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Protein Science (2003), 12:666-671.
Copyright © 2003 The Protein Society

ACCELERATED COMMUNICATION

Tightly winding structure of sequential model peptide for repeated helical region in Samia cynthia ricini silk fibroin studied with solid-state NMR

Yasumoto Nakazawa, Mie Bamba, Satoko Nishio and Tetsuo Asakura

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184–8588, Japan

Reprint requests to: Tetsuo Asakura, Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan; e-mail: asakura{at}cc.tuat.ac.jp; fax: 81-42-383-7733

(RECEIVED November 7, 2002; FINAL REVISION December 18, 2002; ACCEPTED December 18, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0239203.


   Abstract
TOP
 Abstract
Introduction
 Results and discussion
 Materials and methods
 References
 
There are many kinds of silks from silkworms and spiders with different structures and properties, and thus, silks are suitable to study the structure-property relationship of fibrous proteins. Silk fibroin from a wild silkworm, Samia cynthia ricini, mainly consists of the repeated similar sequences by about 100 times where there are alternative appearances of the polyalanine (Ala)12–13 region and the Gly-rich region. In this paper, a sequential model peptide, GGAGGGYGGDGG(A)12GGAGDGYGAG, which is a typical sequence of the silk fibroin, was synthesized, and the atomic-level conformations of Gly residues at the N- and C-terminal ends of the polyalanine region were determined as well as that of the central Ala residue using 13C 2D spin diffusion solid-state nuclear magnetic resonance (NMR) under off-magic angle spinning. In the model peptide with {alpha}-helical conformation, the torsion angle of the central Ala residue, the 19th Ala, was determined to be ({phi}, {psi}) = (-60°, -50°), which was a typical {alpha}-helical structure, but the torsion angles of two Gly residues, the 12th and 25th Gly residues, which are located at the N- and C-terminal ends of the polyalanine region, were determined to be ({phi},{psi}) = (-70°, -30°) and ({phi},{psi}) = (-70°, -20°), respectively. Thus, it was observed that the turns at both ends of polyalanine with {alpha}-helix conformation in the model peptide are tightly wound.

Keywords: Structure of Samia cynthia ricini silk fibroin; 2D spin diffusion NMR under off-magic angle spinning; {alpha}-helix of polyalanine region; determination of torsion angles


   Introduction
TOP
 Abstract
 Introduction
Results and discussion
 Materials and methods
 References
 
Silks are a wide class of fibrous proteins with properties that intrigue scientists ranging from structural engineers to polymer chemists, physicists, and biomedical researchers (Gosline et al. 1999). There are many kinds of silks from silkworms and spiders with different structures and properties, and thus, silks are suitable to study the structure-property relationship of fibrous proteins. Samia cynthia ricini is a wild silkworm and the amino-acid composition of the silk fibroin is different from that of the silk fibroin from the domesticated silkworm, Bombyx mori. The sum of Gly and Ala residues is 82%, which is similar to B. mori silk fibroin (73%), but the relative composition of Ala and Gly is reversed (Asakura and Murakami 1985; Zhou et al. 2000); the proportion of Gly residues is greater in B. mori silk fibroin, while the content of the Ala residues is greater in S. c. ricini silk fibroin. S. c. ricini silk fibroin mainly consists of about 100 times repeated similar sequences with alternative appearances of the (Ala)12–13 region and the Gly-rich region (Asakura et al. 1999a). Here, the polyalanine sequences such as (Ala)12–13 or (Ala)12 are defined as poly-Ala in this paper. This primary structure is similar to the structure of dragline (major ampullate) silk from spider but the length of poly-Ala is shorter: (Ala)5–6 (Xu and Lewis 1990; Hinman and Lewis 1992).

From 13C solution nuclear magnetic resonance (NMR) studies of S. c. ricini silk fibroin, it is evaluated that ~90% of the Ala residues form a poly-Ala region with {alpha}-helical structure, and fast exchange in the NMR time scale between helix and coil forms in the poly-Ala region has been observed with increasing temperature (Asakura and Murakami 1985; Asakura et al. 1988). On the other hand, the Gly carbonyl peaks, which were split into the primary structure, did not change during the transition, suggesting that the solution structure of the Gly-rich region is mainly in random-coil state. However, we recently found that the underscored Gly residue in the Gly-Gly-(Ala)12–13 sequence of S. c. ricini silk fibroin was incorporated into the helix structure (Nakazawa and Asakura 2002). A more detailed structure of both poly-Ala and Gly-rich regions of the silk fibroin before spinning is required to clarify the fiber formation mechanism.

In general, X-ray diffraction is one of the most practical methods to obtain the detailed structure for single-crystal samples, and this method has provided the molecular structures of numerous proteins in atomic level. However, it is necessary to prepare a single crystal of proper size for the purpose and thus X-ray diffraction is not suitable to determine the structure of amorphous and un-oriented samples in the solid state. On the other hand, the chemical shift interaction and dipolar interaction, which are mainly observed in solid state NMR for half-spin nuclei, have been used for the determination of solid-state structures. Especially, two dimensional (2D), spin-diffusion, solid-state NMR is a powerful method to obtain the relative orientation of two chemical shift tensors of 13C-labeled sites in the local molecular framework. When two carbonyl carbons of the neighboring residues in peptide were 13C labeled, the torsion angles, {phi} and {psi}, of the amino-acid residue could be determined. Actually, the 2D spin-diffusion NMR under off-magic angle spinning has been used to determine the torsion angles of Ala and Gly residues of the peptide, (Ala-Gly)15 as a model peptide of the crystalline domain of B. mori silk fibroin and a new structural model for B. mori silk fibroin before spinning in the solid state has been proposed (Asakura et al. 2001).

In this paper, solid state NMR, especially 13C, 2D, spin-diffusion, solid-state NMR under off-magic angle spinning, was used for the determination of the torsion angles, {phi} and {psi} of the Gly residues at the N- and C- terminal ends of the poly-Ala region along with that of the central Ala residue in the sequential model peptide, GGAGGGYGGDGG(A)12– GGAGDGYGAG, which is a typical sequence containing the poly-Ala region of the silk fibroin.


   Results and discussion
TOP
 Abstract
 Introduction
 Results and discussion
Materials and methods
 References
 
13C CP/MAS NMR spectra of S. c. ricini silk fibroin film and GGAGGGYGGDGG(A)12–GGAGDGYGAG
Figure 1Go shows 13C CP/MAS (cross polarization/magic angle spinning) NMR spectra of the model peptide of S. c. ricini silk fibroin, GGAGGGYGGDG(A)12GGAGDGYGAG after dialysis of the 9M LiBr solution against water (Fig. 1AGo) and after trifluoroacetic acid (TFA) treatment (Fig. 1BGo), together with the film of S. c. ricini silk fibroin prepared from the silk fibroin stored in the silkgland (Fig. 1CGo). Here "TFA treatment" means that the peptide was dissolved in TFA and then precipitated in diethylether. The chemical shifts of the spectrum (Fig. 1AGo), 20.2 ppm for Ala Cß, 48.7 ppm for Ala C{alpha}, and 171.9 ppm for Ala carbonyl carbons, clearly show that the conformation is ß-sheet structure (Asakura et al. 1999b). On the other hand, the model peptide after TFA treatment takes {alpha}-helical conformation judging from the chemical shifts of the Ala residues in the spectrum (Fig. 1BGo), 15.3 ppm for Ala Cß, 52.3 ppm for Ala C{alpha}, and 175.9 ppm for Ala carbonyl carbons. The latter chemical shift values are the same as those of S. c. ricini silk fibroin film (Fig. 1CGo). Thus, it is concluded that both conformations of the silk fibroin film and the sequential model peptide after TFA treatment are {alpha}-helix.



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  		Figure 1. 13C CP/MAS nuclear magnetic resonance spectra of the model peptide of S. c. ricini silk fibroin, GGAGGGYGGDG(A)12GGAGDGYGAG, after dialysis of the 9M LiBr solution against water (A) and after TFA treatment of the same peptide (B), together with S. c. ricini silk fibroin film (C).

 
Determination of the torsion angle of the 19th Ala residue in GGAGGGYGGDGG(A)5[1-13C] A18[1-13C]A19(A)5GGAGDGYGAG
In Figure 2Go, the 2D spin-diffusion NMR spectra (only the carbonyl region is expanded) of the model peptide, GGAGGGYGGDGG(A)5[1-13C]A18[1-13C]A19(A)5GGAGDGYGAG, after TFA treatment (Fig. 2AGo) and after dialysis of the 9M LiBr solution against water (Fig. 2BGo) are shown, together with the calculated spectra of the Ala residue with the torsion angles ({phi}, {psi}) = (-60°, -50°), (Fig. 2CGo), and ({phi}, {psi}) = (-150°, 150°) (Fig. 2DGo). For the calculation of the 2D spin-diffusion NMR spectra, it is necessary to obtain the principal values of the chemical shift tensors of the Ala carbonyl carbon atoms and therefore these values were determined using slow MAS experiments described elsewhere (Asakura et al. 2001).



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  		Figure 2. Two-dimensional spin-diffusion, 13C, solid-state nuclear magnetic resonance spectra (only the carbonyl region is expanded) of the model peptide, GGAGGGYGGDGG(A)5[1-13C]A18[1-13C]A19(A)5GGAGDGY GAG, after TFA treatment (A), and after dialysis of the 9-M LiBr solution against water, (B) are shown together with the calculated spectra of the Ala residue with the torsion angles ({phi}, {psi}) = (-60°, -50°) (C), and ({phi}, {psi}) = (-150°, 150°) (D).

 
The remarkable change was observed between the spectra (Fig. 2A,BGo); the spectrum (Fig. 2AGo) has strong off-diagonal intensity compared with the spectrum (Fig. 2BGo), which emphasizes the diagonal intensity. To examine the dependence of the spectral pattern on the torsion angle of the Ala residue, the spectra were calculated as a function of the torsion angles {phi} and {psi} of the Ala residue for each 30° in the region of -180°<{phi}<0° and -180°<{psi}<180° as shown in Figure 3Go. The calculated spectral patterns change remarkably depending on the {phi} and {psi} values, suggesting that the torsion angles can be determined with high precision. Through more detailed spectral calculations in typical {alpha}-helical or ß-sheet regions of a Ramachandran map, the torsion angles ({phi}, {psi}) of the Ala19 residue at the center of the poly-Ala region could be determined to be ({phi}, {psi}) = (-60°, -50°) and (-150°, 150°), respectively, as shown in Figure 2Go, C and D. The error in the angle determination was ±5° and ±10°, respectively. The torsion angles of the Ala19 residue in the model peptide with {alpha}-helical form determined here are essentially the same as the reported angles, ({phi}, {psi}) = (-60°, -45°) of the Ala residue for [1-13C]Ala-silk fibroin film from S. c. ricini using the 2D DOQSY NMR measurements (van Beek et al. 2000).



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  		Figure 3. Ramachandran map of the two-dimensional spin-diffusion nuclear magnetic resonance spectral patterns calculated as a function of torsion angles {phi} and {psi} of the Ala residue for each 30° in the region of -180°<{phi}<0° and -180°<{psi}<180°.

 
Determination of the torsion angles of the 12th Gly residue in GGAGGGYGGD[1-13C]G11[1-13C] G12(A)12GGAGDGYGAG and the 25th Gly residue in GGAGGGYGGDGG(A)11[113C]A24[1-13C]G25GAGDGYGAG.
Recently, the solution 13C NMR has been applied to analysis of the conformation of Gly residues, which are located closely at the poly-Ala region in [1-13C]Gly- S. c. ricini silk fibroin extracted from the silkgland (Nakazawa and Asakura 2002). It was observed that some of such Gly residues were incorporated into the {alpha}-helix conformation of the poly-Ala region. Therefore, 2D spin-diffusion NMR was also applied to determination of the torsion angles of these Gly residues of the peptide, GGAGGGYGGDGG(A)12G GAGDGYGAG as a model of the silk fibroin structure before spinning.

As shown in Figure 4Go, 2DGo spin diffusion NMR spectra were observed for two kinds of the model peptides, GGAGGGYGGD[1-13C]G11[1-13C]G12(A)12GGAGDGY GAG (Fig. 4AGo) and GGAGGGYGGDGG(A)11[1-13C]A24 [1-13C]G25GAGDGYGAG (Fig. 4BGo), respectively, after TFA treatment. The spectral patterns are slightly different from that of the 19th Ala residue in GGAGGGYGGD GG(A)5[1-13C]A18[1-13C]A19(A)5GGAGDGYGAG (Fig. 2AGo). To clarify the origin of the difference, the 2D spin-diffusion NMR spectra were calculated as a function of torsion angles {phi} and {psi} of the Gly residue for each 30° in the region of -180°<{phi}<0° and -180°<{psi}<180° (data not shown). However, the Ramachandran maps of the calculated spin-diffusion patterns were similar to Figure 3Go. Thus, the difference between Figures 2AGo and 4AGo or 4BGo is a result of the difference in the torsion angles rather than the difference between Gly and Ala residues. There are four candidates that satisfy the observed spectra, Figure 4, A and BGo; ({phi}, {psi}) = (-60°, -30°), (60°, 30°), (-120°, 90°) and (120°, -90°). It is necessary to select the torsion angles of the Gly residues by considering further structural information. In our previous papers (Asakura et al. 1999b; Ashida et al. 2002), the Gly C{alpha} chemical shift was used to select possible torsion angles of Gly residues. By using the observed Gly C{alpha} chemical sift value, 44 ppm, only the torsion angle, ({phi}, {psi}) = (-60°, -30°) was selected among the four candidates. Then, in order to determine the torsion angle more precisely, the root-mean-squared deviations (RMSD), {chi}2, between the observed and calculated 2D spin-diffusion NMR spectra, were calculated as a function of the torsion angles, {phi} and {psi} of the Gly residue in the {alpha}-helical region such as ({phi}, {psi}) = (-50° ~-90°, -10° ~-50°) (data not shown) (Ashida et al. 2002). Finally, the torsion angles of the Gly12 and Gly25 residues were determined to be ({phi}, {psi}) = (-70°, -30°) and ({phi}, {psi}) = (-70°, -20°), respectively, as shown in Figure 4Go. The error in the angle determination was ±10°. Thus, the torsion angles of the Gly residues at both N- and C-terminals of the poly-Ala region were slightly different from the torsion angle of the Ala residue at the center of the poly-Ala region in the model system before spinning. It has been observed that the final turn at the ends of the {alpha}-helix is tightly wound because of the appearance of the conformation such as 310 helix ; ({phi}, {psi}) = (-49°, -26°) (Creighton 1993). The {psi} values of the Gly residues, -30° or -20°, determined here are the same as that of 310 helix, but the {phi} value, -70° seems to be significantly different from -49° of 310 helix. Thus, it is likely that the N- and C-terminal ends of Poly-Ala with typical {alpha}-helix conformation in S. c. ricini silk fibroin have tightly wound conformation.



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  		Figure 4. Two-dimensional spin diffusion nuclear magnetic resonance spectra of two kinds of the model peptides: (A) GGAGGGYGGD [1-13C]G11[1-13C]G12(A)12GGAGDGYGAG and (B)GGAGGGYGG- DGG(A)11[1-13C]A24[1-13C]G25GAGDGYGAG. The calculated spectra of the Gly residues with the torsion angles (C) ({phi}, {psi}) = (-70°, -30°) and (D)({phi}, {psi}) = (-70°, -20°), respectively, are also shown.

 
After dialysis of the 9M LiBr solution of the model peptide against water, the peptide was precipitated. The 2D spin-diffusion NMR spectra of the peptides, GGAGGGY GG-D[1-13C]G11[1-13C]G12(A)12GGAGDGYGAG and GG AGGGYGGDGG(A)11[1-13C]A24[1-13C]G25GAGDGYGAG show typical patterns of ß-sheet structure as shown in Figure 2BGo. Thus, the Gly residues at both N- and C-terminals of the poly-Ala region were incorporated into ß-sheet structure of the poly-Ala region. In our previous paper (Asakura et al. 1999a), the structure of 13C- and 15N-labeled S. c. ricini silk fibers after spinning was determined with 13C and 15N CP NMR for the blocks of the oriented silk fibers by changing the angles of the oriented silk fiber axis and the magnetic field. 65% of Gly residues in the silk fiber was in the ß-sheet structure. Thus, the incorporation of the Gly residues at the terminal ends into ß-sheet structure of the poly-Ala region that was observed for the model peptide is in agreement with the previous structural study on S. c. ricini silk fiber.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and discussion
 Materials and methods
References
 
The model peptides of S. c. ricini silk fibroin containing the poly-Ala region were synthesized by the solid phase method.

GGAGGGYGGDGG(A)12GGAGDGYGAG, GGAGGGYGGD GG(A)5[1-13C]A18[1-13C]A19(A)5GGAGDGYGAG, GGAGGG YGGD[1-13C]G11[1-13C]G12(A)12GGAGDGYGAG GGAGGG YGGDGG(A)11[1-13C]A24[1-13C]G25GAGDGYGAG.

After synthesis, "TFA treatment" was tried in order to transform the structure of the peptide into the structure of the silk fibroin before spinning. Namely, the model peptides were dissolved in TFA and then precipitated in diethylether. The peptides were dried under vacuum at room temperature. The 2D spin-diffusion NMR spectra were obtained with Varian Unity INOVA 400 NMR spectrometer and 7-mm{phi}, Jakobsen-type, double-tuned MAS probe at off-magic angle condition ({theta}m-7°) and sample spinning of 6 kHz at room temperature. Therefore, the scaling factor of the 2D spin-diffusion spectra is 1/2 (3 cos2 [{theta}m-7°]-1) = 0.198. Other NMR experimental conditions are the same as those in our previous paper (Asakura et al. 2001).


   Acknowledgments
 
T.A. acknowledges support from the Program for Promotion of Basic Research Activities for Innovative Biosciences, Japan.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Asakura, T., and Murakami, T. 1985. NMR of silk fibroin. 4. Temperature- and urea-induced helix-coil transition of the -(Ala)n- sequence in philosamia cynthia ricini silk fibroin protein monitored by 13C NMR spectroscopy. Macromolecules 18: 2614–2619.[CrossRef]

Asakura, T., Kashiba, H., and Yoshimizu, H. 1988. NMR of silk fibroin. 8. 13C NMR analysis of the conformation and the conformational transition of Philosamia cynthia ricini silk fibroin protein on the basis of Bixon-Scheraga-Lifson theory. Macromolecules 21: 644–648.[CrossRef]

Asakura, T., Ito, T., Okudaira, M., and Kameda, T. 1999a. Structure of alanine and glycine residues of Samia cynthia ricini silk fibers studied with solid-state 15N and 13C NMR. Macromolecules 32: 4940–4946.[CrossRef]

Asakura, T., Iwadate, M., Demura, M., and Williamson, M.P. 1999b. Structural analysis of silk with 13C NMR chemical shift contour plots. Int. J. Biol. Macromol. 24: 167–171.[CrossRef][Medline]

Asakura, T., Ashida, J., Yamane, T., Kameda, T., Nakazawa, Y., Ohgo, K., and Komatsu, K. 2001. A repeated ß-turn structure in poly(Ala-Gly) as a model for silk I of Bombyx mori silk fibroin studied with two-dimensional spin-diffusion NMR under off magic angle spinning and rotational echo double resonance. J. Mol. Biol. 306: 291–305.[CrossRef][Medline]

Ashida, J., Ohgo, K., Komatsu, K., Kubota, A., and Asakura, T. 2002. Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)10 using solid-state NMR methods. J. Biomol. NMR (in press).

Creighton, T.E., 1993. Proteins. Structures and molecular properties. 2nd ed., p. 187–188. W.H. Freeman and Company, NY.

Gosline, J.M., Guerette, P.A., Ortlepp, C.S., and Savage, K.N. 1999. The mechanical design of spider silks: From fibroin sequence to mechanical function. J. Exp. Biol. 202: 3295–3303.[Abstract]

Hinman, B.M. and Lewis, R.V. 1992. Isolation of a clone encoding a second dragline silk fibroin. J. Biol. Chem. 267: 19320–19324.[Abstract/Free Full Text]

Nakazawa, Y. and Asakura, T. 2002. Heterogeneous exchange behavior of Samia cynthia ricini silk fibroin during helix-coil transition studied with 13C NMR. FEBS Lett. 529: 188–192.[CrossRef][Medline]

van Beek, J.D., Beaulieu, L., Schafer, H., Demura, M., Asakura, T., and Meier, B.H. 2000. Solid-state NMR determination of the secondary structure of Samia cynthia ricini silk. Nature 405: 1077–1079.[CrossRef][Medline]

Xu, M., and Lewis, R.V. 1990. Structure of a protein superfiber: Spider dragline silk. Proc. Natl. Acad. Sci. 87: 7120–7124.[Abstract/Free Full Text]

Zhou, C., Confalonieri, F., Medina, N., Zivanovic, Y., Esnault, C., Yang, T., Jacquet, M., Janin, J., Duguet, M., Perasso, R., et al. 2000. Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res. 28: 2413–2419.[Abstract/Free Full Text]


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