Presence of β-Turn Structure in Recombinant Spider Silk Dissolved in Formic Acid Revealed with NMR

Spider dragline silk is a biopolymer with excellent mechanical properties. The development of recombinant spider silk protein (RSP)-based materials with these properties is desirable. Formic acid (FA) is a spinning solvent for regenerated Bombyx mori silk fiber with excellent mechanical properties. To use FA as a spinning solvent for RSP with the sequence of major ampullate spider silk protein from Araneus diadematus, we determined the conformation of RSP in FA using solution NMR to determine the role of FA as a spinning solvent. We assigned 1H, 13C, and 15N chemical shifts to 32-residue repetitive sequences, including polyAla and Gly-rich regions of RSP. Chemical shift evaluation revealed that RSP is in mainly random coil conformation with partially type II β-turn structure in the Gly-Pro-Gly-X motifs of the Gly-rich region in FA, which was confirmed by the 15N NOE data. In addition, formylation at the Ser OH groups occurred in FA. Furthermore, we evaluated the conformation of the as-cast film of RSP dissolved in FA using solid-state NMR and found that β-sheet structure was predominantly formed.

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Stably Express Spider Flagelliform Silk Protein in Bombyx Mori Cell Line by PiggyBac Transposon-Derived Vector

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.332-334.779 ◽

2011 ◽

Vol 332-334 ◽

pp. 779-782 ◽

Cited By ~ 1

Author(s):

Yuan Song Zhang ◽

Tian Fu Zhao ◽

Ai Chun Zhao ◽

Masao Nakagaki

Keyword(s):

Cell Line ◽

Blot Analysis ◽

Spider Silk ◽

Silk Protein ◽

Transgenic Silkworm ◽

Helper Plasmid ◽

Antibiotic Selection ◽

Spider Silk Protein ◽

Piggybac Vector ◽

Turn Structure

Silkworm BmN4 cells were transfected with the helper plasmid and the piggyBac vector( piggyBac-FLAG) in which was inserted with the spider flagelliform silk expression cassette. Via antibiotic selection, most cells showed stable DsRed-expression. Immuno blot analysis showed that flagelliform silk protein of spider was expressed stably in BmN4 cells. Circular dichroism spectra indicated the existence of β-turn structure in recombinant spider flagelliform protein. The present results suggested that transgenic silkworm directly secreting functional spider silk protein in cocoon by using piggyBac system is feasible.

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Mechanical Properties of Spider Silk for Use As a Biomaterial: Molecular Dynamics Investigations

Volume 3: Advanced Materials: Design, Processing, Characterization, and Applications ◽

10.1115/imece2020-23951 ◽

2020 ◽

Author(s):

Atul Rawal ◽

Kristen L. Rhinehardt ◽

Ram V. Mohan

Keyword(s):

Mechanical Properties ◽

Molecular Dynamics ◽

Spider Silk ◽

Complete Separation ◽

Silk Protein ◽

Silk Proteins ◽

Silk Fibers ◽

Spider Silk Protein ◽

Β Chain ◽

Smd Simulations

Abstract Even though silkworm are the most dominant type of silk fibers used for commercial applications, spider silk has a definitive role in biomedical applications due to its biocompatibility and excellent mechanical properties as biomaterials. In recent years, recombinant production of the silk proteins at a larger scale has found new interest. Spider silk composites with a combination of a variety of other biomaterials have also been used to improve properties such as bio-compatibility, mechanical strength and controlled degradation. [1] A major constituent of spider silk fibers, are spidroin proteins. These are made up of repetitive segments flanked by conserved non-repetitive domains. The fiber proteins consist of a light chain and a heavy chain that are connected via a single disulfide bond. [2] Present paper employed steered molecular dynamics (SMD) as the principal method of investigating the mechanical properties of these nanoscale spider silk protein 3LR2, with a residual count of 134 amino acids. [3]. SMD simulations were performed by pulling on β-chain of the protein in the x-direction, while holding the other fixed. The focus of this paper is to investigate the mechanical properties of the nanoscale spider silk proteins with lengths of about 4.5nm in a folded state, leading to understanding of their feasibility in bio-printing of a composite spider silk biomaterial with a blend of various other biomaterials such as collagen. An in-depth insight into the fraying and tensile deformation and structural properties of the spider silk proteins are of innovative significance for a multitude of biomedical engineering applications. A calculated Gibbs free energy value of 18.59 kCal/mol via umbrella sampling corresponds with a complete separation of a single chain from a spider silk protein in case of fraying. Force needed for complete separation of the chain from the spider silk protein is analyzed, and discussed in this paper. It is found that the protein molecule undergoes a tensile stretch at strain rates of ≅ 11.65. An elastic modulus of 20.136 GPa, calculated via simple SMD simulations by subjecting the silk β-chain to a tensile stretch is also presented.

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Conformational Transition of Regenerated Spider Silk in Water

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.796.107 ◽

2013 ◽

Vol 796 ◽

pp. 107-111

Author(s):

Ye Mei Zhang ◽

Zhi Juan Pan

Keyword(s):

Mechanical Properties ◽

High Performance ◽

Spider Silk ◽

Conformational Transition ◽

Ph Value ◽

Random Coil ◽

Spider Dragline Silk ◽

Turn Structure ◽

Α Helix ◽

Spider Silks

Spider silks have excellent mechanical properties, which can even compare with some high-performance synthetic materials. Although as reported, the impressive mechanical properties are closely related to the primary amino acid sequence, the conformation that molecular chains form is also an important determinant. In this paper, effects of solvent, pH value, temperature, centrifugation and concentrating on the secondary structure of regenerated Ornithoctonus huwenna spider dragline silk protein aqueous solution were investigated by circular dichroism. Spidroin solutions prepared from different LiBr solutions had a distinct combination of secondary structures. The increasing temperature and concentrating can promote the formation of β-sheet structure. While centrifugation was opposite, which elevate the content of β-turn structure. Circular dichroic spectra quantitatively verified an increased α-helix structure content but a decrease of random coil and β-turn structure content with the increasing of pH value.

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