Dragline Silk Of Nephila Clavipes Research Articles

Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate.

Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH `locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 Å resolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised `lock-and-trigger' mechanism for spider silk formation is presented.

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.

The conformational analysis of spider dragline silks is difficult because of the amorphous character of the silks. In this article, the fractions of several conformations were determined for three 47-mer peptides, (Glu)4(Ala)6GlyGly12Ala13Gly14GlnGlyGlyTyrGlyGlyLeuGlySerGlnGly25Ala26Gly27-ArgGlyGlyLeuGlyGlyGlnGly35Ala36Gly37(Ala)6(Glu)4, with three underlined 13C-labeled blocks using a 13C CP/MAS NMR method. The conformations of the 13C-labeled sites change significantly depending on the location of the labeled blocks when treated with trifluoroacetic acid, low pH, and freeze-drying. The conformations of Ala36 and Gly37 residues are strongly influenced by the specific conformation of the (Ala)6 sequence at the C-terminal side, but those of other residues, Ala13 and Gly14, and Ala 26 and Gly27, are basically not influenced by the conformations of (Ala)6. Through hydration of the β-sheet peptide, sharp peaks with random coil could be observed depending on the position of the residue, and this result could be interpreted via the change in the Ramachandran map obtained from the molecular dynamics simulation.

Influence of Water on Protein Transitions: Morphology and Secondary Structure

Fibrous protein secondary structural transitions are affected by bound water. A family of specially designed recombinant spider silk-like block copolymers with a gradient of block length, hydrophobilicity, and degree of crystallinity was biosynthesized and characterized to demonstrate the effect of water on the protein structural transitions. These proteins were inspired by the genetic sequences found in the dragline silk of Nephila clavipes, comprising an alanine-rich hydrophobic block, A, a glycine-rich hydrophilic block, B, and a C-terminus or a His-tag, H. Because the A-block is hydrophobic and the B-block is hydrophilic, the spider silk-like block copolymers behave as amphiphilic molecules and self-assemble into various structures in water solution. We employ time-resolved Fourier transform infrared (FTIR) spectroscopy to assign the origin of specific secondary structural transitions during heating. A transition from random coils to β-turns dominates during a lower temperature glass transition (of plasticized protein) mediated by the removal of bound water. Once the protein is in the dry solid state, further heating causes the now-dry protein to undergo the glass transition to the liquid state through conversion of α-helices into β-turns. The structural transitions during protein glass transitions are intrinsic to the amorphous region of protein and are hardly affected by protein hydrophobicity, block length, or crystallinity. The self-assembly morphology of the spider silk-like block copolymers, investigated by scanning electron microscopy, indicates that the large-scale morphology is stable during heating through both the lower and upper temperature glass transitions.

Influence of Water on Protein Transitions: Thermal Analysis

We have developed a methodology using advanced thermal analysis to characterize the role of water in a specially synthesized family of recombinant spider silk-like block copolymers. These proteins were inspired by the genetic sequences found in the dragline silk of Nephila clavipes, comprising an alanine-rich hydrophobic block, A; a glycine-rich hydrophilic block, B; and a C-terminus or a His-tag, H. This family of proteins serves as a model system in which the hydrophobicity is controlled by A and B block lengths, allowing systematic comparison of water effects within the family. Temperature-modulated differential scanning calorimetry and thermogravimetric analyses were employed to capture the glass to rubber transition, Tg, in water-cast protein films. Modeling of the solid and liquid state heat capacity baselines allows us to determine the critical role played by bound water which plasticizes and stabilizes the protein through interchain bonding. In samples containing bound water, two sequential glass transitions, Tg(1) and Tg(2), were observed during heating. The lower temperature glass transition, Tg(1), is related to conformational change induced by bound water removal, the hydrophobicity of the protein sequences, and the crystallinity of the protein. The higher temperature glass transition, Tg(2), is characteristic of the dry protein. The binding energy of water to protein compares favorably to ligand–water binding affinities. The energy absorbed by evaporating water depends upon the volume fraction of the hydrophilic B-block.

Mechanical and physical properties of recombinant spider silk films using organic and aqueous solvents.

Spider silk has exceptional mechanical and biocompatibility properties. The goal of this study was optimization of the mechanical properties of synthetic spider silk thin films made from synthetic forms of MaSp1 and MaSp2, which compose the dragline silk of Nephila clavipes. We increased the mechanical stress of MaSp1 and 2 films solubilized in both HFIP and water by adding glutaraldehyde and then stretching them in an alcohol based stretch bath. This resulted in stresses as high as 206 MPa and elongations up to 35%, which is 4× higher than the as-poured controls. Films were analyzed using NMR, XRD, and Raman, which showed that the secondary structure after solubilization and film formation in as-poured films is mainly a helical conformation. After the post-pour stretch in a methanol/water bath, the MaSp proteins in both the HFIP and water-based films formed aligned β-sheets similar to those in spider silk fibers.

13C solid‐state NMR study of the 13C‐labeled peptide, (E)8GGLGGQGAG(A)6GGAGQGGYGG as a model for the local structure of Nephila clavipes dragline silk (MaSp1) before and after spinning

We prepared the water soluble model peptide, (E)(8) GGLGGQGAG(A)(6) GGAGQGGYGG, to throw light on the local structure of spidroin 1 (MaSpl) protein in spider dragline silk of Nephila clavipes before and after spinning. Solution (13) C NMR showed that the conformation of the peptide in aqueous solution was essentially random coil. Solid-state NMR was used to follow conformation-dependent (13) C chemical shifts in (13) C selectively labeled versions of the peptide. The peptide lyophilized from an aqueous solution at neutral pH (hereafter referred to as "without acid treatment)"was used to mimic the state of the spidroin stored in the spider's silk gland while the peptide precipitated from the acidic solution ("with acid treatment") was used to simulate the role of acid treatment in inducing conformation change in the natural spinning process. In without acid treatment, the fraction of random coil conformation was lowest in the N-terminal region (residues 15-18) when compared with the C-terminus. The conformational change produced by the acid treatment occurred in the sequence, G(15) AG(A)(6) GGAG(27), interposed between pairs of Gly residues pairs, Gly(12,13), and Gly(29,30). The acid treated peptide showed a remarkable decrease in the fraction of random coil conformation from A(20) to A(23) in the poly-Ala region when compared with the peptide without acid treatment. These observations taken together suggest that the peptide can be used as a model for studying the localization of the conformation change in spider silk fibroin in the natural spinning and the role of acid treatment in this process.

Composition and Hierarchical Organisation of a Spider Silk

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.

The effect of genetically engineered spider silk-dentin matrix protein 1 chimeric protein on hydroxyapatite nucleation

Spider silks exhibit remarkable mechanical properties while dentin matrix protein 1 provides controlled nucleation and hydroxyapatite growth. In the present work, these two attributes were combined via genetic engineering to form a chimera, a clone encoding consensus repeats from the major protein in the spider dragline silk of Nephila clavipes fused to the carboxyl terminal domain of dentin matrix protein 1 (CDMP1). The objective was to exploit the self-assembly and material properties of silk proteins with controlled hydroxyapatite (HA) formation from CDMP1, for novel biomaterial composites. The purified recombinant protein retained native-silk like self-assembly properties and β-sheet structure when formed into films and treated with methanol. When the chimeric protein in solution was incubated with CaCl 2, the secondary structure shifted from random coil to α-helix and β-sheet, due to the interactions between the CDMP1 domain and Ca 2+. The control protein without the CDMP1 domain did not undergo a similar transition. Films formed from the recombinant protein were mineralized using simulated body fluids and induced the formation of calcium-deficient carbonated HA, Ca 10(PO 4) 6(OH) 2 based on SEM, EDS, FTIR and TEM analysis. This mineral phase was not formed on the films formed from the control spider silk protein without the CDMP1 domain. Considering the osteoconductivity of HA and the novel material features of spider silks, these new hybrid systems offer potential as biomaterials for a number of potential applications.

Structure of Characteristic Sequences in Nephila clavipes Dragline Silk (MaSp1) Studied with 13C Solid State NMR

Structure of Characteristic Sequences in Nephila clavipes Dragline Silk (MaSp1) Studied with 13C Solid State NMR

Production and purification of recombinant DP1B silk-like protein in plants

Spider dragline silk of Nephila clavipes consists of two highly repetitive proteins, spidroin 1 and spidroin 2. To develop a plant platform for production of recombinant silk-like protein, two plant-optimized DP1B genes were synthesized to mimic spidroin 1. DP1B-8P encodes for a 64 kD DP1B silk-like protein and DP1B-16P for a 127 kD DP1B silk-like protein. Both genes have been introduced into Arabidopsis for leaf-based production driven by the 35S promoter and for seed-specific production driven by the β-conglycinin α’ subunit promoter, respectively. They have also been expressed in somatic soybean embryos under the control of the β-conglycinin α’ subunit promoter. The results demonstrate the synthesis and accumulation of DP1B silk-like protein in leaves and seeds of Arabidopsis, as well as in somatic soybean embryos. They suggest that seeds are the more preferred tissue for DP1B production since they offer higher production yield and quality. In addition, a simplified protocol for purifying DP1B from plant tissue is discussed.

A 45 Å equatorial long period in dry dragline silk of Nephila clavipes

A small-angle peak has been found by X-ray scattering measurements on the dry dragline silk of Nephila clavipes. The weak peak, located on the equatorial axis, yields a long period of approximately 45Å. Probably it corresponds to the lateral spacing of the crystals and is determined kinetically during the formation of the nanofibers.

Investigation of the nanofibrillar morphology in silk fibers by small angle X-ray scattering and atomic force microscopy

Small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) measurements have been shown to be consistent with the presence of nanofibrils in the cocoon silk of Bombyx mori and the dragline silk of Nephila clavipes. The transverse dimensions and correlation lengths range from ⪢59 to 220 nm and in the axial direction from ⪢80 to 230 nm. Also, the two-dimensional Fourier transforms of the height profiles of AFM topographic images of interior surfaces of B. mori follow a power law approximately the same as that for the Porod region of the SAXS data. In this manner, the AFM can be used to help remove ambiguity about the scatterers responsible for SAXS patterns.

Fiber Morphology of Spider Silk: The Effects of Tensile Deformation

The fiber morphology of the dragline silk of Nephila clavipes has been investigated by the detailed analysis of wide-angle X-ray diffraction (WAXD) patterns. WAXD gives the crystal lattice dimensions, the orientation distribution, the crystalline fraction, and an estimate of the crystal size. It is found that the crystals are very small and well oriented. The mean (minimum) crystal dimensions are 2 × 5 × 7 nm, and the angle, φ, between the molecular chains in the crystals and the fiber axis has a full width at half-maximum (fwhm) of 15.7° and an orientation function f = 0.981. The X-ray crystallinity is in the range 10−15%, and the amorphous diffraction is divided 60:40 between an isotropic ring and an oriented halo with fwhm 30°. This means one-third of the material is in the oriented amorphous state, with a chain orientation of fwhm 43° and f = 0.87. When the fiber is extended up to 10%, the orientation of the crystals increases as predicted for affine deformation at constant volume. There is no observa...

Aspects of the Morphology of the Silk ofBombyx Mori

Abstract Atomic force microscopy was used to examine the exterior surface of as-spun cocoon silk, raw silk, and degummed silk as well as the interior surface of peeled degummed silk of Bombyx mori. The images yielded a broad variety of features with a wide range of dimensions. They are qualitatively similar to those of the dragline silk of Nephila clavipes. Examination of the surface roughness, the surface profiles, and the Fourier transforms of the profiles showed the surface of the degummed silk to be quantitatively similar to the silk of N. clavipes. The average roughness is 4 nm. The average dimensions of the features are approximately 100 to 150 (> 2000 in peeled samples) nm axially and 50 to 300 nm laterally. They appear to emerge from and to enter the surface, to vary in width, and to twist about one another in an irregular manner. In the peeled degummed silk they extend more regularly for long distances in approximately the axial direction. In some cases they appear to be oriented at different small angles with respect to the fiber axis in different layers. Taken together with other data, they suggest the presence of large fibrillar structures. The same suggestion also applies to similar structures in N. clavipes.

Solid-State 13C NMR of Nephila clavipes Dragline Silk Establishes Structure and Identity of Crystalline Regions

13 C CP/MAS NMR spectra of dragline silk of Nephila clavipes show unambiguously that the alanine residues are present in the β-sheet conformation. Proton T 1ρ data show that the heterogeneities in the silk must be small. Two motional environments have been detected for the alanine residues. These data verify that dragline silk is composed of crystalline regions of polyalanine β-sheets in a more flexible matrix

New internal structure of spider dragline silk revealed by atomic force microscopy

Atomic force microscopy was used to study the three-dimensional nanometer-scale structure of the dragline silk of Nephila clavipes from microtomed sections of the silk. Contrary to a previously proposed model of randomly distributed protein crystallites interspersed in amorphous regions, a highly organized skin-core structure of the fiber was observed. The skin appeared to be thin with no discernible distinct features. The core consists of pleated fibril-like structures, which are arranged in two concentric cylinders. Upon stretching, the pleats were smoothed out substantially. The mechanical properties of spider silk can quite straightforwardly be related to the newly observed structures.

Nephila Clavipes Dragline Silk: Approaches to a Recombinantly Produced Silk Protein

Spider silks exhibit an unusual combination of strength and toughness that distinguishes them from other natural and synthetic fibers. Silk proteins perform a key natural function as structural fibers, to absorb impact energy from flying insects without breaking. They dissipate energy over a broad area and balance stiffness, strength and extensibility (1,2). In addition to their unusual mechanical properties and visual lustre, silks also exhibit interesting interference patterns within the electromagnetic spectrum (3), unusual viscometric patterns related to processing (4), and piezoelectric properties (3,5,6). These properties suggest they would be good candidates for high performance fiber and composite applications. However, the spider is not capable of producing sufficient quantities of proteins to enable thorough evaluation of their potential. Consequently, we are pursuing recombinant DNA techniques to clone and express adequate quantities of recombinant spider silk for these studies.