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Protein Science (2005), 14:2654-2657. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Evidence from 13C solid-state NMR spectroscopy for a lamella structure in an alanine–glycine copolypeptide: A model for the crystalline domain of Bombyx mori silk fiber

Tetsuo Asakura, Yasumoto Nakazawa, Erika Ohnishi and Fumika Moro

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 June 3, 2005; FINAL REVISION July 23, 2005; ACCEPTED July 25, 2005)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
13C high-resolution solid-state NMR coupled with selective 13C isotope-labeling of different Ala one methyl carbons was used to clarify the structure of (AG)15 peptide in the silk II structure as a model for the crystalline domain of Bombyx mori silk fiber. At the inner part of the peptide, the fraction of the peak at 16.6 ppm of the Ala C{beta} resonance assigned to {beta}-turn structure increased at 11th and 19th positions. These data indicate the appearance of the most probable lamellar structure having a turn structure at these two positions, although the position of turn was distributed along the chain.

Keywords: Bombyx mori silk; solid-state NMR; lamellar structure; (Ala-Gly)15; stable isotope labeling peptide

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051525505.


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
Bombyx mori silk fibroin can assume two distinct structures in the solid state: silk I before spinning; and silk II after spinning. The corresponding structures have been investigated by X-ray fiber diffraction (Marsh et al. 1955; Fraser et al. 1966; Fraser and MacRae 1973; Asakura et al. 1985; Okuyama et al. 1988; Takahashi et al. 1999), electron diffraction (Lotz et al. 1974; Okuyama et al. 1988), conformational energy calculations (Lotz and Cesari 1979; Fossey et al. 1991), infrared spectroscopy (Fraser and MacRae 1973; Magoshi et al. 1979; Asakura et al. 1985), and 13C and 15N cross-polarization magic angle spinning (CP/MAS) NMR (Saito et al. 1984; Asakura et al. 1985; Nicholson et al. 1993; Demura et al. 1998; Asakura et al. 2001b). Despite a long history of studying silk I, its structure determination has proven difficult because any attempt to induce a macroscopic orientation of the sample for X-ray diffraction, electron diffraction, or solid-state NMR readily causes a conversion of the silk I conformation to the silk II form (Saito et al. 1984; Asakura et al. 1985; Ishida et al. 1990; Asakura et al. 1997, 1999). Recently, we have resolved the molecular conformation of silk I as a "repeated {beta}-turn type II," using solid-state NMR methods such as 2D spin-diffusion NMR under off magic angle spinning, Rotational Echo Double Resonance (REDOR), and 13C chemical shift data (Asakura et al. 2001a).

Concerning the structure of silk II, Marsh et al. (1955) were the first to propose an anti-parallel {beta}-sheet model based on a fiber diffraction study of native B. mori silk fiber. Recently, Takahashi et al. (1999) reported a more detailed X-ray fiber diffraction analysis of B. mori silk fibroin based on 35 quantified intensities. After considering previously proposed models for the silk II form in terms of the experimentally derived R-factor, Takakashi et al. (1999) proposed that two anti-polar anti-parallel {beta}-sheet structures are statistically stacked with different orientations, occupying the crystal site with a ratio of 1:2. Even though the local protein conformation is still the basic {beta}-sheet as proposed by Marsh et al., the refined silk II model accounts for the stacking of the {beta}-sheet planes in two different arrangements. In the structural analysis of B. mori silk fibroin, poly (alanyl-glycine) (poly [AG]), or alanine-glycine copolypeptides (AG)n has been used for spectroscopic studies as the model system.

In our previous papers (Asakura and Yao 2002; Asakura et al. 2002), we found that the 13C resonances of the Ala C{beta} positions in the CP/MAS NMR spectra of both B. mori silk fibroin fiber in the silk II form and (AG)15 are broad and asymmetric, which reflects a heterogeneous structure. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. For example, the deconvolution of (AG)15 shown in Figure 1Go is 27% distorted {beta}-turn (16.7 ppm), 46% {beta}-sheet (alternating Ala residues; 19.1 ppm), and 27% {beta}-sheet (parallel Ala residues; 22.4 ppm). Panitch et al. (1997) produced (AG)64 from recombinant Escherichia coli, and presented SANS and WAXS evidence for the chain-folded lamella structural model of (AG)64 in the silk II form. The structure consists of polar anti-parallel {beta}-sheets with repetitive folding through {gamma}-turns from every eighth amino acid (including the fold), stacking with like surfaces together. Indirect evidence for a folded anti-parallel {beta}-sheet model with a lamellar structure has also been recently reported for a drying hydrogel of regenerated silk fibroin that slowly developed a silk II structure (Valluzzi and Jin 2004). In our previous paper (Asakura and Yao 2002), the Ala methyl carbon in the 13C CP/MAS NMR spectrum of the short peptide, G(AG)3 with silk II form showed no broad peak at 16.7 ppm and only two peaks at a lower field corresponding to the {beta}-sheet peak region. However, the 16.7-ppm peak was newly observed for a longer peptide, (AG)n (n = 9 and 12), as well as the two peaks at a lower field observed for G(AG)3. The fraction of the peak intensity of 16.7 ppm at the Ala methyl region was slightly smaller for (AG)9 compared with (AG)12 and (AG)15, and the fraction was almost the same in the latter two peptides. These data may indicate that G(AG)3 is too short to form such a structure. In addition, (AG)15 is enough to consider the local structure because the pattern is the same in the case of (AG)25. It has been shown that high-resolution solid-state NMR study coupled with selective stable isotope labeling of the peptide samples can provide a great deal of information on the detailed structures of silk fibroins in the solid state.



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  		Figure 1. Expanded Ala C{beta} peaks in the 13C CP/MAS NMR spectrum of natural abundance (AG)15 peptide.

 
In this paper, using 13C high-resolution solid-state NMR coupled with selective 13C isotope labeling of the Ala methyl groups, we show that (AG)15 in the silk II structure has the folded lamella structure.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
Figure 2AGo shows 13C CP/MAS NMR spectra of the expanded Ala methyl region of (AG)15 peptides with the 13C isotope labeling of different Ala one methyl carbons, which were synthesized with a solid phase method. The spectra presented in Figure 2AGo are difference spectra obtained by subtracting natural abundance spectra from the 13C-labeled peptide. This subtraction provides local structural information for each specific 13C-labeled Ala residue. The asymmetric peaks were observed for every Ala methyl carbon, as reported previously for natural abundance (AG)15 in the silk II form. The peak at 22.4 ppm has been assigned to the Ala methyl groups aligned in parallel, while the peak at 19.1 ppm corresponds to those pointing in opposite directions. The 16.7 ppm was assigned to the distorted {beta}-turn, as mentioned above. Figure 2AGo shows clearly that the peak patterns change largely depending on the labeled position of the Ala residue, indicating a heterogeneous local structure along the peptide chain. To discuss the heterogeneous local structure quantitatively, a 2D spin-diffusion NMR spectrum of (AG)8A[1-13C]G18[1-13C]A19G(AG)5 was observed under off the magic angle condition as shown in Figure 3Go (left) (Asakura et al. 2002). The simulation was performed by assuming exclusively {beta}-sheet structure ({phi},{psi} = –150°, 150°) for the Ala19 residue (Fig. 3Go, center). This could not reproduce the observed one, but the spectrum was reproduced by assuming 66% {beta}-sheet and 34% distorted {beta}-turn very well (Fig. 3Go, right). Here, the peak broadening at 16.7 ppm reflects the distribution of the {phi},{psi} angles because of the appearance of many conformations at the turn site. Therefore, the fraction of each conformation was evaluated from the Ala C{beta} chemical shift reported previously, and the peak pattern calculated by assuming each conformation was superimposed in the simulation (Fig. 3Go, right). Here, we discuss the behavior of the peak at 16.7 ppm, which reflects the turn position of the folded lamella structure. The distorted {beta}-turn structure component of the peak at 16.7 ppm evaluated by the peak decomposition was plotted against the labeled position in Figure 2BGo. The fraction at residue 1 was 70%, indicating that the turn conformation of the N-terminal residue cannot form the expected {beta}-sheet structure. The fraction decreased markedly at residue 3 and decreased gradually toward the inner part of the chain. This composition change decreases to form the {beta}-sheet structure.



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  		Figure 2. (A)Expanded Ala C{beta} peaks in the 13C CP/MAS NMR spectra of 15 [3-13C](AG)15 peptides with different [3-13C]Ala labeling sites. These silk II peptides were dissolved in formic acid and then dried in air. The gray areas are assigned to the distorted {beta}-turn structure. (B) The relative intensity of the 16.7-ppm peak was calculated from these spectra by changing the 13C Ala labeling site in (AG)15.

 


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  		Figure 3. 2D spin-diffusion 13C solid-state NMR spectrum (carbonyl region) of (AG)8A[1-13C]G18[1–13C]A19G(AG)5 (left). The calculated spectra are shown by assuming the Ala15 residue with exclusively {beta}-sheet structure ({phi},{psi}) = (–150°, 150°) (center), and by assuming the Ala15 residue with 77% {beta}-sheet and 23% distorted {beta}-turn structures (right).

 
The fraction of the peak at 16.7 ppm increases suddenly at the 9th position and at the 11th position, indicating the appearance of the folded lamella structure with a {beta}-turn at these positions. After the 11th position, the fraction decreased. Moving from the 11th to the 17th position, the fraction of the peak at 16.7 ppm was constant before reaching a new maximum at the 19th position. Thereafter, the fraction increased from a low at the 25th position to a final high at the 29th position (C terminus). Thus, the most probable lamellar structure is shown in Figure 4Go. As in poly(AG) reported by Panitch et al. (1997), eight amino acid residues contribute to the {beta}-sheet structure from residues 11 to 19. However, these authors proposed {gamma}-turns as the conformation for the turn position, but {beta}-turns seem more likely as the repeated {beta}-turn type II structure is found in (AG)15 before formic acid treatment. Taking into account the increase in the variations of structure at the N-terminal and C-terminal ends, we suggest that two or three Ala residues in each terminus are not incorporated into the {beta}-sheet structure of AG sequences.



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  		Figure 4. The most probable lamella structure of (AG)15 with silk II structure by assuming the presence of two turn parts at the 11th and 19th positions exclusively.

 

   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
Preparation of the stable isotope-labeled model peptides
The natural abundance and 13C-labeled model peptides, (AG)15, were synthesized by the solid-phase method (Asakura et al. 2001a). After synthesis, the samples were dissolved in formic acid and dried, the structure was {beta}-sheet by judging from the 13C CP/MAS NMR chemical shifts of the Ala residue.

13C CP/MAS NMR
The 13C CP/MAS NMR experiments were performed on a Chemagnetics Infinity 400 MHz spectrometer with a 13C operating frequency of 100.04 MHz. Samples were spun at a rate of 10 kHz. The number of acquisitions was 8000, and the recycle delays were 5 sec. A 50-kHz radiofrequency field strength was used for 1H-13C decoupling the acquisition period of 12.8 msec. A 90° pulse width of 5 µsec with 1 msec CP contact time was employed. Phase cycling was used to minimize artifacts. 13C chemical shifts were calibrated indirectly through the adamantane methylenes peak observed at 28.8 ppm relative to TMS at 0 ppm.

2D spin diffusion 13C solid-state NMR under off magic angle condition
The 2D spin-diffusion NMR spectra were obtained using a Varian Unity INOVA 400 NMR spectrometer with a 7-mm Jakobsen-type double-tuned MAS probe at off magic angle condition ({theta}m – 7°) at room temperature. The sample spinning rate was 6 kHz (±3 Hz). The scaling factor of the 2D spin-diffusion spectra is 1/2 (3cos2 ({theta}m – 7°) –1) = 0.198. The mixing times were set to be 2 sec. They were optimized for spin diffusion between intramolecular-specific carbon atoms of selectively isotope-labeled Ala and Gly residues or both, but with no spin diffusion among carbon atoms in difference molecules. The contact time was set to 2 msec using the variable-amplitude CP technique (Peersen et al. 1993). About 400 scans with a recycle delay of 2 sec were accumulated for every T1 value in the 2D experiment. The recycle delay of 2 sec was carefully determined from the 1D OMAS spectra under several recycle times. The principal values of the chemical shift tensors for the carbonyl carbon nuclei of the 13C-labeled Ala and Gly residues were determined by analysis of the spinning sidebands under slow MAS conditions (Herzfeld and Berger 1980; Asakura et al. 2001a) using a Chemagnetics Infinity 400 spectrometer.


   Acknowledgments
 
T.A. acknowledges Dr. D. Knight (Oxford University), Prof. E. Atkins (Univ. of Massachusetts), and Prof. T. Gullion (West Virginia Univ.) for stimulating discussions. Dr. Jun Ashida (Varian Technologies Japan) is acknowledged for the useful suggestions and solid-state NMR measurements. T.A. also acknowledges support from the Program for Promotion of Basic Research Activities for Innovative Biosciences, Japan.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 References
 
Asakura, T. and Yao, J. 2002. 13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by streching. Protein Sci. 11: 2706–2713.[Abstract/Free Full Text]

Asakura, T., Kuzuhara, A., Tabeta, R., and Saito, H. 1985. Conformation characterization of Bombyx mori silk fibroin in the solid state by high-frequency 13C cross polarization-magic angle spinning NMR, X-ray diffraction, and infrared spectroscopy. Macromolecules 18: 1841–1845.[CrossRef]

Asakura, T., Minami, M., Shimada, R., Demura, M., Osanai, M., Fujito, T., Imanari, M., and Ulrich, S., A. 1997. 2H-labeling of silk fibroin fibers and their structural characterization by solid-state 2H NMR. Macromolecules 30: 2429–2435.[CrossRef]

Asakura, T., Iwadate, M., Demura, M., and Williamson, M.P. 1999. 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. 2001a. A repeated {beta}-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]

Asakura, T., Yamane, T., Nakazawa, Y., Kameda, T., and Ando, K. 2001b. Structure of Bombyx mori silk fibroin before spinning in solid state studied with wide angle X-ray scattering and 13C cross-polarization/magic angle spinning NMR. Biopolymers 58: 521–525.[Medline]

Asakura, T., Yao, J., Yamane, T., Umemura, K., and Ulrich, A.S. 2002. Heterogeneous structure of silk fibers from Bombyx mori resolved by 13C solid-state NMR spectroscopy. J. Am. Chem. Soc. 124: 8794–8795.[CrossRef][Medline]

Demura, M., Minami, M., Asakura, T., and Cross, T.A. 1998. Structure of Bombyxmori silk fibroinbasedonsolidstateNMRorientational constraints and fiber diffraction unit cell parameters. J.Am.Chem.Soc. 120: 1300–1308.[CrossRef]

Fossey, A.S., Nemethy, G., Gibson, D.K, and Scheraga, A.H. 1991. Conformational energy studies of {beta}-sheets of model silk fibroin peptides. I. Sheets of poly(Ala-Gly) chains. Biopolymers 31: 1529–1541.[CrossRef][Medline]

Fraser, R.D.B. and MacRae, T.P. 1973. Conformations of fibrous proteins and related synthetic polypeptides. Academic Press, New York.

Fraser, R.D., MacRae, T.P., and Stewart, F.H. 1966. Poly-l-alanylglycyl-l-alanylglycyl-l-serylglycine: A model for the crystalline regions of silk fibroin. J. Mol. Biol. 19: 580–582.[Medline]

Herzfeld, J. and Berger, A.E. 1980. Sideband intensities in NMR spectra of sample spinning at the magic angle. J. Chem. Phys. 73: 6021–6030.[CrossRef]

Ishida, M., Asakura, T., Yokoi, M., and Saito, H. 1990. Solvent- and mechanical-treatment-induced conformational transition of silk fibroins studied by high-resolution solid-state 13C NMR spectroscopy. Macromolecules 23: 88–94.[CrossRef]

Lotz, B. and Cesari, C.F. 1979. The chemical structure and the crystalline structures of Bombyx mori silk fibroin. Biochimie 61: 205–214.[Medline]

Lotz, B., Brack, A., and Spach, G. 1974. {beta} structure of periodic copolypeptides of L-alanine and glycine. Their relevance to the structure of silks. J. Mol. Biol. 87: 193–203.[Medline]

Magoshi, J., Mizuide, M., Magoshi, Y., Yakahashi, K., Kubo, M., and Nakamura, S. 1979. Physical properties and structure of silk. VI. Conformational changes in silk fibroin induced by immersion in water at 2 to 130°C. J. Polym. Sci. Polym. Phys. Ed. 17: 515–520.[CrossRef]

Marsh, R.E., Corey, R.B., and Pauling, L. 1955. An investigation of the structure of silk fibroin. Biochem. Biophys. Acta 16: 1–34.[Medline]

Nicholson, L.K., Asakura, T., Demura, M., and Cross, T.A. 1993. A method for studying the structure of uniaxially aligned biopolymers using solid-state nitrogen-15 NMR: Application to Bombyx mori silk fibroin fibers. Biopolymers 33: 847–861.[CrossRef][Medline]

Okuyama, K., Takahashi, K., Nakajima, Y., Hasegawa, Y., Hirabayashi, K., and Nishi, N. 1988. Analysis of silk I structure by X-ray and electron diffraction methods. J. Seric. Sci. Jpn. 57: 23–30.

Panitch, A., Matsuki, K., Cantor, E., Cooper, S., Atkins, E., Fournier, M., Mason, T., and Tirrell, D. 1997. Poly(L-alanylglycine): Multigram-scale biosynthesis, crystallization, and structural analysis of chain-folded lamellae. Macromolecules 30: 42–49.[CrossRef]

Peersen, O.B., Wu, X.L., Kustanovich, I., and Smith, S.O. 1993. Variable-amplitude cross-polarization MAS NMR. J. Magn. Reson. Ser. A 104: 334–339.[CrossRef]

Saito, H., Tabeta, R., Asakura, T., Iwanaga, Y., Shoji, A., Ozaki, T., and Ando, I. 1984. High-resolution 13C NMR-study of silk fibroin in the solid-state by the cross-polarization magic angle spinning method— conformational characterization of silk-I and silk-II type forms of Bombyx mori fibroin by the conformation-dependent 13C chemical-shifts. Macromolecules 17: 1405–1412.[CrossRef]

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