Lamellar Structure in Alanine-Glycine Copolypeptides Studied by Solid-State NMR Spectroscopy: A Model for the Crystalline Domain of Bombyx mori Silk Fibroin in Silk II Form.

Bombyx mori silk fibroin (SF) fibers with excellent mechanical properties have attracted widespread attention as new biomaterials. However, the structural details are still not conclusive. Here, we propose a lamellar structure for the crystalline domain of the SF fiber based on structural analyses of the Ala Cß peaks in the 13C cross-polarization/magic angle spinning NMR spectra of (Ala-Gly)m (m = 9, 12, 15, and 25) and 13C selectively labeled (Ala-Gly)15 model peptides. Namely, three Ala Cß peaks with relative intensities of 1:2:1 obtained by deconvolution were assigned to two kinds of ß-sheet and a ß-turn, which are interpreted as a lamellar structure formed by repetitive folding using ß-turns every eighth amino acid, for which the basic structure is (Ala-Gly)4 in an antipolar arrangement. The dynamics and intermolecular arrangement were further studied using 13C solid-state spin-lattice relaxation time observations and the rotational echo double resonance experiments, respectively.

Packing Structure of Antiparallel ß-Sheet Polyalanine Region in a Sequential Model Peptide of Nephila clavipes Dragline Silk Studied Using 13C Solid-State NMR and MD Simulation.

Packing structures of polyalanine regions, which are considered to be the reason for the extremely high strength of spider dragline silks, were studied using a series of sequential peptides: (Glu)4GlyGlyLeuGlyGlyGlnGlyAlaGly(Ala)nGlyGlyAlaGlyGlnGlyGlyTyrGlyGly(Glu)4 (n = 3-8) using 13C solid-state NMR spectroscopy. The conformations of (Ala)n in the freeze-dried peptides changed gradually with increasing n from random coils to α-helices with partial antiparallel ß-sheet (AP-ß) structures. Conversely, all the insolubilized peptides, n = 6-8 after low-pH treatment and n = 4-8 after formic acid/methanol treatment, formed AP-ß structures with significant amounts of staggered packing arrangements. These results are different from previously obtained results for pure alanine oligopeptides, that is, AP-ß (Ala)n formed rectangular packing for less than n = 6 but staggered packings for n ≥ 7. The 13C-labeled peptides were also used to confirm the staggered packing arrangements from NMR dynamics. Furthermore, a MD simulation supported the observed results.

Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations.

Samia cynthia ricini is one of the wild silkworms and its silk fibroin (SF) consists of alternatively repeating poly-l-alanine (PLA) sequences as crystalline domain and glycine-rich sequences as noncrystalline domain; the structure is similar to those of spider silk and other wild silkworm silks. In this paper, we proposed a new staggered model for the packing arrangement of the PLA sequence through the use of the Cambridge Serial Total Energy Package program and a comparison of the observed and calculated chemical shifts of the PLA sequence with the Gauge Including Projector Augmented Wave method. The new model was supported by the interatomic distance information from the cross peaks of Ala Cß dipolar-assisted rotational resonance (DARR) spectrum of the PLA sequences in S. c. ricini SF fiber. In addition, three 13C NMR peaks observed in the ß-sheet region were assigned to the carbons with different environments in the same model, but not assigned to different ß-sheet structures.

Packing arrangement of 13C selectively labeled sequence model peptides of Samia cynthia ricini silk fibroin fibers studied by solid-state NMR.

Samia cynthia ricini (S. c. ricini) is one of the wild silkworms. Their silk fibroins have been paid attention as potentially valuable biomedical materials as well as Bombyx mori silk fibroins, but detailed information on the packing arrangement of the fibers is still not currently well understood at a molecular level. In this study, 34 mer model peptides, GGAGGGYGGDGG(A)12GGAGDGYGAG with different 13C labeled positions have been synthesized as a typical sequence of the primary structure of S. c. ricini silk fibroins made up of tandemly repeated sequences of polyalanine as the crystalline region and glycin-rich sequences as the non-crystalline region. The heterogeneous structure was obtained from the determination of the fraction of several conformations depending on the position of the Ala residue by 13C cross polarization/magic angle spinning NMR. The packing arrangement was studied by 13C dipolar assisted rotational resonance NMR and packing in a staggered arrangement rather than a rectangular arrangement of this peptide with an anti-parallel ß-sheet structure was clarified, which is in good agreement with our previous report on the packing arrangement of (Ala)7 with an anti-parallel ß-sheet structure.

Glycerin-Induced Conformational Changes in Bombyx mori Silk Fibroin Film Monitored by (13)C 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, (13)C 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 ß-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.

NMR study of the structures of repeated sequences, GAGXGA (X = S, Y, V), in Bombyx mori liquid silk.

The silk fibroin stored in the silk gland of the Bombyx mori silkworm, called "liquid silk", is spun out and converted into the silk fiber with extremely high strength and high toughness. Therefore it is important to determine the silk structure before spinning called Silk I at an atomic level to clarify the fiber formation mechanism. We proposed the repeated type II ß-turn structure as Silk I in the solid state with the model peptide (AG)15 and several solid state NMR techniques previously. In this paper, the solution structure of native "liquid silk" was determined with solution NMR, especially for tandem repeated sequences with (GAGXGA)n (X = S, Y, V) and GAASGA motifs in the B. mori silk fibroin. The assignment of the (13)C, (15)N, and (1)H solution NMR spectra for the repetitive sequence motifs was achieved, and the chemical shifts were obtained. The program, TALOS-N, to predict the backbone torsion angles from the chemical shifts of proteins was applied to these motifs with (13)Cα, (13)Cß, (13)CO, (1)Hα, (1)HN, and (15)N chemical shifts. The twenty-five best matches of torsion angles (ϕ, φ) were well populated and mainly fell into the regions for typical type II ß-turn structures in the (ϕ, φ) map for the GAGXGA (X = S, Y, V) motifs. In contrast, (ϕ, φ) plots for motif GAASGA were scattered, indicating that the motif is in a disordered structure. Furthermore, inter-residue HN-Hα NOE cross peaks between i-th and (i+2)th residues in GAGXGA (X = S, Y, V) motifs were observed, supporting the repeated type II ß-turn structure. Thus, we could show the presence of the repeated type II ß-turn structure in "liquid silk".

Chain-Folded Lamellar Stacking Structure of the Crystalline Fraction of Bombyx mori Silk Fibroin with Silk II Form Studied by 2D 13C-13C Homonuclear Correlation NMR Spectroscopy.

The structure of Bombyx mori silk fibroin (SF) is a subject of significant interest due to its remarkable physical properties; however, its atomic-level structure is still not conclusive. We previously proposed a lamellar stacking structure for the crystalline fraction (Cp) with ß-turns occurring every eighth amino acid. In this study, we took the following steps: At first, a model of the chain-folded lamellar stacking structure in antipolar and antiparallel ß-sheet layers was constructed. Then, dipolar-assisted rotational resonance solid-state NMR spectra were observed to determine the effective internuclear distance (rj,keff) for the uniformly 13C-labeled Cp fraction sample. By comparing the experimentally obtained rj,keff (obs) values with the calculated rj,keff (calc) values from our structural model, a fairly good correlation between the observed and calculated values of the internuclear distances was obtained with a standard deviation of 0.37 Å. This supports the existence of the chain-folded lamellar stacking structure in the SF fiber. These findings contribute to our understanding of the atomic-level structure of SF and its exceptional properties.

Infrared-A Irradiation-induced Inhibition of Human Keratinocyte Proliferation and Potential Mechanisms.

Infrared-A (IRA), which can penetrate deeply into the human skin, is a major component of solar radiation and is recognized to promote photoaging of human dermis. To our knowledge, however, the cellular and molecular consequences of human epidermis exposure to IRA have not been clarified. Thus, we investigated whether IRA inhibits the proliferation of normal human epidermal keratinocytes (NHEKs). IRA irradiation ed in cell cycle arrest at G1 and a dose-dependent reduction in the proliferation of NHEKs. We found that mechanistic target of rapamycin complex 1 (mTORC1) was initially inactivated during IRA irradiation due to the formation of stress granules (SGs), and this inactivation was maintained for at least 6 h after irradiation due to Akt dephosphorylation. Furthermore, repeated exposure of human skin equivalents to IRA led to marked thinning of the epidermal cell layer. In conclusion, IRA irradiation inhibits mTORC1 activity possibly through two molecular mechanisms involving SG formation in the early-phase and subsequent Akt dephosphorylation. This sequential mechanism seems to cause G1 cell cycle arrest and a reduction in cell proliferation, supporting the hypothesis that the decreased proliferation of basal keratinocytes that occurs during skin aging might be partly attributable to IRA radiation.

Conformational change of 13C-labeled 47-mer model peptides of Nephila clavipes dragline silk in poly(vinyl alcohol) film by stretching studied by 13C solid-state NMR and molecular dynamics simulation.

For determination of the conformation of irregular sequences in glycine-rich region of the Nephila clavipes spider dragline silk, the combination of 13C selectively labeled model peptides for the typical primary structure and their 13C solid-state NMR observations is very useful (T. Asakura et al. Macromolecules. 51 (2018) 3608-3619). However, spiders produce the fiber through the stretching process in nature and therefore, it is difficult to study conformational change by stretching as mimic using the model peptides because these are generally in the powder form. In this paper, 13C selectively labeled three model peptides, (Glu)4(Ala)6GlyGly12Ala13Gly14GlnGlyGlyTyrGlyGlyLeuGlySerGlnGly25Ala26Gly27ArgGly-GlyLeuGlyGlyGlnGly35Ala36Gly37(Ala)6(Glu)4 with three underlined 13C labeled blocks and their poly(vinyl alcohol) blend films were prepared and the conformational changes of these peptides were monitored by stretching of the films using 13C solid-state NMR. In addition, the molecular dynamics simulation was done to evaluate change in the conformation of the sequence by stretching theoretically. The fractions of ß-sheet of Ala36 and Gly37 residues in glycine-rich region adjacent to the C-terminal (Ala)6 sequence increased significantly by stretching compared with those of other 13C labeled Ala and Gly residues.

Structural Analyses of Alanine Trimer and Tetramer Crystals with Antiparallel and Parallel ß-Sheet Structures Using Solid-State 1H Spin-Diffusion 2D Correlation NMR Spectroscopy.

Poly-l-alanine (PLA) sequences are key elements of the crystalline domains of spider dragline and wild silkworm silks. In the present work, 1H spin-diffusion two-dimensional (2D) correlation NMR spectra were observed for selectively deuterated (Ala)3 and (Ala)4 crystals to develop the analytical method for the structure of PLA sequences. The build-up curves of the cross peaks for three kinds of 1H pairs in selectively deuterated (Ala)3 and (Ala)4 crystals were observed to obtain spin-diffusion rate constant k j, k from relaxation master equations P i, j(τm). The k j, k values subsequently lead to effective interproton distance r j, keff (obs) values for individual proton-proton pairs, which include intra- and intermolecular contributions. The r j, keff (obs) values were compared to r j, keff (calc) values obtained from the experimentally determined atomic coordinates of antiparallel (AP) ß-sheet (Ala)3 and (Ala)4 and parallel (P) ß-sheet of (Ala)3 and (Ala)4 crystals. The agreement between the r j, keff (obs) and r j, keff (calc) values was good for AP ß-sheet (Ala)3 and (Ala)4 crystals but poor for P ß-sheet (Ala)3 and (Ala)4 crystals. These deviations were obtained from the interproton distances of the interchain contributions due to different packing arrangements. The packing arrangements of the PLA region are important when considering the relevant structure and the mechanical properties of silks.

Quantitative Analysis of Solid-State Homonuclear Correlation Spectra of Antiparallel ß-Sheet Alanine Tetramers.

Poly-l-alanine (PLA) sequences are a key element in the structure of the crystalline domains of spider dragline silks, wild silkworm silks, antifreeze proteins, and amyloids. To date, no atomic-level structures of antiparallel (AP)-PLA longer than Ala4 have been reported using the single-crystal X-ray diffraction analysis. In this work, dipolar-assisted rotational resonance solid-state NMR spectra were observed to determine the effective internuclear distances of 13C uniformly labeled alanine tetramer with antiparallel (AP) ß-sheet structure whose atomic coordinates are determined from the X-ray crystallographic analysis. Initial build-up rates, R j, k, were obtained from the build-up curves of the cross peaks by considering the internuclear distances arising in the master equation. Subsequently, experimentally obtained effective internuclear distances, reffj, k(obs), were compared with the calculated reffj, k(calc) values obtained from the X-ray crystallographic data. Fairly good correlation between reffj, k(obs) and reffj, k(calc) was obtained in the range of 1.0-6.0 Å, with the standard deviation of 0.244 Å, without considering the zero-quantum line-shape functions. It was further noted that the internuclear distances of intermolecular contributions provide details relating to the molecular packing in solid-state samples. Thus, the present data agree well with AP-ß-sheet packing but do not agree with P-ß-sheet packing.

Parallel ß-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 ß-sheet structures (AP-Ala3 and AP-Ala4, respectively) together with alanine tripeptide with parallel ß-sheet structures (P-Ala3) have been determined, but alanine tetrapeptide with a parallel ß-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.

Rebamipide increases the mucin-like glycoprotein production in corneal epithelial cells.

PURPOSE: Dry eye is a multifactorial disease of tears and the ocular surface due to tear deficiency or excessive tear evaporation. Tear film instability is due to a disturbance in ocular surface mucin leading to a dysfunction of mucin, resulting in dry eye. In this study, we examined the effect of rebamipide, an anti-ulcer agent, on glycoconjugate production, as an indicator of mucin-like glycoprotein in cultured corneal epithelial cells. Further, we investigated the effect of rebamipide on the gene expression of membrane-associated mucins. METHODS: Confluent cultured human corneal epithelial cells were incubated with rebamipide for 24 h. The glycoconjugate content in the supernatant and the cell extracts was measured by wheat germ agglutinin-enzyme-linked lectin assay combined gel-filtration method. In the experiment on mucin gene expression, cultured human corneal epithelial cells were collected at 0, 3, 6, and 12 h after administration of rebamipide. Real-time quantitative polymerase chain reaction was used to analyze the quantity of MUC1, MUC 4, and MUC16 gene expression. RESULTS: Rebamipide significantly increased the glycoconjugate contents in the supernatant and cell extract. In the mucin gene expression in the cells, rebamipide increased MUC1 and MUC4 gene expression, but did not increase MUC16 gene expression. CONCLUSIONS: Rebamipide promoted glycoconjugate, which has a property as a mucin-like glycoprotein, in human corneal epithelial cells. The increased production was mediated by MUC1 and MUC4 gene expression.

Determination of accurate 1H positions of an alanine tripeptide with anti-parallel and parallel ß-sheet structures by high resolution 1H solid state NMR and GIPAW chemical shift calculation.

The accurate (1)H positions of alanine tripeptide, A(3), with anti-parallel and parallel ß-sheet structures could be determined by highly resolved (1)H DQMAS solid-state NMR spectra and (1)H chemical shift calculation with gauge-including projector augmented wave calculations.

Lamellar structure in poly(ala-gly) determined by solid-state NMR and statistical mechanical calculations.

Lamellar structure of poly(Ala-Gly) or (AG)n in the solid was examined using 13C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1-13C]Gly14[1-13C]Ala15- and [1-13C]Gly18[1-13C]Ala19 of (AG)15 were examined by two-dimensional (2D) 13C spin diffusion NMR in the solid state. In addition five doubly labeled [15N,13C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3-13C] in each of the successive Ala residues were utilized for REDOR and 13C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted beta-turns with a large distribution of the torsion angles and antiparallel beta-sheets. The relative proportion of the distorted beta-turn form was evaluated by examination of 13C CP/MAS NMR spectra of [3-13C]Ala-(AG)15. In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13C and 15N nuclei which were also interpreted in terms of a combination of beta-sheets and beta-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cbeta 13C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.