NMR Characterization of Silk Proteins - ACS Symposium Series (ACS

Chapter 13 NMR Characterization of Silk Proteins 1

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Tetsuo Asakura , Makoto Demura , Atsuo Uyama , Katsuaki Ogawa, Keiichi Komatsu , Linda K. Nicholson , and Timothy A. Cross 4

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Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan Department of Bio Research and Development, Tokyo Rikakikai Company Ltd., Ina-machi, Saitama 362, Japan Research Institute for Biological Science, Katakura Company Ltd., 4—5—25 Chuo, Matsumoto, Nagano 390, Japan Sericultural Science Research Institute, Shinjuku-ku, Tokyo 109, Japan Department of Chemistry, Florida State University, Tallahassee, FL 32306

Downloaded by MONASH UNIV on September 13, 2013 | http://pubs.acs.org Publication Date: December 3, 1993 | doi: 10.1021/bk-1994-0544.ch013

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Structures of Bombyx mori silk fibroin have been studied in solution, in silkworm and in the solid state by means of solution and solid C and N NMR spectroscopies. The silk fibroin yields very sharp C NMR signals in aqueous solution and in silkworm, indicating the fast segmental motion of the main chain in spite of a fairly high molecular weight, 3 x 10 . This makes detailed sequential and conformational analyses of the silk fibroin possible. The structure of the silk fiber in the solid state was studied with N CP NMR and N isotope-labeled silk fibroins on the basis of the chemical shift tensors in detail. The torsion angles of the glycine, alanine and tyrosine residues were determined. 13

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In the class Insecta, many species produce long silken filaments (1). Among them, a well-known silk from Bombyx mori has excellent properties which are suitable for clothing and recently, for carrier of the immobilization of enzymes (2). In this paper, we will describe the structure of silk fibroin, mainly B.mori silk fibroin in aqueous solution, in silkworm and in the solid state studied with ^ C and 15

N

NMR spectroscopies. Especially, a new method is developed in which solid state 15N

NMR spectra obtained from uniaxially aligned molecules placed with the axis of alignment both parallel and perpendicular to the applied magneticfield(3). Results from the application of this method to the B.mori silkfibroinprovide structural detail that is consistent with currently accepted structural models based on fiber diffraction studies. Experimental Materials B.mori and a wild silkworm, Philosamia cynthia ricini larvae were reared with an artificial diet in our laboratory. The isotope labeling of B. mori silkfibroinwas achieved biosynthetically through the use of an artificial diet supplemented with the isotope enriched amino acids (4) or by the cultivation of the posterior silk glands

0097-6156/94/0544-0148$06.00/0 © 1994 American Chemical Society In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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extracted from the larvae in the culture medium containing the isotope enriched amino acids (5). The highly isotope labeled silks were obtained by the latter method. The aqueous solution of regenerated silk fibroin from B.mori cocoon was prepared as follows. The cocoons were first degummed twice with 0.5% Marselles soap solution at 100°C for 30 min. to remove another silk protein, silk sericin and then washed with distilled water. The silk was then dissolved in 9 M LiBr at 40°C. After dialysis against distilled water for 4 days, the solution was clarified by spinning in a centrifuge at 10,000 rpm for 30 min. The supernatant was collected and brought gently up to the desired concentration with an electric fan. The powder sample was obtained by freeze-dry treatment of the aqueous solution of silk and then stirred in methanol for 3-4 daystoform the Silk II ( anti parallel β sheet ) conformation. The powder sample of the model compound Boc ( t-butoxy carbonyl )-[l- C]Ala-[ N]Gly-Ala-Gly-OP!ac ( phenyl ester ) was prepared by liquid phase synthesis. The thin sheets of the silk fibroin fibers aligned uniformly were produced with quick-setting bonding reagent and were cut into 8 mm χ 12 mm pieces, stacked together and fixed with the bonding reagenttoform an 8 mm χ 8 mm χ 12 mm block. The block is used for the quantitative analytical approachtoutilize distinct features of the chemical shift and aie line shapes obtained from oriented fibers placed parallel and perpendiculartothe magnetic field for restriction of the number of possible orientations of an individual amide plane (3). 13


NMR Characterization of Silk Proteins

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NMR Measurements JEOL GX 400 and EX 270 spectrometers were used for C CP, C CP/MAS, N CP and N CP/MAS NMR measurements of silk fibroins in the solid state. A home built solid state NMR spectrometer assembled around an Oxford Instruments 400/89 magnet and a Chemagnetics data acquisition system at Florida State University was also used for the ^ N CP NMR measurement of the silk. JEOL GX 270, FX 200 and FX 90Q spectrometers were used for C and N NMR observations of the silkfibroinin solution and in silkworm. Experimental conditions were described elsewhere (3-12). 1 3

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Results and Discussion In vivo and Solution l^C NMR Using C high resolution solution NMR, we can easily observe the silk protein directly in silkworm under non-destructive condition as well as the aqueous solution of the silk (Figure 1). Surprisingly, high resolution * C NMR spectra are observed and are assigned to triglyceride, trehalose and silk protein in the silkworm. High mobility of these components gives high resolution NMR, but other components do not give any NMR spectra because of their low mobility. The NMR spectrum of the pupa shows the peaks due to triglyceride and trehalose, and therefore the peaks of the amino acid residues of the silk protein are easily assigned in the spectra of both silkworms . In the NMR spectrum of B.mori silkworm, the spectral pattern is different from that of P.cricini silkworm, mainly in the resonance region of the carbons of the Ala residue. The shapes of the peaks from the Ala residue from P.cricini silkworm are doublets or asymmetric, indicating the presence of both α-helical and random coil conformations in the silkfibroinas reported previously (6,7). However, the corresponding Ala peaks of B.mori are all singlets and the peak positions coincide with those of the low-field component of the Ala Cβ and of the high-field component of the Ala Ο* and C=0 of P.c.ricini. These data indicate that there is no α-helical portion in the silkfibroinstored in the silk gland of B.mori and the conformation is essentially random coil. Thus, it is 1 3

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In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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SILK POLYMERS: MATERIALS SCIENCE AND BIOTECHNOLOGY

possible to obtain structural information of the silkfibroinfrom the 13 C NMR directly. The aqueous solution of the silk fibroin prepared from Bjnori cocoon also gives high resolution 13c NMR spectrum, indicating very fast segmental motion of the main chain characterized by a very small correlation time, on the order of Ι Ο s. at room temperature (8). The information of the amino acid sequence of the silk fibroin can be obtained from the carbonyl region (4). Sixteen peaks are observed (Figure 2). The assignment is performed using [l-l C]Ala, [l- C]Gly or [l^N]Gly labeled silk fibroins, from a comparison of the silk spectrum with those of the crystalline fraction after chymotrypsin hydrolysis and of the model polypeptides such as ( Ala-Ala-Gly ) and ( Ala-Gly ) . We assigned these peaks at the pentapeptide sequence level as shown in Figure 2. The relative intensities of the main six peaks are nearly equal, indicating that main sequence of silk fibroin is GlyAla-Gly-Ala-Gly-Ser, that is, the alternative copolypeptides of Gly. The content of this sequence is about 70% of B.mori silk fibroin. 10

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Solid State NMR Recent advances in the application of solid state NMR spectroscopy to uniformly aligned biopolymers have opened a window through which to view the detailed structure of biological macromolecules. Atomic resolution structural details are obtained from solid state NMR data in the form of bond orientations, which yield the relative positions of specific atoms within the molecule. A new method is developed in which solid state l^N NMR spectra obtained from uniaxially aligned molecules placed with the axis of alignment both parallel and perpendicular to the magnetic field are analyzed to yield die orientations of specific molecular bonds (3). A diagram of the scheme used to analyze spectral data to obtain high resolution structural details is shown in Figure 3. Analytical expressions are derived which utilize spectral features read from *^N chemical shift anisotropy lineshapes to calculate a discrete number of possible orientations for a specific site. Figure 4 shows observed and simulated CP NMR spectra of a block of oriented [^N] Ala silkfibroinfibersplaced both parallel and perpendicular to the applied magnetic field along with the powder pattern of the silk fibroin. From the simulation, the Euler angles, otp and βρ, which express therelativeorientations of the l^N chemical shift anisotropy (CSA) principal axis system (PAS) and the fiber axis system (FAS) frames of reference were determined. In addition, the angles C I D N C and P D N C for the model compound, Boc-[l-l C]Ala-[ N]Gly-Ala-Gly-OPac , were experimentally determined where C I D N C and P D N C are the Eular angles which express the relative orientations of the l^N CSA PAS and the N-C bond direction (Molecular Symmetry Axis, MSA system frames of reference) (3). Using these Eular angles, the angles, ΘΝΗ and 9NC, between the NH and N C bond direction and the fiber axis, respectively, are obtained as follows. 3

COSONX

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= cosPFCOsPDNx+sinPFCOsapcosaDNxsinPDNx +sinPFsinaFsinaDNxsinPDNx

where C I D N X and βοΝχ are the Eular angles for transformation from CSA PAS to MAS system and X is Η or C. A similar l^N solid state NMR analysis was applied to [l^NJGly and [l^NJTyr silkfibroinfibers in order to determine the angles,ΘΝΗ and 6NC. Details have been reported for the former [l^NJGly silk sample (3). Determination of the torsion angles, ψ and ψ, of the Gly, Ala and Tyr residues of


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ASAKURA ET AL.

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Figure 1. C NMR spectra of the silk gland portion of the mature larva (A) and of the abdomen of the pupa (B) of P.c.ricini. The spectrum of the mature larva of B.mori is also shown (C).

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Figure 2. Assignment of the carbonyl region in B.mori silk fibroin spectrum.


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Figure 3. Diagram of the scheme used to analyze spectral data to obtain high resolution structural details

B.mori silk fibroin is possible with two sets of ΘΝΗ and 6 N C values obtained for successive nuclei in the chain, where ψ describes the rotation about the N - C bond while ψ describes the rotation about the C - C bond. Figure 5 shows the relationship between φ or ψ and the orientations of the relevant bonds. The definitions of the symbols used here are described in the reference 9. Several numbers of possible torsion angle solutions are obtained. The C CP/MAS NMR (10,11) and N CP/MAS NMR data (12) of the silkfibroincan be used to reduce the solutions. The final ψ and ψ values obtained here are -141 ± 5° and 147 ± 5° for the Gly residue, -139 ± 5° and 146 ± 5° for the Ala residue , and -139 ± 5° and 147 ± 5° for the Tyr residue, respectively. a

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Figure 4. Observed and simulated CP NMR spectra of a block of oriented [1*N] Ala silk fibroin fibers placed both parallel (A) and perpendicular (B) to the applied magnetic field along with the powder pattern of the silk fibroin (C).

Figure 5. Relationship between φ or ψ and the orientation of the relevant bonds. In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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Acknowledgments This work has been funded, in part, through the Grant-in-Aid of the Ministry of Education, Science, and Culture of Japan ( grant no. 04556007 ). T.A. gratefully acknowledges support of the Yamada Science Fundation,Japan. T.A. also extends thanks to Toyo Seikan Co., R. & D. Group/Yokohama, Japan for use of their JEOL EX-270 spectrometer. References and Notes 1.Asakura T. and Kaplan D.L., Silk Production and Processing, Encyclopedia of Agricultural Science, Ed. by Arntzen C.J. Academic Press in press. 2.Asakura T., Kitaguchi M., Demura M., Sakai H.and Komatsu K., J. Appl. Polym. Sci., 46, 49 ( 1992 ). and references therein. 3.Nicholson L.K., Asakura T., Demura M.and Cross T.A., Biopolymers in press. 4.Asakura T., Watanabe Y. and Itoh T., Macromolocules 17, 2421 ( 1984). 5. Asakura T., Sakaguchi R., Demura M., Manabe T., Uyama Α., Ogawa K. and Osanai M., Biotechnol. Bioeng., 41, 245 (1993) and references therein. 6. Asakura T., Suzuki H. and Watanabe Y., Macromolecules 16, 1024 ( 1983 ). 7. Asakura T., Kashiba H. and Yoshimizu H., Macromolecules 21, 644 ( 1988 ). 8. Asakura T., Watanabe Y., Uchida A. and Minagawa H., Macromolecules 17,1075 ( 1984 ). 9. Teng.Q., Nicholson L.K. and Cross T.A., J. Mol. Biol. 218, 607 ( 1991 ). 10.SaitoH.,Tabeta R., Asakura T., Iwanaga Y., Shoji Α., Ozaki T. and Ando I., Macromolecules 17,1405 ( 1984 ). 11.Asakura T., Kuzuhara Α., Tabeta R. and Saito H., Macromolecules 18, 1841 (1985). 12.Asakura T., Miyashita N., Demura M., Yoshimizu H., Ando I. and Shiibashi T., Rept. Prog. Polym. Phys. Japan. 33, 633 ( 1990 ). R E C E I V E D May

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NMR Characterization of Silk Proteins - ACS Symposium Series (ACS

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