Towards Understanding Silk Fiber Structure: 13C Solid State NMR Studies of the Packing Structures of Alanine Oligomers before and after Trifluoroacetic Acid Treatment.

Polyalanine (poly-A) sequences with tightly packed antiparallel β sheet (AP-β) structures are frequently observed in silk fibers and serve as a key contributor to the exceptionally high fiber tensile strength. In general, the poly-A sequence embedded in the amorphous glycine-rich regions has different lengths depending on the fiber type from spiders or wild silkworms. In this paper, the packing structures of AP-β alanine oligomers with different lengths were studied using 13C solid-state NMR as a model of the poly-A sequences. These included alanine oligomers with and without the protection groups (i.e., 9-fluorenylmethoxycarbonyl and polyethylene glycol groups at the N- and C-terminals, respectively). The fractions of the packing structures as well as the conformations were determined by deconvolution analyses of the methyl NMR peaks. Trifluoroacetic acid was used to promote the staggered packing structures, and the lineshapes changed significantly for oligomers without the protected groups but only slightly for oligomers with the protected groups. Through NMR analysis of the 3-13C singly labeled alanine heptamer and refined crystal structure of the staggered packing units, a possible mechanism of the staggered packing formation is proposed for the AP-β alanine heptamer.

[1]  A. Rawal,et al.  DNP NMR spectroscopy reveals new structures, residues and interactions in wild spider silks. , 2019, Chemical communications.

[2]  A. Jouraku,et al.  A study of the extraordinarily strong and tough silk produced by bagworms , 2019, Nature Communications.

[3]  T. Asakura,et al.  Changes in the Local Structure of Nephila clavipes Dragline Silk Model Peptides upon Trifluoroacetic Acid, Low pH, Freeze-Drying, and Hydration Treatments Studied by 13C Solid-State NMR. , 2018, Biomacromolecules.

[4]  A. Naito,et al.  Dynamics of Alanine Methyl Groups in Alanine Oligopeptides and Spider Dragline Silks with Different Packing Structures As Studied by 13C Solid-State NMR Relaxation , 2018, Macromolecules.

[5]  A. Naito,et al.  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. , 2018, The journal of physical chemistry. B.

[6]  A. V. D. Vaart,et al.  Uncovering the structure-function relationship in spider silk , 2018 .

[7]  Xungai Wang,et al.  Structural Comparison of Various Silkworm Silks: An Insight into the Structure-Property Relationship. , 2018, Biomacromolecules.

[8]  T. Asakura,et al.  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 , 2018 .

[9]  Junichi Sugahara,et al.  NMR Investigation about Heterogeneous Structure and Dynamics of Recombinant Spider Silk in the Dry and Hydrated States , 2017 .

[10]  A. Naito,et al.  Packing Arrangements and Intersheet Interaction of Alanine Oligopeptides As Revealed by Relaxation Parameters Obtained from High-Resolution 13C Solid-State NMR. , 2017, The journal of physical chemistry. B.

[11]  T. Asakura,et al.  Distinct solvent- and temperature-dependent packing arrangements of anti-parallel β-sheet polyalanines studied with solid-state 13C NMR and MD simulation. , 2017, Physical chemistry chemical physics : PCCP.

[12]  A. Naito,et al.  Packing arrangement of 13C selectively labeled sequence model peptides of Samia cynthia ricini silk fibroin fibers studied by solid-state NMR. , 2017, Physical chemistry chemical physics : PCCP.

[13]  A. Naito,et al.  Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations. , 2017, Biomacromolecules.

[14]  C. Glorieux,et al.  Investigation of synthetic spider silk crystallinity and alignment via electrothermal, pyroelectric, literature XRD, and tensile techniques. , 2017, Macromolecular materials and engineering.

[15]  Qing Meng,et al.  The correlation between the length of repetitive domain and mechanical properties of the recombinant flagelliform spidroin , 2017, Biology Open.

[16]  M. Burghammer,et al.  Nanoscale Structural Features in Major Ampullate Spider Silk. , 2017, Biomacromolecules.

[17]  A. Naito,et al.  Parallel β-Sheet Structure of Alanine Tetrapeptide in the Solid State As Studied by Solid-State NMR Spectroscopy. , 2016, The journal of physical chemistry. B.

[18]  M. Auger,et al.  Major Ampullate Spider Silk with Indistinguishable Spidroin Dope Conformations Leads to Different Fiber Molecular Structures , 2016, International journal of molecular sciences.

[19]  R. Reis,et al.  Fabrication and characterization of Eri silk fibers-based sponges for biomedical application. , 2016, Acta biomaterialia.

[20]  K. Numata,et al.  Crystal structure and physical properties of Antheraea yamamai silk fibers: Long poly(alanine) sequences are partially in the crystalline region , 2015 .

[21]  M. Elices,et al.  Persistence and variation in microstructural design during the evolution of spider silk , 2015, Scientific Reports.

[22]  C. Holland,et al.  Identification and classification of silks using infrared spectroscopy , 2015, Journal of Experimental Biology.

[23]  M. Williamson,et al.  Analysis of the Structure of Bombyx mori Silk Fibroin by NMR , 2015 .

[24]  J. Yarger,et al.  Molecular dynamics of spider dragline silk fiber investigated by 2H MAS NMR. , 2015, Biomacromolecules.

[25]  K. Nishimura,et al.  Intermolecular Packing in B. mori Silk Fibroin: Multinuclear NMR Study of the Model Peptide (Ala-Gly)15 Defines a Heterogeneous Antiparallel Antipolar Mode of Assembly in the Silk II Form , 2015 .

[26]  S. Kundu,et al.  Silk proteins for biomedical applications: Bioengineering perspectives , 2014 .

[27]  David L Kaplan,et al.  Structure-function-property-design interplay in biopolymers: spider silk. , 2013, Acta biomaterialia.

[28]  T. Asakura,et al.  The Silk I and Lamella Structures of (Ala-Gly)15 as the Model of Bombyx mori Silk Fibroin Studied with Solid State NMR , 2014 .

[29]  K. Nishimura,et al.  Difference in the structures of alanine tri- and tetra-peptides with antiparallel β-sheet assessed by X-ray diffraction, solid-state NMR and chemical shift calculations by GIPAW. , 2014, Biopolymers.

[30]  Tetsuo Asakura,et al.  Elucidating silk structure using solid-state NMR , 2013 .

[31]  Janelle E. Jenkins,et al.  Characterizing the secondary protein structure of black widow dragline silk using solid-state NMR and X-ray diffraction. , 2013, Biomacromolecules.

[32]  Joydip Kundu,et al.  An emerging functional natural silk biomaterial from the only domesticated non-mulberry silkworm Samia ricini. , 2013, Macromolecular bioscience.

[33]  T. Asakura,et al.  Silk structure studied with nuclear magnetic resonance. , 2013, Progress in nuclear magnetic resonance spectroscopy.

[34]  Manuel Elices,et al.  Sequential origin in the high performance properties of orb spider dragline silk , 2012, Scientific Reports.

[35]  M. Williamson,et al.  Two different packing arrangements of antiparallel polyalanine. , 2012, Angewandte Chemie.

[36]  Andrew M. Smith,et al.  Decoding the secrets of spider silk , 2011 .

[37]  Janelle E. Jenkins,et al.  Solid-state NMR evidence for elastin-like beta-turn structure in spider dragline silk. , 2010, Chemical communications.

[38]  Janelle E. Jenkins,et al.  Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks. , 2010, Biomacromolecules.

[39]  Janelle E. Jenkins,et al.  Determining secondary structure in spider dragline silk by carbon-carbon correlation solid-state NMR spectroscopy. , 2008, Journal of the American Chemical Society.

[40]  Janelle E. Jenkins,et al.  Solid-state NMR investigation of major and minor ampullate spider silk in the native and hydrated states. , 2008, Biomacromolecules.

[41]  Thierry Lefèvre,et al.  Protein secondary structure and orientation in silk as revealed by Raman spectromicroscopy. , 2007, Biophysical journal.

[42]  A. I. Kishore,et al.  Orientational order of Australian spider silks as determined by solid‐state NMR , 2006, Biopolymers.

[43]  T. Asakura,et al.  Structural analysis of alanine tripeptide with antiparallel and parallel beta-sheet structures in relation to the analysis of mixed beta-sheet structures in Samia cynthia ricini silk protein fiber using solid-state NMR spectroscopy. , 2006, Journal of the American Chemical Society.

[44]  Fritz Vollrath,et al.  Characterization of the protein components of Nephila clavipes dragline silk. , 2005, Biochemistry.

[45]  Thierry Lefèvre,et al.  Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy. , 2004, Biomacromolecules.

[46]  T. Asakura,et al.  Structure and structural changes of the silk fibroin from Samia cynthia ricini using nuclear magnetic resonance spectroscopy. , 2004, Macromolecular bioscience.

[47]  C. Michal,et al.  A DECODER NMR study of backbone orientation in Nephila clavipes dragline silk under varying strain and draw rate. , 2004, Biomacromolecules.

[48]  C. Michal,et al.  Strain Dependent Local Phase Transitions Observed during Controlled Supercontraction Reveal Mechanisms in Spider Silk , 2004 .

[49]  T. Asakura,et al.  Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods. , 2003, Journal of the American Chemical Society.

[50]  T. Asakura,et al.  Tightly winding structure of sequential model peptide for repeated helical region in Samia cynthia ricini silk fibroin studied with solid‐state NMR , 2003, Protein science : a publication of the Protein Society.

[51]  R. Young,et al.  Analysis of structure/property relationships in silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks using Raman spectroscopy , 2003 .

[52]  B. Meier,et al.  The molecular structure of spider dragline silk: Folding and orientation of the protein backbone , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[53]  T. Asakura,et al.  Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR. , 2002, Biochemistry.

[54]  A. Ghosh,et al.  Purification and characterization of fibroin from the tropical Saturniid silkworm, Antheraea mylitta. , 2001, Insect biochemistry and molecular biology.

[55]  A. I. Kishore,et al.  Solid-state NMR relaxation studies of Australian spider silks. , 2001, Biopolymers.

[56]  F Vollrath,et al.  X-ray diffraction on spider silk during controlled extrusion under a synchrotron radiation X-ray beam. , 2000, Biomacromolecules.

[57]  Hideki Sezutsu,et al.  Dynamic Rearrangement Within the Antheraea pernyi Silk Fibroin Gene Is Associated with Four Types of Repetitive Units , 2000, Journal of Molecular Evolution.

[58]  Oskar Liivak,et al.  Supercontraction and Backbone Dynamics in Spider Silk: 13C and 2H NMR Studies , 2000 .

[59]  Fritz Vollrath,et al.  In Situ X-ray Diffraction during Forced Silking of Spider Silk , 1999 .

[60]  Z. Shao,et al.  Analysis of spider silk in native and supercontracted states using Raman spectroscopy , 1999 .

[61]  L. Jelinski,et al.  Rotational-echo Double-resonance in Complex Biopolymers: a Study of Nephila Clavipes Dragline Silk , 1998, Journal of biomolecular NMR.

[62]  R. Lewis,et al.  Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non‐silk‐like “spacer regions” , 1998, Protein science : a publication of the Protein Society.

[63]  David T. Grubb,et al.  Fiber Morphology of Spider Silk: The Effects of Tensile Deformation , 1997 .

[64]  M. Williamson,et al.  NMR study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C-NMR chemical shift contour plots , 1997 .

[65]  B. Meier,et al.  Local Structure in Spider Dragline Silk Investigated by Two-Dimensional Spin-Diffusion Nuclear Magnetic Resonance† , 1996 .

[66]  L W Jelinski,et al.  Molecular Orientation and Two-Component Nature of the Crystalline Fraction of Spider Dragline Silk , 1996, Science.

[67]  Y. Termonia.  Molecular modeling of spider silk elasticity , 1994 .

[68]  L. Jelinski,et al.  Solid-State 13C NMR of Nephila clavipes Dragline Silk Establishes Structure and Identity of Crystalline Regions , 1994 .

[69]  M B Hinman,et al.  Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. , 1992, The Journal of biological chemistry.

[70]  A. Camerman,et al.  L‐Alanyl‐L‐alanyl‐L‐alanine: Parallel pleated sheet arrangement in unhydrated crystal structure, and comparisons with the antiparallel sheet structure , 1991, Biopolymers.

[71]  R. Lewis,et al.  Structure of a protein superfiber: spider dragline silk. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[72]  M. Denny,et al.  The structure and properties of spider silk , 1986 .

[73]  D. Torchia.  The measurement of proton-enhanced carbon-13 T1 values by a method which suppresses artifacts , 1978 .

[74]  A. Camerman,et al.  The structure of the tripeptide l-alanyl-l-alanyl-l-alanine , 1975 .

[75]  G. Han,et al.  9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group , 1970 .

[76]  A Elliott,et al.  Structure of beta-poly-L-alanine: refined atomic co-ordinates for an anti-parallel beta-pleated sheet. , 1967, Journal of molecular biology.

[77]  S. G. Smith,et al.  Comparative studies of fibroins. I. The amino acid composition of various fibroins and its significance in relation to their crystal structure and taxonomy. , 1960, Journal of molecular biology.