Stretching-Induced Conformational Transition of [3-¹³C]Ser- and [3-¹³C]Tyr-Antheraea yamamai Silk Fibroin before Spinning Investigated with ¹³C Solid-State NMR Spectroscopy

The conformational transition of [3-13C]Ser- and [3-13C]Tyr-Antheraea yamamai silk fibroin before spinning induced by stretching was investigated with 13C CP/MAS NMR spectroscopy. The α-helix content of the silk fibroin before stretching was found to be 31.6% based on the Ala and Ser peaks. With increasing stretching ratio, the α-helix and the random coil Ala Cβ peaks decreased gradually, while the β-sheet peak was observed at a stretching ratio of ×5 and increased rapidly upon further stretching. For Ser residue, the α-helix peak decreased monotonically with increasing stretching ratio, but the random coil peak increased slightly till the stretching ratio of ×5 and then decreased. A small β-sheet peak was observed before stretching and then increased rapidly starting from the stretching ratio of ×7. In contrast, a gradual decrease of random coil peak and an increase of β-sheet peak were observed for the Tyr residue. The results of this investigation may be helpful for further studies of fiber formation mechanism in A. yamamai and in the future design of artificial silk materials

Determination of Local Structure of ¹³C Selectively Labeled 47-mer Peptides as a Model for Gly-Rich Region of <i>Nephila clavipes</i> Dragline Silk Using a Combination of ¹³C Solid-State NMR and MD Simulation

For the first time, we elucidate the complex structure of the Gly-rich regions in <i>Nephila clavipes</i> dragline silk through synergistic experimental and theoretical studies. First, the ¹³C selectively labeled 47-mer peptides selected from the glycine (Gly)-rich region of <i>N. clavipes</i> dragline silk were synthesized. The ¹³C CP/MAS NMR spectra were analyzed to determine the fractions of the conformations of individual Gly and Ala residues through ¹³C conformation-dependent chemical shifts and peak deconvolution. By comparing the ¹³C solid-state NMR spectra of several simple model peptides, the presence of 3₁ helix in the 47-mer peptides was disproved, and the (Ala)₆ regions were shown to form β-sheet structure in the staggered arrangement. Although the fraction of β-sheet components tended to increase and the fraction of random coil component decrease toward both chain ends, significant change in the fractions was observed depending on the amino acid position. These results were successfully rationalized through molecular dynamics simulation

Unusual Dynamics of Alanine Residues in Polyalanine Regions with Staggered Packing Structure of Samia cynthia ricini Silk Fiber in Dry and Hydrated States Studied by ¹³C Solid-State NMR and Molecular Dynamics Simulation

Recently, the wild silkworm and spider dragline silks have been paid considerable attention as potentially valuable biomedical materials. Samia cynthia ricini is one of the wild silkworms and the primary structure of the silk fibroin (SF) consists of tandemly repeated polyalanine (poly-A:(A)_12,13). Here, we report the unusual dynamical character observed in Ala Cβ groups in the poly-A region which forms an antiparallel-β-sheet structure with a staggered packing arrangement. The ¹³C spin–lattice relaxation (<i>T</i>₁’s) and spin–spin relaxation times (<i>T</i>₂’s) of Ala Cβ peaks in S. c. ricini SF fibers were observed in dry and hydrated states. The lowest field peak in Ala Cβ of the poly-A region showed 2 times longer <i>T</i>₁ value and shorter correlation time than the other Ala Cβ peaks of the staggered packing structure, suggesting unusually fast hopping in methyl groups. Molecular dynamics simulations indicated that two of the Ala Cβ carbons out of eight existing in the unit cell of the staggered packing structure exhibited the fastest hopping motion in spite of the shortest Cβ–Cβ distance, indicating a geared hopping motion. <i>T</i>₂ values of the hydrated and dry Ala Cβ peaks showed a similar value, indicating that the backbone motion of S. c. ricini SF fiber is not significantly affected by hydration

Packing Arrangements and Intersheet Interaction of Alanine Oligopeptides As Revealed by Relaxation Parameters Obtained from High-Resolution ¹³C Solid-State NMR

Alanine oligopeptides provide a key structure of the crystalline domains of the silks from spiders and wild silkworm and also the sequences included in proteins such as antifreeze proteins and amyloids. In this paper, the local dynamics of alanine oligopeptides, (Ala)₃, (Ala)₄, and (Ala)₆ were examined by high-resolution ¹³C solid-state NMR. The ¹³C spin–lattice relaxation times (<i>T</i>₁’s) for the Cβ4 carbons of antiparallel (AP)-β-sheet (Ala)₄ significantly prolonged and the correlation time was estimated as 3.6 × 10^–11 s which was shorter than those of other carbons in the AP-β-sheet (Ala)₄ (2.8 × 10^–10 s). The <i>T</i>₁ values for the Cβ carbons of (Ala)₆ showed significantly longer correlation time (8.8 × 10^–9 s) than those of AP-β-sheet (Ala)₄. It is thus revealed that AP-β-sheet (Ala)₆ exhibited stronger intersheet interaction than those of AP-β-sheet (Ala)₄. The ¹³C spin–spin relaxation times (<i>T</i>₂’s) for the Cβ4 carbons showed longer than those of the other Cβ1–3 carbons of AP-β-sheet (Ala)₄. <i>T</i>₂ values of Cβ carbons reflect the slow time-scale (∼70 kHz) backbone motions. The C-terminal forms strong hydrogen bonds with water molecules and thus the backbone motion is slower than ∼70 kHz, while the central backbone motions are faster than ∼70 kHz in the AP-β-sheet (Ala)₄

Effect of Water on the Structure and Dynamics of Regenerated [3-¹³C] Ser, [3-¹³C] , and [3-¹³C] Ala-<i>Bombyx mori</i> Silk Fibroin Studied with ¹³C Solid-State Nuclear Magnetic Resonance

The effects of water on the structure and dynamics of natural and regenerated silk fibroin (SF) samples were studied using ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy. We prepared different types of SF materials, sponges, and fibers with different preparation methods and compared their NMR spectra in the dry and hydrated states. Three kinds of ¹³C NMR techniques, r-INEPT, CP/MAS, and DD/MAS, coupled with ¹³C isotope labeling of Ser, Tyr, and Ala residues were used. In the hydrated sponges, several conformations, that is, Silk I* and two kinds of β-sheets, A and B, random coil, and highly mobile hydrated random coil were observed, and the fractions were determined. The fractions were remarkably different among the three sponges but with only small differences among the regenerated and native fibers. The increase in the fraction of β-sheet B might be one of the structural factors for preparing stronger regenerated SF fiber

Mixture of Rectangular and Staggered Packing Arrangements of Polyalanine Region in Spider Dragline Silk in Dry and Hydrated States As Revealed by ¹³C NMR and X‑ray Diffraction

For the first time, we determined the relative percentages of “rectangular” and “staggered” packing arrangements in the crystalline polyalanine regions with antiparallel β-sheet structure within spider dragline silk fiber from <i>Nephila clavata</i> (NCF) and recombinant silk protein (RSP). The methods used included X-ray diffraction and ¹³C NMR coupled with selective ¹³C isotope labeling of the Ala Cβ carbons. From deconvolution analyses of the Ala Cβ peaks in the ¹³C CP/MAS NMR spectra, the relative percentages of the rectangular arrangements in [3-¹³C]­Ala-NCF were determined to be 49 ± 8% and 40 ± 7% in the dry and hydrated states, respectively, and in [3-¹³C]­Ala-RSP 62 ± 11% and 81 ± 5% in the dry and hydrated states, respectively. Thus, the packing structure changed significantly between the two spider silks and also between the two physical states. The use of NMR was critical in this analysis; from X-ray diffraction patterns alone it would have been difficult to obtain these quantitative data

NMR Investigation about Heterogeneous Structure and Dynamics of Recombinant Spider Silk in the Dry and Hydrated States

Spider silks continue to attract researchers because of their excellent mechanical properties and supercontraction behavior. In this paper, the structure and dynamics of recombinant spider silk protein (RSP) were characterized using ¹³C CP/MAS, ¹³C DD/MAS, and ¹³C refocused-INEPT NMR spectroscopies in the dry and hydrated states. The fractions of several structures of RSP with helical, random coil, and β-sheet polyalanine sequences were determined from the CP/MAS NMR spectra in the dry state. The CP/MAS NMR spectra changed to very simple one with dominant β-sheet Ala peaks by hydration due to a significant loss in CP signals of the other mobile carbons. On the contrary, only sharp mobile peaks, and both mobile and immobile peaks could be observed in the refocused-INEPT and DD/MAS NMR spectra, respectively. The cis/trans proportion of the Gly–Pro bond was also determined. Our measurements provide new insight into understanding the supercontraction phenomenon of spider silks