Hydrogen-Bonding Structure of Serine Side Chains in Bombyx mori and Samia cynthia ricini Silk Fibroin Determined by Solid-State 2H NMR

Kameda, Tsunenori; Ohkawa, Yasunobu; Yoshizawa, Keiko; Naito, Junko; Ulrich, and Anne S.; Asakura, Tetsuo

doi:10.1021/ma990554q

Cited by 45 publications

(73 citation statements)

References 37 publications

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“…About 75% of all serine residues occur within the sequence of the repetitive region (65), which forms crystalline domains. It was also found that ϳ75% of the serine residues in fibroin interact with carbonyl groups on adjacent chains to form intermolecular hydrogen-bonding networks in the fibroin fiber (66). The remaining serine residues in B. mori fibroin and serine residues in sericin likely contribute to intermolecular hydrogen bonding related to sericin coating the two fibroin fibers.…”

Section: Construction Of Recombinant Expression Plasmid Pet21a(ϩ)-srcn-mentioning

confidence: 97%

Cloning, Expression, and Assembly of Sericin-like Protein

Huang

Valluzzi

Bini

et al. 2003

Journal of Biological Chemistry

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Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to ␤-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited applegreen birefringence, indicating long-range order in the array of ␤-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. Silk of Bombyx mori is composed of two kinds of proteins: the fibroin which is synthesized in the posterior silk gland and the sericins produced by the middle silk gland. The sericins bind two fibroin fibers together in the anterior region of the gland as the fibers are spun to form the cocoon. Sericins represent a family of proteins whose molecular mass ranges from 20 to 310 kDa (2, 3) and are characterized by an unusually high serine content, 38.1 mol % (4). Sericins are soluble in hot water, which makes it possible to degum the silk fibroin threads.Two genes encode sericins, Ser1 and Ser2 (5-8). The different molecular weight sericins are the products of different splicing events at the transcript level. Some conformation studies with sericins from the silk gland of B. mori or regenerated sericin from B. mori cocoons suggested random coil with some ␤ structure (9, 10). However, the samples used in these studies were mixtures of the various native sericin proteins, therefore the structure of the individual proteins is unknown and the contribution of specific sequences toward secondary structure is not confirmed. Sericin model peptides such as poly(L-serine) (11-16), poly(O-benzyl-L-serine) (12), and poly(O-acetyl-L-serine) (13,14) were synthesized for structural feature characterization. The ability of poly(L-serine) to fold into a ␤ conformation depended on molecular weight. Low molecular weight poly(L-serine) (molecular weight 650) was soluble and adopted a random coil conformation in aqueous solution, whereas high molecular weight poly(L-serine) (molecular weight 105,000) formed ␤-sheets and was insoluble in water (11). However, homopolypeptides like poly(L-serine) do not match native sericin in terms of amino acid composition, sequence, and molecular weight. Therefore, to better understand sericin structure, interactions between sericin and fibroin, and the biological relevance of sericin in fiber structure, high molecular weight pure sericin-like proteins are required.Sericin 1 encoded by Ser 1 consists of 70 repeats of the 38-amino acid motif (5): SSTGSSSNTDSNSNSVGSSTS-GGSSTYGYSSNSRDGSV. The molecular mass of sericin 1 proteins is 76 -284 kDa. The 38-amino acid repeat motif exhibits a high content of hydrophilic amino acids: 44.7% serine, 10.5% threonine, and 7.9% tyrosine, very close to the average amino acid composition of serici...

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Section: Construction Of Recombinant Expression Plasmid Pet21a(ϩ)-srcn-mentioning

confidence: 97%

Cloning, Expression, and Assembly of Sericin-like Protein

Huang

Valluzzi

Bini

et al. 2003

Journal of Biological Chemistry

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“…In particular, solid-state NMR has been applied to silkworm silk to investigate molecular orientation using isotopically labeled amino acids: 13 C, 11,19-23 15 N, 20,23 and 2 H to study the hydrogen bonding structure. 24 Amino acid composition of silk varies, however, from silkworm to spider, and a high degree of variation has been noted between individuals of the same species of spider. 2,25,26 A strong relationship between structure and function in silk would suggest that silk from different species of spiders and different silk types may vary in amino acid composition and molecular properties.…”

Section: Introductionmentioning

confidence: 99%

Orientational order of Australian spider silks as determined by solid‐state NMR

et al. 2006

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A simple solid-state NMR method was used to study the structure of 13 C-and 15 Nenriched silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. Carbon-13 and 15 N spectra from alanine-or glycine-labeled oriented dragline silks were acquired with the fiber axis aligned parallel or perpendicular to the magnetic field. The fraction of oriented component was determined from each amino acid, alanine and glycine, using each nucleus independently, and attributed to the ordered crystalline domains in the silk. The relative fraction of ordered alanine was found to be higher than the fraction of ordered glycine, akin to the observation of alanine-rich domains in silk-worm (Bombyx mori) silk. A higher degree of crystallinity was observed in the dragline silk of N. edulis compared with A. keyserlingi, which correlates with the superior mechanical properties of the former #

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“…However, it is not straightforward to clarify the influence of Tyr on the structural characteristics of the silk fibroin chain, because the presence of several Tyr residues in model peptides has been found to destroy the ordered structure and lead to a mixture of distortedˇ-sheet and distortedˇ-turn conformations. 27,28,32 The latter is characterized by a large distribution of torsion angles around an average conformation of the type IIˇ-turn. Concerning the distortedˇ-sheet, some variety in the intermolecular packing arrangements further contributes to the large distribution in torsion angles.…”

Section: Introductionmentioning

confidence: 99%

Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid‐state NMR and molecular mechanics on a model peptide prepared as silk I and II

Asakura

Suita

Kameda

et al. 2004

Magnetic Reson in Chemistry

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105

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The influence of the bulky and H-bonding Tyr side-chain on its Ala- and Gly-rich environment in Bombyx mori silk fibroin was examined by (13)C cross-polarization magic angle spinning (CP/MAS), static (2)H and (19)F NMR and molecular mechanics calculations. Model peptides of the type (AG)(15) were synthesized with Tyr in a number of different positions, precipitated under conditions favoring either of the two characteristic protein conformations, and the resulting structures were assigned from their (13)C chemical shifts. Dialysis of native fibroin or the simple (AG)(15) peptide from a 9 M LiBr solution against water produces silk I (the structure of silk before spinning), whereas drying from formic acid yields silk II (fibrous structure after spinning). We found that the introduction one or more Tyr into (AG)(15) can have a dramatic effect not only on the local backbone conformation but also on the long-range intermolecular chain packing in the samples. The antiparallel beta-sheet conformation of silk II is able readily to accommodate a single Tyr residue. Interestingly, the beta-turn conformation of silk I only remains stable when Tyr is positioned near the chain terminus in (AG)(12)YG(AG)(2), but the conformation is driven towards silk II when Tyr is located in the central region of (AG)(7)YG(AG)(7). The role of H-bonding was tested by replacing Tyr with Phe or 4F-Phe, which are no longer compatible with silk I and fully induced a silk II conformation. In the presence of several Tyr residues a mixture of distorted beta-sheet and beta-turn conformations was obtained, regardless of the precipitation conditions. Static (2)H NMR of ring-deuterated [3',5'-(2)H(2)]Tyr located in the central region of (AG)(7)YG(AG)(7) showed that the side-chain is immobilized in both silk I and II, which was also observed by static (19)F NMR of the 4F-Phe analogue. To visualize the local packing around the Tyr side-chain, molecular mechanics calculations were performed on a mixture of (AG)(4) and AGAGYGAG, starting from either the beta-turn type II or the antiparallel beta-sheet structure. The resulting structures show that the intermolecular chain arrangement is significantly affected by Tyr, thus explaining the long-range packing effects in the semi-crystalline regions of silk fibers compared with the crystalline regions that are devoid of Tyr.

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Hydrogen-Bonding Structure of Serine Side Chains in Bombyx mori and Samia cynthia ricini Silk Fibroin Determined by Solid-State ²H NMR

Cited by 45 publications

References 37 publications

Cloning, Expression, and Assembly of Sericin-like Protein

Cloning, Expression, and Assembly of Sericin-like Protein

Orientational order of Australian spider silks as determined by solid‐state NMR

Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid‐state NMR and molecular mechanics on a model peptide prepared as silk I and II

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