Ala-Gly

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Tetsuo Asakura - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of a Ca binding-amphipathic silk-like protein and peptide with the sequence (Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4 with potential for bone repair
    Soft Matter, 2012
    Co-Authors: Aya Nagano, Hirohiko Sato, Yumi Tanioka, Yasumoto Nakazawa, David W. Knight, Tetsuo Asakura
    Abstract:

    Bombyx mori silk fibroin with the main sequence (Ala-Gly-Ser-Gly-Ala-Gly)n is a promising scaffold for bone regeneration not only on account of its excellent mechanical property as a structural matrix, but also for its slow biodegradability with adequate control of hydroxyapatite (HAP) deposition. Seeking to develop a material that might stimulate bone regeneration, we prepared a recombinant calcium binding-amphipathic silk-like protein [(Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4]4 by expression in E. coli. We also prepared the homologous peptide (Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4 by solid phase synthesis. The poly-L-glutamic acid was introduced into both protein and peptide because this sequence is involved in HAP-nucleating domains of bone sialoprotein. The recombinant protein was shown to bind relatively large quantities of Ca2+ ions in solution by a spectrophotometric assay and in the solid state by X-ray photoelectron spectroscopy. Changes in the electronic structure and local conformation of the peptide resulting from Ca2+ binding were studied using 13C solution NMR, especially 13C chemical shifts. We obtained evidence that Ca2+ bound to the poly-L-glutamic acid domains but not to the predominantly hydrophobic (Ala-Gly-Ser-Gly-Ala-Gly)4 domains. A remarkable conformational change induced by adsorption of the synthetic peptide on the HAP surface was also demonstrated using 13C solid state NMR.

  • Local conformation of serine residues in a silk model peptide, (Ala–Gly–Ser–Gly–Ala–Gly) 5 , studied with solid-state NMR:REDOR
    Polymer Journal, 2010
    Co-Authors: Yu Suzuki, Tetsuo Asakura
    Abstract:

    Local conformation of serine residues in a silk model peptide, (Ala–Gly–Ser–Gly–Ala–Gly) 5 , studied with solid-state NMR:REDOR

  • Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)_10 using solid-state NMR methods
    Journal of Biomolecular NMR, 2003
    Co-Authors: Jun Ashida, Kosuke Ohgo, Kohei Komatsu, Ayumi Kubota, Tetsuo Asakura
    Abstract:

    Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), ^13C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of ^13C=O isotope-labeled (Ala-Gly-Gly)_10. The torsion angles were determined to be (φ, ψ) = (−90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 3_1 helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3_1 helix of (Ala-Gly-Gly)_10 does not change by formic acid treatment although (Ala-Gly)_15 easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3_1 helix conformation of (Ala-Gly-Gly)_10 is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)_10 were also determined as (φ, ψ) = (−90°, 150° ) and this peptide is also considered to take 3_1 helix conformation.

  • the role of irregular unit gaas on the secondary structure of bombyx mori silk fibroin studied with 13c cp mas nmr and wide angle x ray scattering
    Protein Science, 2002
    Co-Authors: Tetsuo Asakura, Rena Sugino, Tatsushi Okumura, Yasumoto Nakazawa
    Abstract:

    Bombyx mori silk fibroin is a fibrous protein whose fiber is extremely strong and tough, although it is produced by the silkworm at room temperature and from an aqueous solution. The primary structure is mainly Ala-Gly alternative copolypeptide, but Gly-Ala-Ala-Ser units appear frequently and periodically. Thus, this study aims at elucidating the role of such Gly-Ala-Ala-Ser units on the secondary structure. The sequential model peptides containing Gly-Ala-Ala-Ser units selected from the primary structure of B. mori silk fibroin were synthesized, and their secondary structure was studied with 13C CP/MAS NMR and wide-angle X-ray scattering. The 13C isotope labeling of the peptides and the 13C conformation-dependent chemical shifts were used for the purpose. The Ala-Ala units take antiparallel β-sheet structure locally, and the introduction of one Ala-Ala unit in (Ala-Gly)15 chain promotes dramatical structural changes from silk I (repeated β-turn type II structure) to silk II (antiparallel β-sheet structure). Thus, the presence of Ala-Ala units in B. mori silk fibroin chain will be one of the inducing factors of the structural transition for silk fiber formation. The role of Tyr residue in the peptide chain was also studied and clarified to induce "locally nonordered structure."

  • Chain-folded lamellar structure and dynamics of the crystalline fraction of Bombyx mori silk fibroin and of (Ala-Gly-Ser-Gly-Ala-Gly)n model peptides.
    International Journal of Biological Macromolecules, 1
    Co-Authors: Tetsuo Asakura, Tatsuya Ogawa, Akira Naito, Michael P Williamson
    Abstract:

    Abstract Solid-state NMR is a powerful analytical technique to determine the composite structure of Bombyx mori silk fibroin (SF). In our previous paper, we proposed a lamellar structure for Ala-Gly copolypeptides as a model of the crystalline fraction in Silk II. In this paper, the structure and dynamics of the crystalline fraction and of a better mimic of the crystalline fraction, (Ala-Gly-Ser-Gly-Ala-Gly)n (n = 2–5, 8), and 13C selectively labeled [3-13C]Ala-(AGSGAG)5 in Silk II forms, were studied using structural and dynamical analyses of the Ala Cβ peaks in 13C cross polarization/ magic angle spinning NMR and 13C solid-state spin-lattice relaxation time (T1) measurements, respectively. Like Ala-Gly copolypeptides, these materials have lamellar structures with two kinds of Ala residues in β-sheet, A and B, plus one distorted β-turn, t, formed by repetitive folding using β-turns every eighth amino acid in an antipolar arrangement. However, because of the presence of Ser residues at every sixth residue in (AGSGAG)n, the T1 values and mobilities of B decreased significantly. We conclude that the Ser hydroxyls hydrogen bond to adjacent lamellar layers and fix them together in a similar way to Velcro®.

Yasumoto Nakazawa - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of a Ca binding-amphipathic silk-like protein and peptide with the sequence (Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4 with potential for bone repair
    Soft Matter, 2012
    Co-Authors: Aya Nagano, Hirohiko Sato, Yumi Tanioka, Yasumoto Nakazawa, David W. Knight, Tetsuo Asakura
    Abstract:

    Bombyx mori silk fibroin with the main sequence (Ala-Gly-Ser-Gly-Ala-Gly)n is a promising scaffold for bone regeneration not only on account of its excellent mechanical property as a structural matrix, but also for its slow biodegradability with adequate control of hydroxyapatite (HAP) deposition. Seeking to develop a material that might stimulate bone regeneration, we prepared a recombinant calcium binding-amphipathic silk-like protein [(Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4]4 by expression in E. coli. We also prepared the homologous peptide (Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4 by solid phase synthesis. The poly-L-glutamic acid was introduced into both protein and peptide because this sequence is involved in HAP-nucleating domains of bone sialoprotein. The recombinant protein was shown to bind relatively large quantities of Ca2+ ions in solution by a spectrophotometric assay and in the solid state by X-ray photoelectron spectroscopy. Changes in the electronic structure and local conformation of the peptide resulting from Ca2+ binding were studied using 13C solution NMR, especially 13C chemical shifts. We obtained evidence that Ca2+ bound to the poly-L-glutamic acid domains but not to the predominantly hydrophobic (Ala-Gly-Ser-Gly-Ala-Gly)4 domains. A remarkable conformational change induced by adsorption of the synthetic peptide on the HAP surface was also demonstrated using 13C solid state NMR.

  • the role of irregular unit gaas on the secondary structure of bombyx mori silk fibroin studied with 13c cp mas nmr and wide angle x ray scattering
    Protein Science, 2002
    Co-Authors: Tetsuo Asakura, Rena Sugino, Tatsushi Okumura, Yasumoto Nakazawa
    Abstract:

    Bombyx mori silk fibroin is a fibrous protein whose fiber is extremely strong and tough, although it is produced by the silkworm at room temperature and from an aqueous solution. The primary structure is mainly Ala-Gly alternative copolypeptide, but Gly-Ala-Ala-Ser units appear frequently and periodically. Thus, this study aims at elucidating the role of such Gly-Ala-Ala-Ser units on the secondary structure. The sequential model peptides containing Gly-Ala-Ala-Ser units selected from the primary structure of B. mori silk fibroin were synthesized, and their secondary structure was studied with 13C CP/MAS NMR and wide-angle X-ray scattering. The 13C isotope labeling of the peptides and the 13C conformation-dependent chemical shifts were used for the purpose. The Ala-Ala units take antiparallel β-sheet structure locally, and the introduction of one Ala-Ala unit in (Ala-Gly)15 chain promotes dramatical structural changes from silk I (repeated β-turn type II structure) to silk II (antiparallel β-sheet structure). Thus, the presence of Ala-Ala units in B. mori silk fibroin chain will be one of the inducing factors of the structural transition for silk fiber formation. The role of Tyr residue in the peptide chain was also studied and clarified to induce "locally nonordered structure."

Çetin Tasseven - One of the best experts on this subject based on the ideXlab platform.

Wolfgang Hohne - One of the best experts on this subject based on the ideXlab platform.

  • amino acid sequence of the small cyanogen bromide peptide of thermitase a thermostable serine proteinase from thermoactinomyces vulgaris relation to the subtilisins
    International Journal of Peptide and Protein Research, 2009
    Co-Authors: Miroslav Baudys, V Kostka, Karel Gruner, Gert Hausdorf, Wolfgang Hohne
    Abstract:

    The following amino acid sequence of the small cyanogen bromide peptide (mol. wt. 5399) of thermitase from Thermoactinomyces vulgaris has been determined: Ala-Thr-Pro-His-Val-Ala-Gly-Val-Ala-Gly-Leu-Leu-Ala-Ser-Gln-Gly-Arg-Ser-Ala-Ser-Asn-Ile-Arg-Ala-Ala-Ile-Glu-Asn-Thr-Ala-Asp-Lys-Ile-Ser-Gly-Thr-Gly-Thr-Tyr-Trp-Ala-Lys-Gly-Arg-Val-Asn-Ala-Tyr-Lys-Ala-Val-Gln-Tyr. The results obtained support the classification of the enzyme as a serine proteinase of the subtilisin type as proposed in a previous paper (1). This partial sequence extending from the serine residue involved in the active site to the C-terminal amino acid of the enzyme shows a 40% homology with the corresponding part of the subtilisin BPN' or subtilisin Carlsberg molecule but a 56% homology as regards conservative amino acid replacements. The secondary structure of this polypeptide fragment, predicted from the data obtained by the method of Chou & Fasman (2) agrees fairly well, within the limit of error of the method, with the structure of the corresponding part of the subtilisin BPN' molecule. Therefore, as expected, no dramatic changes in the spatial structure appear to account for the higher thermostability of thermitase, at least in this area of the polypeptide chain.

  • amino acid sequence of the tryptic sh peptide of thermitase a thermostable serine proteinase from thermoactinomyces vulgaris relation to the subtilisins
    International Journal of Peptide and Protein Research, 2009
    Co-Authors: Miroslav Baudys, V Kostka, Gert Hausdorf, Siegfried Fittkau, Wolfgang Hohne
    Abstract:

    The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val - Val - Gly - Gly - Trp - Asp - Phe - Val-Asp-Asn-Asp-Ser-Thr-Pro-Gln-Asn - Gly - Asn - Gly -His64- Gly - Thr - His -Cys68 - Ala-Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn - Asn - Ser - Thr - Gly - Ile - Ala - Gly - Thr - Ala - Pro - Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-14CH2-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.

Miroslav Baudys - One of the best experts on this subject based on the ideXlab platform.

  • amino acid sequence of the small cyanogen bromide peptide of thermitase a thermostable serine proteinase from thermoactinomyces vulgaris relation to the subtilisins
    International Journal of Peptide and Protein Research, 2009
    Co-Authors: Miroslav Baudys, V Kostka, Karel Gruner, Gert Hausdorf, Wolfgang Hohne
    Abstract:

    The following amino acid sequence of the small cyanogen bromide peptide (mol. wt. 5399) of thermitase from Thermoactinomyces vulgaris has been determined: Ala-Thr-Pro-His-Val-Ala-Gly-Val-Ala-Gly-Leu-Leu-Ala-Ser-Gln-Gly-Arg-Ser-Ala-Ser-Asn-Ile-Arg-Ala-Ala-Ile-Glu-Asn-Thr-Ala-Asp-Lys-Ile-Ser-Gly-Thr-Gly-Thr-Tyr-Trp-Ala-Lys-Gly-Arg-Val-Asn-Ala-Tyr-Lys-Ala-Val-Gln-Tyr. The results obtained support the classification of the enzyme as a serine proteinase of the subtilisin type as proposed in a previous paper (1). This partial sequence extending from the serine residue involved in the active site to the C-terminal amino acid of the enzyme shows a 40% homology with the corresponding part of the subtilisin BPN' or subtilisin Carlsberg molecule but a 56% homology as regards conservative amino acid replacements. The secondary structure of this polypeptide fragment, predicted from the data obtained by the method of Chou & Fasman (2) agrees fairly well, within the limit of error of the method, with the structure of the corresponding part of the subtilisin BPN' molecule. Therefore, as expected, no dramatic changes in the spatial structure appear to account for the higher thermostability of thermitase, at least in this area of the polypeptide chain.

  • amino acid sequence of the tryptic sh peptide of thermitase a thermostable serine proteinase from thermoactinomyces vulgaris relation to the subtilisins
    International Journal of Peptide and Protein Research, 2009
    Co-Authors: Miroslav Baudys, V Kostka, Gert Hausdorf, Siegfried Fittkau, Wolfgang Hohne
    Abstract:

    The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val - Val - Gly - Gly - Trp - Asp - Phe - Val-Asp-Asn-Asp-Ser-Thr-Pro-Gln-Asn - Gly - Asn - Gly -His64- Gly - Thr - His -Cys68 - Ala-Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn - Asn - Ser - Thr - Gly - Ile - Ala - Gly - Thr - Ala - Pro - Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-14CH2-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.