nature.com/scientificreports/ 8 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 Application of NMR to the study of this set of fibers allows exploring the relationship between their short-range microstructural features and the existence (or not) of supercontraction23,31,32. It must be acknowledged that some long-range microstructural features, such as the size or orientation of the β-nanocrystals, might also play a role in the appearance of supercontraction. Long-range microstructural details are outside the scope of NMR, but previous works on the analysis of these feature did not allow reaching any firm conclusion on its influence on the origin of this phenomenon19,43. The study of the NMR spectra of N fibers represents a convenient starting point for the analysis of the whole set of natural and regenerated fibers, since a number of previous works23,32,44–47 have established the main microstructural features of this material. In addition, such a study takes advantage from the possibility of characterizing the fibers both dry and wet. Silkworm silk presents a semicrystalline microstructure in which β-nanocrystals are embedded in an amorphous matrix. In broad terms it is assumed that β-nanocrystals impart silkworm silk its structural integrity and stiffness, while amorphous regions are responsible for its remarkable strain at breaking. The singular sequence of silkworm silk4 is critical for the appearance of the crystals from regions with β-sheet secondary structure, since they are formed from the characteristic motif of sequence –GAGAGS–. The relationship between this motif of sequence and the crystals implies that Ser residues appear mostly in the crystalline phase, Tyr residues in the amorphous phase, and Ala residues can appear in both phases. Immersion in water of silkworm silk fibers leads to a very small contraction, which is not associated with the existence of a ground state (i.e. N fibers does not show proper supercontraction). It was found that the crystallinity of silkworm silk fibers increases when first immersed in water9, but subsequent wetting/drying cycles do not modify either the structure further or lead to an increase in the number of the crystals48. Ser Tyr Ala (c) (b) (a) (d) R-NoPS Hydrated R-3.6PS Hydrated R-3.6PS-SC Hydrated N Hydrated Figure 8. Pie charts of the fractions corresponding to the different conformations of Ser, Tyr and Ala Cβ peaks in the 13C DD/MAS NMR spectra of (a) R-NoPS, (b) R-3.6PS, (c) R-3.6PS-SC and (d) N fiber samples in the hydrated state obtained from the deconvolution of the NMR peaks. The color code is: yellow-random coil; light bluehydrated random coil; redβ-sheet or β-sheet A; pinkβ-sheet B. www.nature.com/scientificreports/ 9 Scientific RepoRts | (2019) 9:2398 | https://doi.org/10.1038/s41598-019-38712-6 As expected from the large proportion of –GAGAGS– motifs in its sequence, silkworm silk shows one of the highest crystallinities (~60%) of all silks48. This high crystallinity is consistent with the β-sheet contributions to the Ala- and Ser-NMR peaks (Figs4 and 7), although the interpretation of these data should take into account that NMR provides information on the secondary structure of the amino acids, but does not distinguish between amino acids present in the amorphous or in the crystalline phase. It is also found that β-sheet A constitutes the major contribution to this secondary structure, which is consistent with its participation in the formation of β-nanocrystals. The absence of significant macroscopic changes in N upon immersion in water is best reflected by the constancy of the random coil and β-sheet contributions in the Tyr-NMR peaks. In contrast, a tendency towards increasing the random coil structure at the expense of the β-sheet structures is observed in the Ala- and Ser-peaks upon wetting. Significant differences are found when the NMR spectra of the R-NoPS samples are compared with those of N. In R-NoPS samples the random coil structure represents more than 50% of the total area of all peaks in the dry state. Upon wetting a significant fraction of the random coil structure is converted into hydrated random coil and into β-sheet. The comparison of the NMR data from the N and R-NoPS fibers suggests that a minimum proportion of random coil structure is required to elicit the elastomeric behaviour of the fibers when tested in water. Additionally, it might also be critical that a sufficient fraction of the random coil becomes hydrated when the material is immersed in water. The requirement of a minimum proportion of random coil to impart the fiber the ability to supercontract is consistent with previous results on regenerated fibers23, in which spinning proceeded from a solution of silk protein in hexafluoroisopropanol (HFIP) and methanol (MeOH) was used as coagulant. The 13C CP/MAS NMR spectra of [3-13C] Ser-, [3-13C] Tyr- and [3-13C] Ala-labeled of these regenerated fibers found a large proportion of β-sheet, comparable to that in N samples, even in the case of as spun (no-stretched) fibers. No supercontraction was found upon immersion of these fibers in water. The conversion of random coil into β-sheets in the wet fibers suggests that the driving force behind the observed microstructural changes in the R-NoPS fibers might be the tendency to increase crystallinity at the expense of the amorphous phase. A comparable tendency to increase the values of crystallinity upon wetting and drying is found in N and explains the effects that degumming exerts on silkworm silk9. When the previous discussion is extended to include the R-3.6PS and R-3.6PS-SC, some regularities are immediately found. In the dry fibers the proportion of β-sheet follows the order: N > R-3.6PS-SC > R-3.6PS > R-NoPS for all three NMR peaks (correspondingly, the proportion of random coil follows the order R-NoPS > R-3.6PS > R-3.6PS-SC > N). When the wet fibers are considered it is found that the proportion of hydrated random coil follows the order: R-NoPS ~ R-3.6PS ~ R-3.6PS-SC > N. Consistently with the previous hypothesis on the origin of the elastomeric behaviour in regenerated fibers upon immersion in water, R-3.6PS and R-3.6PS-SC fibers show a larger proportion of random coil structure compared with N fibers, and the fraction of hydrated random coil is comparable to that of the R-NoPS sample. At this point of the discussion, the emergence of supercontraction in the R-3.6PS samples is probably the most intriguing question of the system formed by the four sets of fibers. The comparison of the natural and regenerated fibers supports the hypothesis that high values of crystallinity may limit the conformational changes induced on the fiber by wetting. In turn, these restrained conformational changes would prevent the N fibers from supercontrating. Consequently, there seems to exist a maximum threshold to the crystallinity of the fibers that allows the appearance of supercontraction. In addition, and although tempting, the comparison of the R-3.6PS samples with the R-NoPS fibers immediately precludes establishing a simple relationship between the proportion of random coil structure and supercontraction. In this context, such a direct relationship would imply that R-NoPS samples should not only supercontract, but also that their percentages of supercontraction should be possibly higher than those of the R-3.6PS samples. Thus, there seems to exist a “sweet spot” in the microstructural organization that allows the emergence of supercontraction. The previous data suggest that the “sweet spot” would be defined by the existence of a lower and an upper limit to the proportion of the amorphous phase. The existence of a lower limit is compatible with the data obtained from the N material, while that of the upper limit is indicated by the results on R-NoPS fibers. Fortunately, the possibility of converting R-NoPS into R-3.6PS-SC fibers through wet-stretching and of interconverting R-3.6PS and R-3.6PS-SC fibers through supercontraction/stretching in air offers the possibility of getting a deeper insight on the conditions required by supercontraction. The NMR data of the dry fibers indicate that the transition from R-NoPS to R-3.6PS-SC samples implies the conversion of random coil structures to β-sheets both in the A and B forms. In addition, the comparison of the R-3.6PS-SC and R-3.6PS spectra indicates that the stretching of the R-3.6PS-SC leads to an increase in the proportion of random coil at the expense of the β-sheet structure. The higher proportion of β-sheet structure after supercontraction found in the R-3.6PS-SC compared to R-3.6PS samples is again compatible with the consideration of the increase in the crystallinity of the material as the major driving force that causes the observed microstructural changes. Another major difference is also found between the R-NoPS and the R-3.6PS-SC samples when the effect of water is considered. As shown in Figs5 and 8 and in TablesS1 and S2 (Supplementary Information) and, in contrast to the conversion of random coil to β-sheet observed from the dry to the wet states of R-NoPS fibers, R-3.6PS-SC only show the interconversion between the β-sheet A and B forms, but no net conversion between random coil and β-sheet structures. Following the previous discussion, the development of a comprehensive scheme that encompasses the behaviour and properties of both natural and regenerated fibers requires providing an explanation for the absence of supercontraction in R-NoPS samples. A possible hypothesis for this behaviour can be found in the principles of the elastomeric behaviour of polymer chains. The one-dimensional constitutive equation of an elastomeric material37,38 is: