Solution NMR Structure and Conformation of Silk Fibroins Stored in

Chapter 11

Downloaded by CORNELL UNIV on November 5, 2017 | http://pubs.acs.org Publication Date (Web): November 2, 2017 | doi: 10.1021/bk-2017-1258.ch011

Solution NMR Structure and Conformation of Silk Fibroins Stored in Bombyx mori and Samia cynthia ricini Silkworms Tetsuo Asakura,1,* Yu Suzuki,2 and Akio Nishimura1 1Department

of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo 184-8588, Japan 2Tenure-Track Program for Innovative Research, University of Fukui, 3-9-1 Bunkyo, Fukui, Fukui 910-8507, Japan *E-mail: [email protected]

The structure of the typical tandem repeated sequence, GSGSGA, of silk fibroin stored in the middle silk glands of Bombyx mori was determined with solution NMR at the atomic level. Type II β-turn structure was found, similar to that determined for the same sequence in Silk I* in the solid state as defined in the text. The structure assumes an approximately random coil conformation but exists in aggregated states. Likewise, the structure of another typical tandem repeated sequence, YGGDGG(A)12GGAG, of silk fibroin stored in the middle silk glands of a wild silkworm, Samia cynthia ricini, was also determined with solution NMR. The polyalanine region assumes the typical α-helix conformation, but at the N-terminal and C-terminal sites of the sequence there is additional intramolecular hydrogen bonding which helps to stabilize the helical structure of the polyalanine region.

Introduction Silks are produced mainly by moth larvae and spiders and are very attractive materials because of their excellent mechanical properties, especially their high strength and toughness (1). It is useful to develop a better understanding of the silk fiber formation mechanism as a means of improving the properties of man-made natural fiber (2). The silk fibroin from domesticated silkworm, Bombyx mori (B. © 2017 American Chemical Society Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


mori), is the most well-studied silk in the world, and much information on its structure before and after spinning (including fiber formation mechanism) has been acquired. However, recent developments in spectroscopic methods have supplied more detailed information on silk structure and sometimes even prompted revisions of some aspects of the structure which have been widely accepted for a long time. Nuclear magnetic resonance, NMR, is one of these spectroscopic methods that can be used to study polymer structure at the atomic level. For example, in our previous paper (3) and review (4), we have pointed out that the Marsh-Pauling model (5), which was proposed (and accepted) for the structure of B. mori silk fibroin fiber, should be revised. In particular, the inter-molecular chain arrangement in the silk structure should be changed from polar to anti-polar β-sheet structure. Here, the methyl groups of Ala residues are only on one side of the sheet in the polar structure (Marsh-Pauling model), but the methyl groups alternately point to both sides of the sheet along the hydrogen bonding direction in the antipolar structure (our model). Thus, whereas in the past the inter-molecular hydrogen bonding pair between two silk fibroin chains was regarded as having anti-parallel β-sheet structure (3, 6), we proposed a heterogeneous β-sheet structure instead (3, 4). As for the B. mori silk fibroin structure before spinning, we recently proposed new type II β-turn coordinates for the main repeated sequence GSGAGA in the polypeptide chain (7). A number of factors are known to affect the silk fiber formation in B. mori silkworm (8). These include changes in pH, ion concentrations, silk concentration, water content, shear stress, and stretching force. We mainly studied stretching force and shear stress because we believe these are the primary external forces for fiber formation. We based our assumptions on the known cocooning behavior of the silkworm (i.e., the larva spinning the silk fiber via head movement to depict the number eight (9)) and the significant change in the cross-sectional area and shapes of the press part in the anterior silk gland just before spinning (10). For example, previously the pH of the B. mori silk gland lumen had been reported to change from approximately 6.9 in the posterior silk gland to 4.9 in the anterior silk gland (11). This significant pH change from neutral to acidic was considered important factors in the fiber formation. However, recent pH determination in silk fibroin in silk gland using concentric ion selective microelectrodes by Domigan et al (12) reported different pH results. They observed a pH gradient from pH 8.2 to 7.2 in the posterior silk gland, a constant pH 7 throughout the middle silk gland, and a pH gradient from 6.8 to 6.2 in the beginning of the anterior silk gland where silk-to-fiber processing occurs. In this paper the concentrated aqueous solution of silk fibroin stored in the middle silk gland is called “liquid silk.” It is important therefore to have a better understanding of the structure of liquid silk, which is a good starting point to figure out the fiber formation mechanism. In vivo 13C solution NMR spectra of B. mori and a wild silkworm, Samia cynthia ricini (S.c. ricini) silk fibroins stored in living silkworms were first reported by us in 1983 together with the spectrum of pupa (Figure 1) (13, 14). The shape of these fifth-larval-stage silkworms and pupa is suitable for direct observation in a 13C NMR sample tube with a 10-mm outer diameter. High-resolution NMR spectra from the liquid silks (Figure 1a,c) could be 192 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


obtained and assigned easily by comparison with the pupa spectrum (Figure 1b). Because the structure of the liquid silk changes easily due to small external forces during the process of preparation from silk glands (as described later), structural determination of liquid silk without any external forces is essential.

Figure 1. 13C solution NMR spectra (expanded spectrafrom 0 ppm to 100 ppm) of the middle silk gland portions of both living S. c. ricini (a) and B. mori (c) mature larvae and of the abdomen of S. c. ricini pupa (b) together with the assignments. This figure was adapted from ref. (13), but with a minor change. Copyright 1983 American Chemical Society.

The conformation and dynamics of the regenerated silk fibroin together with liquid silk prepared from silk gland directly were studied as a function of their concentration in aqueous solution using 13C conformation-dependent chemical shifts (15). The 13C chemical shifts of all carbons of the Gly, Ala, Ser, Tyr, and Val residues (comprising 92.5% of all amino acids) did not change from dilute aqueous solution (at 2.1% concentration) to liquid silk (at a concentration of ca. 30%) and are almost the same as the 13C chemical shifts of random coil Ac-X-NHMe (X = 193 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


G, A, S, Y, V) in the aqueous solution. The 13C T1 spin-lattice relaxation times and NOEs were also observed for these aqueous solutions from 2.1% to 14.5% for regenerated silk fibroin and liquid silk. The mean correlation time of the backbone of the silk fibroin calculated from both 13C T1 and NOE values increased gradually from 0.10 ns (2.1%) to 0.22 ns (liquid silk) with increasing concentration. This suggests that some aggregation of the polypeptide chains occurs, causing the chain motion to decrease as the concentration increases. In addition, the plots of ln(M∞ − Mτ) vs τ for the liquid silk stored in living silkworms were essentially single exponentials for all carbons of the Ala, Gly, Ser, and Tyr residues. This observation suggests the presence of only one dynamic component in the liquid silk (16). (M∞ is the equilibrium amplitude of the fully relaxed spectrum and Mτ is the amplitude of a partially relaxed spectrum at delay time τ.) Thus, these data indicate that the local structure of liquid silk is close to that of a random coil, which undergoes fast segmental motion in aqueous solution even in the silk gland in spite of chain aggregation (16). In this paper, we provide a review of our recent research relating to the solution structures and conformations of the silk fibroins stored in the silk glands of B. mori and S.c. ricini. The solution structure of the S.c. ricini silk fibroin is particularly interesting because its primary structure is very different from that of B. mori. Whereas B. mori silk fibroin is an approximately alternating copolymer of Ala and Gly residues (17), S.c. ricini has both a tandem poly-Ala repeat motif and a Gly-rich motif (18). As a result, S.c. ricini takes on an α-helical structure in the aqueous solution (19–22). The tandem poly-Ala repeat motif and the Gly-rich motif are similar to the primary structure of spider drag-line silk (1, 8, 23, 24) although the length of poly-Ala here is longer than that of the spider silk.

Experimental The silk glands containing liquid silk were prepared from the fifth instar larvae of B. mori and S.c. ricini silkworms. The glands were soaked in a dish filled with distilled H2O so that the liquid silk could be gently removed from the silk glands. The liquid silk was then soaked in fresh distilled H2O to remove another silk protein, silk sericin, from the liquid silk surface in a mild manner. The liquid silk was carefully placed in a 5-mm NMR tube together with a sealed capillary containing D2O for NMR 2H lock while avoiding the application of any external forces to the liquid silk. There is no significant difference between two silk fibroins in these sample preparations for the solution NMR although the shapes of the silk gland are considerably different. A uniformly 13C-labeled liquid silk was also prepared by feeding U−13C D-glucose (99% enrichment, CIL, USA) in addition to an artificial diet fed to silkworm larvae from the fourth to sixth days of the fifth instar (4, 23). The liquid silks prepared for NMR measurements were stored at 4 °C prior to their use for the NMR experiments. All NMR experiments were carried out at 10 °C on a Bruker Avance 750 spectrometer equipped with a 5-mm inverse triple-resonance probe head with three-axis gradient coils, operating at a 1H frequency of 750 MHz. The chemical 194 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


shifts were indirectly referenced to TSP used as an external reference for 1H and 13C and to liquid NH3 for 15N. Resonance assignments for the 1H signals were initially accomplished by 2D TOCSY and 2D NOESY experiments and confirmed by hetero-nuclear NMR experiments. 1H, 13C, and 15N sequential resonance assignments were obtained by 2D double resonance and 3D double and triple resonance through bond-correlation experiments. Torsion angle constraints for the main chain were derived from database analysis of the chemical shifts (13Cα, 13Cβ, 13CO, 1Hα, 1HN, and 15N) of the backbone atoms using TALOS-N program (25, 26). Energy minimization using MOPAC was performed in order to obtain a final structural model.

Results Bombyx mori For the determination of the solution structure of silk fibroin at atomic level, we determined the torsion angles of the liquid silk using solution NMR (7). Torsion angle constraints for the main chain were derived from the backbone chemical shifts with the program TALOS-N. Moreover, inter-residue NOE cross-peaks, whose intensities are inversely proportional to the sixth power of inter-proton distances, were analyzed to obtain spatial inter-proton distance information.

Spectral Assignments for the Repeated Sequence Motifs Figure 2a shows the TOCSY spectrum used to assign the peaks of natural abundant B. mori liquid silk to the Gly, Ala, Ser, Tyr and Val residues. Figure 2b provides the sequential assignments in the HN/Hα region of the NOESY spectrum for the tandem partial sequence, GAGSGAG, with selected portions of the 1H15N HSQC and 1H-13C HSQC spectra of the natural-abundance liquid silk. The spectra show relatively sharp and fairly well-separated cross-peaks despite the very high molecular weight of the silk fibroin. This feature and the repetitive sequence made assignments of the 1H, 15N, and 13C peaks possible for the residues in the repetitive motifs by multidimensional NMR. Sequential assignments for each repetitive motif were also accomplished by analyzing the Hα/HN region in the NOESY spectrum. The 15N and 13C chemical shifts were assigned by 1H-15N and 1H-13C HSQC, and the consistency of the 1H chemical shift assignments was confirmed. The H-chain of the B. mori silk fibroin with 5263 residues consists of 12 tandem repeats in the primary structure (17). There are several repeated sequence motifs including 428, 144, and 31 copies of the hexapeptides GAGSGA, GAGYGA, and GAGVGA, respectively, and 41 copies of tetrapeptide GAAS. For convenience, a more detailed structural determination has been performed for the most abundant sequence, GAGSGA. 195 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. (a) Assignments for the Gly, Ala, Ser, Tyr, and Val peaks in the TOCSY spectrum and (b) sequential assignments in the HN/Hα region of the NOESY spectrum for GAGSGAG with portions of the 1H−15N HSQC and 1H−13C HSQC spectra of B. mori liquid silk. This figure is reproduced from ref. (7). Copyright 2014 American Chemical Society.

Torsion Angle (Φ, φ) Map Obtained Using TALOS-N The chemical shift values of Ala 13Cα and 13Cβ carbons indicated that these motifs have neither the typical α-helix nor the β-sheet structures (27). To evaluate the unique conformation of the repetitive motifs, we employed the backbone torsion angles prediction program, TALOS-N from NIH (25, 26). TALOS-N is a database system for empirical prediction of backbone torsion angles (Φ, φ) using a combination of six types of backbone chemical shifts and sequence information. Figure 3a shows the (Φ, φ) plots of the 25 closest database matches predicted for the GAGSGAG motif obtained using TALOS-N program. This result shows that the torsion angles (Φ, φ) of the residues (A2-G7) fall into the average torsion angles of the two center residues (residues i+1 and i+2) of the type II β-turn structure observed in the structure database, (Φi+1, φi+1) = (-61 ±13˚, 136 ± 11˚) and (Φi+2, φi+2) = (80 ±16˚, 5 ± 20˚) (25). Figure 3b shows the structural models constructed using the average torsion angles for the best matches (Φ, φ) 196 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


for the repeated GAGSGAG motif. The models for GAGSGAG show hydrogen bond formation between the HN of i-th and the CO of (i+3)th residues, which characterize the β-turn structure.

Figure 3. (a) the 25 best matches for torsion angles (Φ, ψ) for the GAGSGAG motifs obtained using the TALOS-N program; and (b) a structural model constructed using the averaged (Φ, ψ) in the circle for each motif. Hydrogen bonds are assumed to exist between the HN of the i-th and the CO of the (i+3)-th residues for the GAGSGAG motifs. This figure is reproduced from ref. (7). Copyright 2014 American Chemical Society. Thus, all the data are consistent with a strong preference for repeated type II β-turn structures for the typical tandem repeated sequences (GAGSGA)n of B. mori silk fibroin. This type II β-turn structure is essentially the same as Silk I* structure in the solid state (Figure 4) (4, 27–29), which is locally similar to the conformations preferred by random coil peptides in the aqueous solution (30) but is more ordered; in particular, it contains many hydrogen bonds that serve to hold it together (Figure 4) (27–29). Interestingly, molecular dynamics (MD) simulations of model peptides in explicit water indicate that the torsion angles of the Gly, Ala, and Ser residues in a repeated AGSGAG sequence with type II β-turn structure in water are close to the values expected at energy minima (30). Therefore, the amplitude and width of the structural fluctuations of Gly, Ala, and Ser residues may be considered relatively large for random coil conformation in the dilute aqueous solution, but the fluctuation decreases due to aggregation of the silk fibroin molecules with increasing concentration (16), consistent with an increased population of Silk I*. In other words, the highly concentrated silk 197 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


solution contained in the middle silk gland has residues in energetically favored conformations close to average random coil values but forms a hydrogen-bonded network that keeps it in a repeated type II β-turn structure.

Figure 4. Packing structure of poly(AG) chains with type II β-turn as a model for Silk I*form. Dotted lines denote intra- and intermolecular hydrogen bonds. The unit lattice values, a, b, and c, were obtained from X-ray diffraction data. This figure is reproduced from ref. (28). Copyright 2005 American Chemical Society.

Samia cynthia ricini The S.c. ricini silk fibroin consists of 93 tandem repeats of a polyalanine (poly-Ala) region flanked by Gly-rich regions. The length of the poly-Ala is distributed from 10 to 14, indicating a narrow distribution of length (18). The conformation of the liquid silk has been studied by 13C in vivo and solution NMR (13, 14, 19–22), and the poly-Ala region gave a single peak due to rapid conformational exchange between the α-helix and random coil. As the temperature was increased, the main Ala CO and Ala Cα peaks shifted to higher field; meanwhile, the main Ala Cβ peak shifted to lower field due to an increase in the random coil fraction (19–21). These were the same shift tendency observed for urea-induced helix-coil transition of S.c. ricini liquid silk (13). The proportion of 198 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


helix component obtained from the NMR spectrum was quantitatively consistent with that obtained from circular dichroism (CD) (19). A typical amino acid sequence from the Gly-rich region is YGGDGG, which appears 36 times at the N-terminal side, and GGAG appears 65 times at the C-terminal side of the poly-Ala region (17). Thus, the sequence Y1GGDGG6(A)12G19GAG22 can be used as a representative repeat sequence for S.c. ricini silk fibroin. Assignments of 1H, 15N, and 13C resonances for the residues in the repetitive motif were obtained by the same strategy as that applied to B. mori. The sequential assignment in the HN/Hα and HN/HN regions of the NOESY spectrum for the N terminal part, YGGDGGA, was shown in Figure 5.

Figure 5. Sequential assignments in the HN/Ha and HN/HN regions of NOESY spectrum for the N-terminal part, Y1GGDGGA7 of S. c. ricini liquid silk. This figure is reproduced from ref. (31). Copyright 2015 American Chemical Society.

Torsion Angles Derived from the Chemical Shifts The torsion angles for each residue were predicted by TALOS-N as shown in Figure 6. The Φ and φ angles of the poly-Ala region were − 62 ± 2° and −30 ± 11°, respectively (31). These values were similar to those of a typical α-helix, 199 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


where (Φ, φ) = (−60°, −45°). This result confirms that the poly-Ala region of S.c. ricini silk fibroin formed an α-helix in liquid silk. The torsion angles of N and C-terminal residues did not indicate typical secondary structure formation. A model structure was built and energy minimized based on the torsion angles obtained from MOPAC, as shown in Figure 7a (31). The poly-Ala region formed an α-helical structure. In an α-helix in general, the first four NH groups and last four CO groups necessarily lack intra-helical hydrogen bonds and instead are often capped by alternative hydrogen bond partners. At the N-terminal region of the model structure, hydrogen bonds were formed between CO of Gly6 and NH of Ala9 and between the CO of Gly6 and NH of Ala10. These findings demonstrate that Gly6 serves as the N-cap-like motif for the poly-Ala α-helix. At the C-terminal region of the model structure, hydrogen bonds are formed between the CO of Ala16 and NH of Ala21 and between the CO of Ala17 and NH of Gly20, as shown in Figure 7b. These hydrogen bond combinations are characteristic of the Schellman C-cap motif (32). Specifically, the Schellman C-cap motif is defined as a six-residue fragment that is located at the end of an α-helix and exhibits a double hydrogen bond pattern, namely NH of (i + 5) and CO of (i), NH of (i + 4) and CO of (i + 1). The six-residue fragment from Ala16 to Ala21 in the S.c. ricini model structure contains the same combination of hydrogen bonds as the Schellman C-cap motif. The N- and C-cap motifs stabilize the poly-Ala α-helix. Indeed, the capping motifs may play a role in preventing the structural transition from α-helix to β-sheet and fibril formation inside the silkworm body, which would be fatal to the silkworm.

Figure 6. A plot of torsion angles for each residue of the S.c. ricini repeated motif in liquid silk derived from TALOS-N. ♦ and □ denote f and y, respectively. Error bars show the estimated standard deviation provided by TALOS-N. This figure is reproduced from ref. (31). Copyright 2015 American Chemical Society.

200 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 7. (a) An energy minimized model structure of YGGDGG(A)12GGAG built from estimates of the backbone torsion angles using the program TALOS-N. (b) The C-terminal part of the structure forms intra-molecular hydrogen bonds between Ala16 CO and Ala21 HN, Ala17 CO and Gly20 HN, which are characteristic of the Schellman C-cap motif. Details are described in ref. (31). Copyright 2015 American Chemical Society.

Discussion As shown in Figure 1c, the chemical shifts of the well-resolved sharp peaks from Ala Cα, Cβ, and CO carbons of B. mori liquid silk are quite different from the corresponding main Ala peaks of liquid silk stored in the silk gland of S.c. ricini wild silkworm. The latter peaks clearly come from its poly-Ala region; in addition, there is also a small Ala peak coming from isolated Ala residues which is evident in the Ala CO region. The poly-Ala stretches in S.c. ricini are α-helical, whereas the isolated Ala residues are random coil. The chemical shift of the main Ala peak of this latter liquid silk is a good chemical shift reference for α-helix, and the small peak a good chemical shift reference for random coil conformation (15, 19, 20). Thus, the spectral comparison provides clear evidence against the presence of α-helix in the liquid silk from B. mori silkworm. Actually, the X-ray diffraction data of liquid silk dried without using any external force are also inconsistent with the presence of α-helix (33, 34). However, infrared (IR) spectroscopy appeared to demonstrate the presence of α-helix in B. mori liquid silk using automatic analysis carried out by commercial software such as Opus 6.5 software (Bruker Optics Corp., Billerica, MA) (35). Raman spectral analysis of liquid silk reached a similar conclusion (36). The CD spectrum of 201 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


liquid silk from the middle silk gland supported the random coil conformation, but a pattern similar to α-helix was observed at more than about 10% level (37, 38). From the concentration dependence of the Moffitt-Yang parameter b0 in the ORD measurement, Kobayashi et al. (39) reported that the helical content in diluted liquid silk is negligible up to a concentration of 0.1%, but at higher concentration, the helical content increases rapidly. This means that with increasing concentration, the generation of helical structure appears at about 10% concentration, and the percentage increases with increasing silk fibroin concentration. Thus, the assignments of the IR and Raman spectra of the silk fibroin should be done very carefully. Theoretical approach using the co-ordinates of (AGSGAG)n with Silk I* form reported in this work should help provide more exact assignments in the spectroscopic analyses of B. mori silk fibroin stored in the silk gland. As mentioned above, it is difficult to determine the content of Silk I* from the solution NMR. Therefore, this information was determined by solid state NMR for Ser and Ala residues of B. mori liquid silk in the solid state after drying the sample (Figure 8) (40). The determination of the fraction of Ser residues contributing to Silk I* conformation is especially important with respect to the discussion on fiber formation because 68% of all Ser residues are present as the sequence (AGSGAG)n in whole silk fibroin (17). In addition, in the Silk I* model (Figures 3 and 4), the Ser OH group contributes to the stabilization of the Silk I* conformation through intra-molecular hydrogen bond formation (9, 30). The selective 13C labeling of Ser Cβ carbons of B. mori silk fibroin makes it possible to analyze the local conformation of Ser residues in detail as shown in Figure 8a (40, 41). The sharp peak at 60.2 ppm is assigned to Silk I* (broken line) and its content can be determined to be 27%. The percentage of other conformation was 59% for distorted β-turn/random coil and 14% for β-sheet. Because there is no β-sheet in B. mori silk fibroin stored in the silk gland as mentioned above (13, 14), we propose that the observed β-sheet comes from structural transition of Silk I* due to small external forces in the preparation stage to take out the silk fibroin gel from the middle silk gland or at the drying stage to prepare the solid sample. Conversion of Silk I* to β-sheet requires very little external force, implying that the 14% β-sheet observed here is very likely to be Silk I* in the silk gland. Thus, the fraction of Silk I* is 41% (27 + 14) in the absence of external forces. Even this larger estimate is considerably lower than the fraction (68%) of Ser residues in the sequence (AGSGAG)n calculated from the primary structure (17). Thus, many Ser residues in the sequence (AGSGAG)n cannot contribute to Silk I*, implying that most probably only longer (AGSGAG)n sequences contribute to Silk I*. The peak deconvolution of solid state NMR spectrum of Ala residue in B. mori liquid silk after sample drying is also shown in Figure 8b (40). In our previous papers (3, 4, 40, 42, 43), the Ala Cβ peak was deconvoluted by assuming the presence of five peaks. Details of the assignment were described in the published papers. The black solid line in Figure 8b is random coil peak from the non-crystalline domain (45% of whole silk fibroin). For the crystalline domain (55% of whole silk fibroin) deconvoluted as three peaks, the broken line is Silk I* peak, and other two peaks (the grey solid lines) are r: random coil and β: β-sheet structure, respectively. The appearance of Silk I* was also discussed (40). 202 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 8. The deconvolutions of Ser Cβ (a) and Ala Cβ (b) peaks in the 13C CP/MAS NMR spectra of B. mori silk fibroin before spinning in the solid state. The solid lines are the observed and deconvoluted peaks. The Ser Cb peak can be deconvoluted to three peaks (the broken line, Silk I*; the grey solid lines, r: random coil and b: b-sheet structure, respectively). For the Ala Cb peak, the black solid line is the random coil peak from the non-crystalline domain (45% of whole silk fibroin). For the crystalline domain (55% of whole silk fibroin), the broken line is Silk I* peak, and the other two peaks (the grey solid lines) are r: random coil and b: b-sheet structure, respectively. Details of the assignments and deconvolution are described in ref. (40). Copyright 2015 American Chemical Society. Furthermore, we determined the solution structure of S.c. ricini silk fibroin before spinning in native liquid silk (31). The assignments of the 13C, 15N, and 1H solution NMR spectra for the repetitive sequence motif, YGGDGG(A)12GGAG, were achieved, and the corresponding chemical shifts obtained (31). We used the program TALOS-N to predict the backbone torsion angles from the chemical shifts for this motif. The torsion angles obtained indicate: (i) an α-helical structure for the poly Ala region, (ii) an N-cap residue for Gly6 associated by a type I β-turn 203 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


from Gly3 to Gly6 in the N-terminal region, and (iii) a Schellman C-cap motif for the C-terminal region. Amide proton temperature coefficients also confirmed the proposed structure. The amide proton coefficients are more positive than that of a random coil for poly-Ala residues, Gly6, Gly19, and Gly20. NHs from these Gly residues form hydrogen bonds characterized N- and C-cap motifs in the proposed structure. In addition, we studied the fiber formation mechanism of S.c. ricini silk fibroin (44). The three-dimensional architecture of the spinneret of S.c. ricini was reconstructed, and the structural change in the silk fibroin that occurs exclusively at the silk press part was elucidated by the molecular dynamics simulation (44, 45). Readers interested in the experimental details may consult the appropriate references.

Conclusion In this work the structures and the conformations of silk fibroins stored in two silkworms were determined by solution NMR. The torsion angles in the polypeptides were estimated from NMR data (7). Torsion angle constraints for the main chain were derived from the backbone chemical shifts (13Cα, 13Cβ, 13CO, 1Hα, 1HN, and 15N) using the program TALOS-N. Moreover, inter-residue NOE cross-peaks, whose intensities are inversely proportional to the sixth power of inter proton distances, were examined to obtain spatial inter-proton distance information. The sequence Y1GGDGG6(A)12G19GAG22 can be used as a representative repeat sequence of S.c. ricini silk fibroin. Resonance assignments of 1H, 15N, and 13C resonances for the residues in the repetitive motif were obtained using the same strategy as that applied to B. mori. The precise S.c. ricini silk fibroin structure of the repeat motif in liquid silk reported in the present work will hopefully contribute to a better understanding of the mechanism of fibroin processing.

Acknowledgments T.A. acknowledges support by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Supports of Japan (25620169, 26248050) and Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT). We also thank Dr. H. N. Cheng (Southern Regional Research Center, USDA Agricultural Research Service, New Orleans, LA 70124, USA) for discussions.

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