Structure of Bombyx mori Silk Fibroin before Spinning in Silkworm

Chapter 6

Structure of Bombyx mori Silk Fibroin before Spinning in Silkworm

Downloaded by RUTGERS UNIV on February 25, 2016 | http://pubs.acs.org Publication Date: December 10, 2002 | doi: 10.1021/bk-2003-0834.ch006

Tetsuo Asakura, Jun Ashida, and Tsutomu Yamane Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

The silk I structure (the structure of Bombyxmorisilk fibroin before spinning in the solid state) was determined with C two-dimensional (2D) spin-diffusion solid-state NMR, rotational echo double resonance (REDOR) and quantitative use of C CP/MAS NMR chemical shifts. We used C - C double labeled and C - N double labeled model peptides, (AlaGly) in silk I form for solid stateNMRanalyses. The structure was determined to a repeating type II β-turn. The solubility of B. mori silk fibroin in water was examined in the light of the presence of Tyr and Val residues in the repetitive domains of GAGAGYGAGAG and GAGVGYGAGAG sequences. The presence of amorphous domains, TGSSGFGPYVANGGYSGYEYAWSSESDFGT was also considered as the origin of the solubility of silk fibroin in water. The solution structure of silk fibroin in B. mori silkworm is also discussed with previous circular dichroism (CD), optical rotatory dispersion (ORD)andsolutionNMRdata. 13

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The silkworms can produce strong and stiff fibers at room temperature and from an aqueous solution (1). Therefore, it is important to know the structure of the silk fibroin in silkworm in order to understand the mechanism of fiber formation at the molecular level. Two crystalline forms, silk I and silk Π, have been reported as the dimorphs of silk fibroin from B. mori based on several spectroscopic investigations (2). The silk Π structure (the structure of silkfiberafter spinning) wasfirstproposed by Marsh et al. (3) to be an anti-parallel β-sheet, which was subsequently supported by other researchers (1). However, the determination of the silk I structure was difficult because any attempts to induce orientation of the silkfibroinor the model polypeptides with silk I form for studies by X-ray and electron diffraction, causes the silk I form to readily convert to the more

© 2003 American Chemical Society

In NMR Spectroscopy of Polymers in Solution and in the Solid State; Cheng, H. N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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72 stable silk Π form (2). So it is necessary to use more appropriate analytical methods in order to determine the silk I structure at molecular level. Solid state N M R is reported to be one of the suitable techniques for the determination of silk I structure (4). In this study, we attempt to investigate the silk I structure by employing a number of solid -state N M R ( C - 2D sprn-diffusion NMR, REDOR and quantitative use of C CP/MAS N M R chemical shifts) methods (4). The primary structure of B. mon silk fibroin largely consists of repeat sequences of six residues (GlyAlaGlyAlaGlySer^ (1). Therefore, we studied the (AlaGly) sequence as the model for silk I structure. However, the model peptide, (AlaGly) , was found to be insoluble in water although the silk fibroin exists in the solution state in silkworm before spinning. Therefore, we also attempted to clarify the critical amino acids in silk fibroin which may be mainly responsible for its solubility. Recently, Zhou et al. reported its complete amino acid sequence of B. mori silk fibroin (5). The analysis showed that in addition to the repeating sequence of GAGAGS, the primary sequence of silk fibroin also contains G A G A G Y and G A G A G V G Y sequences. These repetitive sequences are flanked by the amorphous region: TGSSGFGPYVANGGYSGYEYAWSSESDFGT, containing both polar and hydrophobic bulky residues. Therefore, the sequential model peptides selected from this primary sequence were also synthesized and the solubility of the peptides in water was examined The structures of these peptides were characterized by solid-state N M R and compared with the (AlaGly) model peptide. The solution structure of silk fibroin in B. mori silkworm will be discussed with reference to previous CD (6), ORD (7) and solution N M R data ( 8,9). 13

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Experimental

Peptide synthesis All the peptides investigated here, were synthesized with the solid phase method by employ F-moc chemistry. The double labeled peptide: C - C and C - N of the (AlaGly)i5 sequence were synthesized for 2D spin-diffusion and REDOR experiments. To achieve purification the samples were dissolved in 9 M LiBr and then dialyzed thoroughly against distilled water for four days. The participated samples were collected and dried. The structures of all the samples were confirmedfromthe C CP/MAS N M R and IR spectra (10). In order to examine the role of specific amino acids on the solubility, peptides with following sequences, 1-4, of silkfibroinwere synthesized. 13

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1 5

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H-GAGAGSGAGAG-OH 1 H-GAGAGYGAGAG-OH 2 H-GAGVGYGAGAG-OH 3 H-TGSSGFGPYVANGGYSGYEYAWSSESDFGT-OH

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73 As described previously, the aqueous solution of silk fibroin was obtained directlyfromthe silkglandofi?. mori.


NMR The 2D spin-diflision N M R spectra were obtained with a Varian OwfylNOVA 400 N M R spectrometer and a 7ηκηφ Jakobsen-type double-tuned M A S probe at off magic angle condition (0^+7°) and with the sample spinning of 6 kHz at room temperature. REDOR experiments were performed on a Chemagnetics CMX-400 spectrometer equipped with a solid-state accessory and a triple-channel magic-angle probe with a 5 mm coil, spirting at 5 kHz. The C CP/MAS N M R measurements were performed on a Chemagnetics CMX-400 spectrometer as described previously (4). 13

Results and Discussion

Silk I structure in the solid state 13

Figure 1 (A) shows the C CP/MAS spectrum of double labeled spectrum of (AlaGly)i5 after dissolving in 9 M LiBr and then diaryzing against water. The high field resonance region (10-70 ppm) was expanded. The observation of chemical shifts at 17.4 ppm for CP and 51.5 ppm for C a carbons of Ala residue, clearly indicate that the structure of the peptide is silk I (10). The C solution N M R spectrum of B. mori silk fibroin obtained directly from the silkgland is also shown in Figure 1(B) for the sake of comparison. The observed C chemical shifts of Ala Cot, Οβ and Gly C a carbons are almost the same between the solid state and the solution state. It may be mentioned that the chemical shifts of Ala C a and Cβ carbons have been more quantitatively used for characterization of the Ala residue in silk I structure (11). The φ and ψ torsion angles satisfy the C a and Οβ chemical shifts, consistent with the values φ = - 8 0 ° to -20°, ψ = 90° to 180°. 13

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φ and ψ angles of Ala and Gly residues in silk I form was determined by the 2D spm7 [1- C]A [1- C]G (AG>7 is shown in Figure 2B along with the simulated spectrum for thetorsionangles ( φ = 70°and ψ = 30° ) of the Gly residue. The simulation was performed for the regions in the Ramachandran map, which were selectedfromthe chemical shift data of silk I obtained 13

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GlyC* Β

Ala C?

Ala C SerC

Set &

JJJ

70

I

60

j 50

1

1

I 40

1

' i 30

1

1

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. 2D 10 ppm from TMS 1

1

l

Figure I *C CP/MAS specirwntf(ÂkG spectrum cfB. mori silkfibroinin aqueous solution obtained directlyfirmthe silkglan (B). The resonance region, 10-70ppm was expanded



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(ppm)

ι

ι

I ,t

i

l

200 190 180 170 160 150

I

·

ι

ι

1 t

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200 190 180 170 160 150

Figure2. The experimental (a) andsimulated (b) 2Dψίη-ώβίΞίοη NMR spectra of [l- C Gtyu U C Ala ] doubfy4abded (AlaGfy) 30 mer (A) and[1- C Ala ,1- C Gty,

Structure of Bombyx mori Silk Fibroin before Spinning in Silkworm

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