NMR of silk fibroin. Carbon-13 NMR study of the chain dynamics and solution structure of Bombyx mori silk fibroin

Abstract

13C NMR spectroscopy has been applied to the chain dynamics of B. mori silk fibroin in solution and the silk fibroin stored in the silk gland of the intact silkworm. All the peaks were sharp except for the C" and C=0 peaks of the Gly residue and the C=0 peak of the Ala residue: a slight splitting was observed for these latter peaks, indicating sensitivity to the amino acid sequence of the silk fibroin. The amino acid composition determined by 13C NMR spectroscopy was very close to that for silk fibroin determined from amino acid analysis, indicating that both domains, i.e., crystalline and noncrystalline, in the fibrous protein were observed in the spectrum. The mean correlation times of the segmental motion, determined from the spin-lattice relaxation times and nuclear Overhauser enhancements of the Ala, Gly, and Ser C" carbons assuming a log x2 distribution model, were of the order of 10-10 s at 40 °C. Moreover, the width parameter, p, in the model was determined to be 11-14, indicating a broad distribution of correlation times. These are typical of a random coil polymer. The rate of internal rotation around the C"-C^bond decreases in the order Ala, Ser, Tyr. The side-chain motion is strongly hindered for the Tyr residue although the pK" value for Tyr OH deprotonation indicates absence of intraor intermolecular hydrogen bonding between the OH group and any amino acid residues of the silk fibroin. A decrease in the Tx values and an increase of the mean correlation times of the segmental motion were observed with increasing concentration. This is discussed in relation to the appearance of the silk I type conformation accompanying the aggregation of the chain.

WISE NMR Characterization of Nanoscale Heterogeneity and Mobility in Supercontracted Nephila clavipes Spider Dragline Silk

“…34 This environment was assigned to a dynamic loose helical structure in silk gland fibroin and is also displays a comparable chemical shift to the silk worm Silk I protein (see Table 1). 35 Observation of this environment in the WISE NMR spectrum of the wet, supercontracted dragline silk indicates a reintroduction of this dynamic loose helical alanine environment when the silk is in contact with water. The 1 H slice taken at this 16.6 ppm 13 C resonance clearly displays a significant narrow component with a ∼5 kHz line width.…”

Theoretical Conformational Analysis on Silk Fibroin Model Polypeptide with Ala-Gly Repeated Sequence

“…29 The CD spectrum of Bombyx mori fibroin solution at the concentration less than 0.1 % has a negative dichroic peaks around 195 nm at 5°C, and at higher temperature, a negative dichroic peaks appeares around 215 nm related to the p-structure. 29 31 and showed that there is no a-helical portion in the silk fibroin stored in the middle silk gland of Bombyx mori and that the helix detected in the Bombyx mori silk fibroin solution is the silk I type rather than the a-helical type.…”

Structure ofBombyx mori silk fibroin before spinning in solid state studied with wide angle x-ray scattering and13C cross-polarization/magic angle spinning NMR

“…On the other hand, broad peaks were observed at 56 ppm (Tyr C␣) and 36 ppm (Tyr C␤) in the spectrum of 3, indicating that the Tyr residues take random coil structure. 26 Thus, (Ala-Gly-Tyr-Gly-Ala-Gly) 5 take ␤-sheet structure with about 30% in the random coil conformation, while the peptide 3-(X-Gly) 15 , where X is Tyr for the 7th, 15th, and 23th residues, and Val for the 11th residue and Ala for other residues-takes mainly random coil conformation.…”