Conformational and orientational transformation of silk proteins in the major ampullate gland of Nephila clavipes spiders

The orientational and conformational transformation of the native liquid silk into a solid fiber in the major ampullate gland of the spider Nephila clavipes has been studied by Raman spectromicroscopy. The spectra show that the conformation of silk proteins in the glandular sac contains several secondary structure elements, which is consistent with intrinsically unfolded proteins. A few alpha-helices are also present and involve some alanine residues located in the polyalanine segments of the spidroin sequence. The conversion of the silk solution in the major ampullate gland appears to be a two-state process without intermediate states. In the first and second limbs of the duct, silk is isotropic and spidroins are generally native-like. beta-Sheets start to develop between the second and the third limb of the duct, suggesting that early beta-sheets are generated by shear forces. However, most of the beta-sheets are formed between the draw down taper and the valve. The early beta-sheets formed upward of the draw down taper might play the role of nucleation sites for the subsequent beta-sheet aggregation. The alignment of the polypeptides chains occurs near the valve, revealing that orientational and conformational changes do not occur simultaneously. Extensional flow seems to be the driving force to produce the orientational order, which in turn is associated with the formation of the major part of the beta-sheets. The slow evolution of the spidroin conformation up to the draw down taper followed by the rapid transformation between the drawn down taper and the valve may be important to achieve the optimal structure of the final fiber.     

Secondary structures and conformational changes in flagelliform, cylindrical, major, and minor ampullate silk proteins. Temperature and concentration effects

Orb weaver spiders use exceptionally complex spinning processes to transform soluble silk proteins into solid fibers with specific functions and mechanical properties. In this study, to understand the nature of this transformation we investigated the structural changes of the soluble silk proteins from the major ampullate gland (web radial threads and spider safety line); flagelliform gland (web sticky spiral threads); minor ampullate gland (web auxiliary spiral threads); and cylindrical gland (egg sac silk). Using circular dichroism, we elucidated (i) the different structures and folds for the various silk proteins; (ii) irreversible temperature-induced transitions of the various silk structures toward beta-sheet-rich final states; and (iii) the role of protein concentration in silk storage and transport. We discuss the implication of these results in the spinning process and a possible mechanism for temperature-induced beta-sheet formation.     

Conformational polymorphism, stability and aggregation in spider dragline silks proteins

Spider silk is spun in a complex and unique process, thought to depend on a hydrophobic conversion of a predominantly disordered to a beta-sheet rich protein structures. To test this hypothesis we monitored the effect of cationic (DOTAC) and anionic (alkyl sulfate) detergents and of (ii) solvent polarity using a series of alcohols on the secondary structure transition in dilute solutions of native spidroin. Our results showed that the detergents hydrophilic head charge and hydrophobic tail length cooperatively induced either a transition to the beta-sheet rich form or a stable helical state. Changing the solvent polarity showed that HFIP and TFE induced formation of stable helical forms whereas MeOH, EtOH and IsoP induced a kinetically driven formation of beta-sheet rich structure.     

Macroscopic fibers self-assembled from recombinant miniature spider silk proteins

Strength, elasticity, and biocompatibility make spider silk an attractive resource for the production of artificial biomaterials. Spider silk proteins, spidroins, contain hundreds of repeated poly alanine/glycine-rich blocks and are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider silk fibers have included solubilization steps in nonphysiological solvents. It is here demonstrated that a miniature spidroin from a protein in dragline silk of Euprosthenops australis can be produced in a soluble form in Escherichia coli when fused to a highly soluble protein partner. Although this miniature spidroin contains only four poly alanine/glycine-rich blocks followed by a C-terminal non-repetitive domain, meter-long fibers are spontaneously formed after proteolytic release of the fusion partner. The structure of the fibers is similar to that of dragline silks, and although self-assembled from recombinant proteins they are as strong as fibers spun from redissolved silk. Moreover, the fibers appear to be biocompatible because human tissue culture cells can grow on and attach to the fibers. These findings enable controlled production of high-performance biofibers at large scale under physiological conditions.     

Synthesis and characterization of multiblock copolymers based on spider dragline silk proteins

Spider dragline silk with its superlative tensile properties provides an ideal system to study the relationship between morphology and mechanical properties of a structural protein. Accordingly, we synthesized two hybrid multiblock copolymers by condensing poly(alanine) [(Ala)(5)] blocks of the structural proteins (spidroin MaSp1 and MaSp2) of spider dragline silk with different oligomers of isoprene (2200 and 5000 Da) having reactive end groups. The synthetic multiblock polymer displayed similar secondary structure to that of natural spidroin, the peptide segment forming a beta-sheet structure. These multiblock polymers showed a significant solubility in the component solvents. Moreover, the copolymer which contains the short polyisoprene segment would aggregate into a micellar-like structure, as observed by TEM.     

Untangling spider silk evolution with spidroin terminal domains

Background  

Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. Due to the highly variable nature of their repetitive sequences, spidroin evolutionary relationships have principally been determined from their non-repetitive carboxy (C)-terminal domains, though they offer limited character data. The few known spidroin amino (N)-terminal domains have been difficult to obtain, but potentially contain critical phylogenetic information for reconstructing the diversification of spider silks. Here we used silk gland expression data (ESTs) from highly divergent species to evaluate the functional significance and phylogenetic utility of spidroin N-terminal domains.     

Araneoid egg case silk: a fibroin with novel ensemble repeat units from the black widow spider, Latrodectus hesperus

Araneoid spiders use specialized abdominal glands to manufacture up to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences that encode egg case fibers (cover silk for the egg case sac) and the secondary structure of these threads have not been previously determined. In this study, MALDI tandem TOF mass spectrometry (MS/MS) and reverse genetics were used to isolate the first egg case fibroin, named tubuliform spidroin 1 (TuSp1), from the black widow spider, Latrodectus hesperus. Real-time quantitative PCR analysis demonstrates TuSp1 is selectively expressed in the tubuliform gland. Analysis of the amino acid composition of raw egg case silk closely aligns with the predicted amino acid composition from the primary sequence of TuSp1, which supports the assertion that TuSp1 represents a major component of egg case fibers. TuSp1 is composed of highly homogeneous repeats that are 184 amino acids in length. The long stretches of polyalanine and glycine-alanine subrepeats, which account for the crystalline regions of minor ampullate and major ampullate fibers, are very poorly represented in TuSp1. However, polyserine blocks and short polyalanine stretches were highly iterated within the primary sequence, and (13)C NMR spectroscopy demonstrated that the majority of alanine was found in a beta-sheet structure in post-spun egg case silk. The TuSp1 repeat unit does not display substantial sequence similarity to any previously described fibroin genes or proteins, suggesting that TuSp1 is a highly divergent member of the spider silk gene family.     

Spidroin N-terminal domain promotes a pH-dependent association of silk proteins during self-assembly

Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD.     

13C NMR of Nephila clavipes major ampullate silk gland.

The major ampullate glands of the spider Nephila clavipes contain approximately 0.2 microliter each of a highly concentrated (approximately 50%) solution of silk fibroin. Therefore, the reservoir of silk in these glands presents an ideal opportunity to observe prefolded conformations of a protein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by 13C NMR spectroscopy. These results were compared to those from silk protein first drawn from the spinneret and then denatured. The 13C NMR chemical shifts, along with infrared and circular dichroism data, suggest that the silk fibroin in the glands exists in dynamically averaged helical conformations. Furthermore, there is no evidence of proline residues in U-(13)C-D-glucose-labeled silk. This transient prefolded "molten fibril" state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of the final beta-sheet structure in the ampullate gland silk fibroin before final silk processing. However, the conformation of silk in the glands appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecting the ampullate glands to the spinnerets play a larger role in silk processing than previously thought.     

Structural conformation of spidroin in solution: a synchrotron radiation circular dichroism study

Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal's spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins' amino acid sequences as well as the gland's environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems.     

pH-Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain

Formation of spider silk from its constituent proteins—spidroins—involves changes from soluble helical/coil conformations to insoluble β-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild‐type (wt) E. australis NT at pH 7.2 and 300 mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5–6 Å into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH 7 and pH 6 and indicate that the biological function of NT depends on conversion between the two conformations.     

Molecular characterization and evolutionary study of spider tubuliform (eggcase) silk protein

As a result of hundreds of millions of years of evolution, orb-web-weaving spiders have developed the use of seven different silks produced by different abdominal glands for various functions. Tubuliform silk (eggcase silk) is unique among these spider silks due to its high serine and very low glycine content. In addition, tubuliform silk is the only silk produced just during a short period of time, the reproductive season, in the spider's life. To understand the molecular characteristics of the proteins composing this silk, we constructed tubuliform-gland-specific cDNA libraries from three different spider families, Nephila clavipes, Argiope aurantia, and Araneus gemmoides. Sequencing of tubuliform silk cDNAs reveals the repetitive architecture of its coding sequence and novel amino acid motifs. The inferred protein, tubuliform spidroin 1 (TuSp1), contains highly homogenized repeats in all three spiders. Amino acid composition comparison of the predicted tubuliform silk protein sequence to tubuliform silk indicates that TuSp1 is the major component of tubuliform silk. Repeat unit alignment of TuSp1 among three spider species shows high sequence conservation among tubuliform silk protein orthologue groups. Sequence comparison among TuSp1 repetitive units within species suggests intragenic concerted evolution, presumably through gene conversion and unequal crossover events. Comparative analysis demonstrates that TuSp1 represents a new orthologue in the spider silk gene family.     

The dimerization mechanism of the N-terminal domain of spider silk proteins is conserved despite extensive sequence divergence

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.     

Characterization and expression of a cDNA encoding a tubuliform silk protein of the golden web spider Nephila antipodiana

Spider silks are renowned for their excellent mechanical properties. Although several spider fibroin genes, mainly from dragline and capture silks, have been identified, there are still many members in the spider fibroin gene family remain uncharacterized. In this study, a novel silk cDNA clone from the golden web spider Nephila antipodiana was isolated. It is serine rich and contains two almost identical fragments with one varied gap region and one conserved spider fibroin-like C-terminal domain. Both in situ hybridization and immunoblot analyses have shown that it is specifically expressed in the tubuliform gland. Thus, it likely encodes the silk fibroin from the tubuliform gland, which supplies the main component of the inner egg case. Unlike other silk proteins, the protein encoded by the novel cDNA in water solution exhibits the characteristic of an alpha-helical protein, which implies the distinct property of the egg case silk, though the fiber of tubuliform silk is mainly composed of beta-sheet structure. Its sequence information facilitates elucidation of the evolutionary history of the araneoid fibroin genes.     

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all ¹H, ¹³C, and ¹⁵N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.     

Spider silk protein refolding is controlled by changing pH

Spidroins, the major silk proteins making up the spider's dragline silk, originate in two distinct tissue layers (A and B) in the spider's major ampullate gland. Formation of the complex thread from spidroins occurs in the lumen of the duct connected to the gland. Using pH-sensitive microelectrode probes, we showed that the spidroins traveling through the gland and duct experience a monotonic decrease in pH from 7.2 to 6.3. In addition, circular dichroism spectroscopy of material extracted from the gland showed a structural refolding concomitant with position in the gland and post-extraction changes in pH. We demonstrate that lowering the pH in vitro causes a dramatic conformational change in the protein from the A zone, converting it irreversibly from a coil to a predominantly beta-sheet structure. Furthermore, amino acid analyses have indicated that there are at least two distinct, though similar, proteins secreted in the A and B zones suggesting a potential factor in the progressive acidification as well as a pH sensitivity of the folding of spidroins in the gland. Thus, we provide, for the first time, a quantitative map of the pH value and position correlated with molecular structural folding in the silk gland characterizing the crucial role that pH plays in spider silk formation.     

Expression and characterization of chimeric spidroins from flagelliform-aciniform repetitive modules

Spiders can produce up to seven different types of silks or glues with different mechanical properties. Of these, flagelliform (Flag) silk is the most elastic, and aciniform (AcSp1) silk is the toughest. To produce a chimeric spider silk (spidroin) Flag_R-AcSp1_R, we fused one repetitive module of flagelliform silk from Araneus ventricosus and one repetitive module of aciniform silk from Argiope trifasciata. The recombinant protein expressed in E. coli formed silk-like fibers by manual-drawing. CD analysis showed that the secondary structure of Flag_R-AcSp1_R spidroin remained stable during the gradual reduction of pH from 7.0 to 5.5. The spectrum of FTIR indicated that the secondary structure of Flag_R-AcSp1_R changed from α-helix to β-sheet. The conformation change of Flag_R-AcSp1_R was similar to other spidroins in the fiber formation process. SEM analysis revealed that the mean diameter of the fibers was around 1 ~ 2 μm, and the surface was smooth and uniform. The chimeric fibers exhibited superior toughness (~33.1 MJ/m³) and tensile strength (~261.4 MPa). This study provides new insight into design of chimeric spider silks with high mechanical properties.     

Containment of Extended Length Polymorphisms in Silk Proteins

The spider silk gene family to the current date has been developed by gene duplication and homogenization events as well as conservation of crucial sequence parts. These evolutionary processes have created an amazing diversity of silk types each associated with specific properties and functions. In addition, they have led to allelic and gene variants within a species as exemplified by the major ampullate spidroin 1 gene of Nephila clavipes. Due to limited numbers of individuals screened to date little is known about the extent of these heterogeneities and how they are finally manifested in the proteins. Using expanded sample sizes, we show that sequence variations expressed as deletions or insertions of tri-nucleotides lead to different sized and structured repetitive units throughout a silk protein. Moreover, major ampullate spidroins 1 can quite dramatically differ in their overall lengths; however, extreme variants do not spread widely in a spider population. This suggests that a certain size range stabilized by purifying selection is important for spidroin 1 gene integrity and protein function. More than one locus for spidroin 1 genes possibly exist within one individual genome, which are homogenized in size, are differentially expressed and give a spider a certain degree of adaptation on silk’s composition and properties. Such mechanisms are shared to a lesser extent by the second major ampullate spidroin gene.