Yugo Tasei

Glycerin-Induced Conformational Changes in Bombyx mori Silk Fibroin Film Monitored by 13C CP/MAS NMR and 1H DQMAS NMR

In order to improve the stiff and brittle characteristics of pure Bombyx mori (B. mori) silk fibroin (SF) film in the dry state, glycerin (Glyc) has been used as a plasticizer. However, there have been very limited studies on the structural characterization of the Glyc-blended SF film. In this study, 13C Cross Polarization/Magic Angle Spinning nuclear magnetic resonance (CP/MAS NMR) was used to monitor the conformational changes in the films by changing the Glyc concentration. The presence of only 5 wt % Glyc in the film induced a significant conformational change in SF where Silk I* (repeated type II β-turn and no α-helix) newly appeared. Upon further increase in Glyc concentration, the percentage of Silk I* increased linearly up to 9 wt % Glyc and then tended to be almost constant (30%). This value (30%) was the same as the fraction of Ala residue within the Silk I* form out of all Ala residues of SF present in B. mori mature silkworm. The 1H DQMAS NMR spectra of Glyc-blended SF films confirmed the appearance of Silk I* in the Glyc-blended SF film. A structural model of Glyc-SF complex including the Silk I* form was proposed with the guidance of the Molecular Dynamics (MD) simulation using 1H–1H distance constraints obtained from the 1H Double-Quantum Magic Angle Spinning (DQMAS) NMR spectra.

Hydration of Bombyx mori silk cocoon, silk sericin and silk fibroin and their interactions with water as studied by 13C NMR and 2H NMR relaxation

The mechanical properties of Bombyx mori silk fibers, such as elasticity and tensile strength, change remarkably upon hydration. However, changes in the local conformation and dynamics of individual amino acid residues and change in the dynamics of water molecules due to hydration are not currently well understood on the molecular level. In this work, the conformations and dynamics of the hydrated Bombyx mori silk fibers, including silk cocoon (SC), silk sericin (SS) and silk fibroin (SF), were determined after sustained immersion in water by using 13C refocused insensitive nuclei enhanced by polarization transfer (INEPT) NMR, 13C cross-polarization/magic angle spinning (CP/MAS) NMR and 13C dipolar decoupled-magic angle spinning (DD/MAS) NMR. The 13C INEPT NMR spectrum reflects their mobile domain, the 13C CP/MAS NMR spectrum their rigid domain, and the 13C DD/MAS NMR spectrum both domains. The mobile domain of the hydrated SC fiber originates mainly from the hydrated SS part and the rigid domain of the hydrated SC fiber from the hydrated SF part. Moreover, the dynamics of mobile water molecules interacting with the silk fiber was studied by 2H solution NMR relaxation measurements in the silk fiber-2H2O system. Using an inverse Laplace transform algorithm, we were able to identify distinct mobile components in the relaxation times for 2H2O. Our measurements provide new insight relating to the characteristics of the hydrated structure of SC, SS and SF fibers, and the water molecules that interact with them in water. The information is relevant in light of current interest in the design of novel silk-based biomaterials which are usually in contact with blood and other body fluids.

13C NMR characterization of hydrated 13C labeled Bombyx mori silk fibroin sponges prepared using glycerin, poly(ethylene glycol diglycidyl ether) and poly(ethylene glycol) as porogens

There is a need to prepare softer and highly flexible Bombyx mori silk fibroin (SF) sponges for the development of biomaterials that are biodegradable and with stiffness that matches sponges and soft tissues. In this paper, we prepared SF sponges using glycerin (Glyc), poly(ethylene)glycol diglycidyl ether (PGDE) and poly(ethylene)glycol (PEG) as porogens. The detailed characterization of the hydrated SF sponges was done using three 13C solid state NMR techniques, viz.,13C refocused insensitive nuclei enhanced by polarization transfer (r-INEPT) NMR, 13C cross polarization/magic angle spinning (CP/MAS) NMR, and 13C dipolar decoupled-magic angle spinning (DD/MAS) NMR. These three NMR methods gave respective information on fast motion, slow motion, and both fast and slow motions for the local structure and dynamics of the hydrated SF sponges. There was no significant difference in the r-INEPT spectra of the three hydrated SF sponges. On the other hand, there were significant differences among the 13C CP/MAS NMR spectra of the three sponges. The fractions of two kinds of β-sheet structure, two kinds of random coil conformations with mobile and immobile motions, and the Silk I* (type II β-turn) conformation were determined for the Ser residues in the 13C DD/MAS NMR spectra. Similarly, the fractions of several conformations were also determined for Tyr, Ala and Gly residues in SF, which showed significant differences among the three hydrated sponges. The relationship between the local structure of these hydrated SF sponges and their mechanical properties was also briefly discussed.

Parallel β-Sheet Structure of Alanine Tetrapeptide in the Solid State As Studied by Solid-State NMR Spectroscopy.

The structural analysis of alanine oligopeptides is important for understanding the crystalline region in silks from spiders and wild silkworms and also the mechanism of cellular toxicity of human diseases arising from expansion in polyalanine sequences. The atomic-level structures of alanine tripeptide and tetrapeptide with antiparallel β-sheet structures (AP-Ala3 and AP-Ala4, respectively) together with alanine tripeptide with parallel β-sheet structures (P-Ala3) have been determined, but alanine tetrapeptide with a parallel β-sheet structure (P-Ala4) has not been reported yet. In this article, first, we established the preparation protocol of P-Ala4 from more stable AP-Ala4. Second, complete assignments of the (13)C, (15)N, and (1)H solid-state NMR spectra were performed with (13)C- and (15)N-labeled Ala4 samples using several solid-state NMR techniques. Then, the structural constraints were obtained, for example, the amide proton peaks of P-Ala4 in the (1)H double-quantum magic-angle spinning NMR spectrum were heavily overlapped and observed at about 7.4 ppm, which was a much higher field than that of 8.7-9.1 ppm observed for AP-Ala4, indicating that the intermolecular hydrogen-bond lengths across strands (N-H···O═C) were considerably longer for P-Ala4, that is, 2.21-2.34 Å, than those reported for AP-Ala4, that is, 1.8-1.9 Å. The structural model was proposed for P-Ala4 by NMR results and MD calculations.

Effect of Water on The Structure and Dynamics of Regenerated [3-13C] Ser, [3-13C] Tyr and [3-13C] Ala-Bombyx mori Silk Fibroin Studied with 13C Solid-State NMR.

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.

Photoirradiation and Microwave Irradiation NMR Spectroscopy

In situ photoirradiation solid-state nuclear magnetic resonance (NMR) spectroscopy is designed for optical irradiation from the top part of a zirconia rotor through a glass cap, which makes it possible to efficiently irradiate the inside of the rotor. This experimental method has made it possible to observe photo-intermediates of sensory rhodopsins, such as sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII), and bacteriorhodopsin (bR) Y185F mutant. In SRI, green light generates M-intermediates, which exhibit positive phototaxis, while blue light generates P-intermediates, which exhibit negative phototaxis. In SRII, green light generates M-intermediates and blue light generates O-intermediates. In Y185F-bR, O-intermediates were first observed using solid-state NMR spectroscopy. The microwave irradiation NMR spectrometer was developed in-house by modification of a commercial NMR spectrometer. A flat long copper ribbon was used as a capacitor and a half turn of copper ribbon at the edge was used as an inductor for the microwave resonance circuit, which was coaxially inserted inside the radiofrequency induction coil and allowed NMR signals to be observed under microwave irradiation conditions. The temperature of N-(4-methoxybenzylidene)-4-butylaniline (MBBA) during microwave irradiation was estimated by measuring the temperature-dependent chemical shifts, whereby different protons were found to indicate significantly different temperatures in the molecule. Liquid crystalline-isotropic phase correlation 2D NMR spectra were observed using pulsed microwave irradiation for rapid temperature jump experiments.

NMR Studies on Silk Materials

A wide range of spectroscopic and imaging techniques have been applied to silk, but the most detailed picture of the structure and dynamics of silk in molecular level has been revealed from NMR, using both solid and solution state measurements. In this review, ten NMR techniques from solution NMR to solid-state NMR used to study the structure of dynamics of silk materials and the obtained typical results were reviewed.

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 ( T1s) and spin-spin relaxation times ( T2s) 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 T1 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. T2 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.

Comparison of the knitted silk vascular grafts coated with fibroin sponges prepared using glycerin, poly(ethylene glycol diglycidyl ether) and poly(ethylene glycol) as porogens:

Development of a small-diameter artificial vascular graft is urgent because existing materials often occlude within a short time. We have shown that small-diameter vascular graft using Bombyx mori silk fibroin is a potential candidate. Silk fibroin grafts are fabricated by coating silk fibroin on the knit tube prepared from silk fibroin fibers. However, there is a serious problem that the coated silk fibroin portion hardens when alcohol is used for insolubilization of the coated silk fibroin. This hardening prevents the desired biodegradation of the coated silk fibroin. In this study, we improved the silk fibroin coating method of the knit silk fibroin tube. Namely, the silk fibroin sponge coating was performed using glycerin, poly(ethylene glycol diglycidyl ether) or poly(ethylene glycol). In addition, silk fibroin grafts were prepared avoiding dryness during the coating process and were kept in the hydrated state until implantation into the abdominal aorta was complete. After implantation of the hydrated silk fibroin grafts, grafts were taken out at two weeks or three months, and histopathological examination was performed. The grafts coated with three types of silk fibroin sponges had a higher tissue infiltration rate than alcohol-treated grafts and were superior in the formation of smooth muscle cell and vascular endothelial cell remodeling. Biodegradations of the silk fibroin grafts prepared using the three types of silk fibroin sponge coatings and alcohol-treated silk fibroin grafts were also examined with protease XIV in vitro, and the grafts were observed by scanning electron microscopy before and 24 h after biodegradation. Faster biodegradations were observed for grafts coated with the three types of silk fibroin sponges. 13C solid-state nuclear magnetic resonance studies showed that the conformation of the silk fibroin sponge prepared using porogen was a random coil with high mobility in the hydrated state. We believe that small-diameter silk fibroin vascular grafts coated with quick biodegradable silk fibroin sponges can be developed based on these findings.

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 (T1s) 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 T1 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 (T2s) for the Cβ4 carbons showed longer than those of the other Cβ1-3 carbons of AP-β-sheet (Ala)4. T2 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.