When is silk repeated

Therefore, herein, further supercontraction—stretching treatment was performed up to three cycles, and the corresponding structural changes were investigated. Also, with the increase in cycle of supercontraction—stretching treatments, a Finally, although the structure of spider dragline silk became increasingly regular with the rise in supercontraction—stretching cycles, mechanical properties remained constant after every cycle of the supercontraction—stretching treatment.

These findings can aid in further understanding the structural changes that are related to the supercontraction of spider dragline silk and provide useful guidance in fabrication of high-performance regenerated or artificial silk fibers. Such files may be downloaded by article for research use if there is a public use license linked to the relevant article, that license may permit other uses. More by Linli Hu. More by Qianying Chen. More by Jinrong Yao. Argiope spiders are not known to be polyploid, thus multiple gene copies per genome is the only explanation for more than two N- or C- terminal region variants in a single individual.

For example, we found seven C-terminal variants in one A. Each A. Likewise, an A. The smaller number of C-terminal variants could be explained by lack of variation in the C-terminal region or by incomplete sampling of variants by PCR survey. ML analysis of sequences from the PCR survey shows that the branch lengths in the repetitive region Figure 4 A are shorter than the branch lengths of the terminal region trees Figure 4 B, C. The majority of N- and C-terminal variants cluster into well-supported, species-specific groups, and intra-specific branch lengths are very short compared to inter-specific branch lengths Figure 4 B, C.

One exception is A. Given the weak clade support, this variant is probably an outlier that is not as homogenized as the other A. The shorter branch lengths of the repetitive region variants tree compared to those of the N- and C-terminal region trees suggest that the repetitive region is the most conserved araneid AcSp1 region Figure 4. Thus concerted evolution and purifying selection both must play a role in the near-perfect homogeneity of Argiope AcSp1 iterated repeats.

Recombination can also affect tests of selection [ 35 ]. Because we could not conclusively determine the exact number of loci within an individual or assign alleles to specific loci, we were unable to ascertain recombination between loci.

Previous work with AcSp1 sequences did not find evidence for multiple loci [ 1 , 19 ]. The lack of variation among A. Alternatively, consistent depletion of aciniform silk may be required to stimulate transcription of multiple AcSp1 loci.

This hypothesis is supported by a significant increase in aciniform-silk dependent web-decorating behavior in three species of Argiope in response to a two-week period of aciniform silk depletion [ 4 ]. Future work could focus on comparing the number of AcSp1 variants expressed by spiders consistently depleted of aciniform silk versus that from spiders that are not depleted.

Survey of individual L. However, the detection of multiple AcSp1 loci in Argiope but not Latrodectus is consistent with the hypothesis that Argiope spiders maintain multiple gene copies as a strategy for efficiently producing large amounts of protein. In contrast with Argiope spiders, Latrodectus spiders use markedly fewer strands of aciniform silk during prey capture [ 3 ] and do not make stabilimenta.

Increased AcSp1 copy number in Argiope spiders may therefore be a strategy for increasing protein production [ 36 ]. Because spidroins are costly, highly expressed proteins [ 37 , 38 ], resource abundance in the form of prey availability may also stimulate aciniform spidroin production in Argiope to prepare for resource scarcity [ 39 , 40 ].

Precedent for this strategy exists. In the bacteria Escherichia coli , multiple copies of rRNA operons provide a competitive advantage by enabling increased growth rates and decreased cell division lag time in environments where resources fluctuate rapidly [ 41 , 42 ]. Previous research has found variants for other spidroins [ 43 — 46 ], and that the dragline spidroin MaSp1 is encoded by multiple loci in several species [ 47 , 48 ].

Unlike A. This difference could indicate functional constraints on the C-terminal coding region of MaSp1 that either differ from or are not acting on AcSp1.

A comparison of structural component predictions from the amino acid sequences of terminal regions across different spidroins would greatly inform our understanding of the contribution of the terminal regions to the evolution of different spider silk types.

In addition, several terminal region variants per individual Argiope genome indicate multiple Argiope AcSp1 loci. Across AcSp1 loci within an individual, we found homogenization of the repetitive region, but variation at the terminal coding regions. We also found evidence for stronger purifying selection in the N-terminal region versus the repetitive or C-terminal region, suggesting that the N-terminal region is the most constrained portion of the aciniform spidroin.

The maintenance of multiple copies of AcSp1 in Argiope genomes underscores the importance of aciniform silk in Argiope ecology and evolution. Indeed, variation between individuals and multiple gene copies within individuals could provide a method for the rapid synthesis of aciniform silk in this genus, and may represent the early stages of the differentiation that led to the extraordinary sequence and functional diversity of spider silks.

The 17 kb fragment was gel purified with the S. Seven plasmid clones with the expected insert size and restriction enzyme digest patterns were end-sequenced with M13 and Sp6 primers to identify orientation of the inserts. Two clones one of each insert orientation were triple-digested with SpeI, XbaI, and XhoI and the fragments were gel-purified. The two clones were also single-digested with BamHI and the largest fragment from each digest 5. The triple digest produced a End-sequencing the subclones revealed that the 2.

The 2. Because the Instead, the The complete contig of the 17 kb genomic fragment was manually assembled with Sequencher 4. Genomic DNA was extracted from single legs removed from four A. Products were directly sequenced. If a chromatograph had overlapping peaks, indicative of heterogeneous amplification, then the product was ligated into pJET 1.

Inserts of the expected size were gel purified and sequenced. If one variant was highly abundant, then additional colonies were PCR amplified and digested with restriction enzymes to identify the abundant variants.

The remaining undigested PCR products containing the rare variant were purified and sequenced. Nucleotide sequences from the PCR fragments from each species were aligned as described below. For variant diagnosis, single nucleotide polymorphisms SNPs that were present in only one individual clone were attributed to Taq polymerase error and that SNP was ignored.

If a sequence had a pattern of polymorphism that was not present in at least one other clone, the sequence was discarded. Neighbor joining trees were then used to visualize highly similar sequences.

With the exception of the cluster for A. Clustered sequences were extracted and aligned to derive the majority rule consensus for that variant. Each variant is therefore supported by at least two sequences. The conserved spidroin N- and C-terminal regions from the complete A. The N- and C-terminal regions were separately aligned with default settings and the alignments were adjusted by eye.

The aa alignments dictated nucleotide alignments. N- and C-terminal encoding region alignments were concatenated for phylogenetic analyses of spidroin paralogs Additional file 1 : Figure S2. Despite potential recombination and convergence in the N- and C-terminal encoding regions, previous research found no conflict between the strongly supported nodes between separate N- and C-terminal trees and that concatenation of the terminal regions provides greater evolutionary resolution [ 12 ].

The 20 repeat units from the complete A. Alignments for the N- and C-terminal encoding sequences obtained from the PCR survey of individual genomes were created as above using diagnosed variants. For each nucleotide alignment, bootstrap and maximum likelihood ML searches for optimal trees were simultaneously conducted over 5, replicates using RAxML 7. Accession numbers given in Additional file 2 : Tables S2—S4. CodonTest [ 54 ] implemented through the Datamonkey webserver [ 55 , 56 ] indicated the Felsenstein F81 [ 57 ] model of codon substitution as the best-fit for all analyzed datasets.

Alignments used in ML analyses are available shown in the additional files. Hayashi CY, Blackledge TA, Lewis RV: Molecular and mechanical characterization of aciniform silk: uniformity of iterated sequence modules in a novel member of the spider silk fibroin gene family. Mol Biol Evol. J Biol Chem.

Zool J Linn Soc-Lond. Article Google Scholar. Behav Ecol. Biol Rev. PubMed Article Google Scholar. Biol Rev Camb Philos Soc. This previous study using VCD spectroscopy provided the overall conformation, while the conformation and dynamics of the repetitive domain as a function of amino acid sequence remained unclear.

In this study, using solution state NMR spectroscopy, we demonstrated the local conformations and dynamics of the repetitive domain as the function of amino acid sequence. Our data clearly demonstrated that the PPII helix population was distributed over the glycine-rich region; whereas, the random coil population was distributed over the polyalanine region. This finding is consistent with the dynamics data, namely, the glycine-rich region has more limited flexibility compared with the polyalanine region.

Here we propose that soluble spider silk occurs in equilibrium with two major populations Schematic illustration in Fig. The soluble precursor of the repetitive domain containing the PPII helix serves as the prefibrillar form of the spider dragline silk. The PPII helix conformation is favorable for protein—protein interactions, such as protein complex assembly The PPII helix interaction in the glycine-rich region is possibly mediated by hydrogen bonds between alpha proton and carbonyl oxygen, similar to the triple helix structure of collagen In the case of Gln in the glycine-rich region, PPII helix interactions are possibly mediated by hydrogen bonds between Gln side chain and carbonyl oxygen of the neighboring residue The PPII helix conformations in the glycine-rich regions between repeat domains enables the ultra-rapid conversion into aligned protein polymers during the silk spinning process, as well as pre-ordering of the soluble spidroins, which helps prevent premature aggregation, even at extremely high concentrations.

The presence of the PPII helix in the glycine-rich region is proposed as a soluble prefibrillar form of spider dragline silk. The polyalanine region is shown in red, and the glycine-rich region is shown in black. Schematic illustration of PPII helix interaction in the glycine-rich region is shown in the box. Interestingly, conformation and dynamics of the recombinant repetitive domain of N. These differences are possibly due to the length of the polyalanine region.

The long polyalanine region 14—15 alanine residues of the repetitive domain of E. As shown in the current study, a shorter polyalanine region 5 alanine residues in N. Our results are in agreement with previous study, which revealed that the conformation of polyalanine region 6—8 alanine residues of native Lactrodectus hesperus dragline silk was random coil In contrast, the longer polyalanine region in E.

The helical structure of the polyalanine in E. In the case of E. Furthermore, the recombinant repetitive domain of E.

The difference in solubility of the repetitive domains can be explained based on the dynamic behavior of the glycine-rich region. The limited flexibility of the glycine-rich region of the repetitive domain from N. On the other hand, the high flexibility of the glycine-rich region of E. Our investigation of the acidic pH effect revealed no conformational changes in the repetitive domains, suggesting that pH changes in the spinning gland do not influence the prefibrillar region of the repetitive domain.

The conformational change in a protein as a function of pH is normally accompanied by protonation and deprotonation of acid and basic groups. Since no acidic residue is available in the repetitive domain, no conformational changes were observed at acidic pH. Previously, volumetric, and spectrophotometric titration studies of native spidroin Nephila edulis demonstrated two apparent p K values: 6. The first p K value was hypothesized to originate from the deprotonation of different residues; whereas, the latter p K value was thought to be related to the deprotonation of Tyr side chain residues The PPII helix conformation is more prevalent in the glycine-rich regions, which present less flexibility than the polyalanine region, which contains more random coils.

The glycine-rich region with the PPII helix population is proposed as the soluble prefibrillar form, which readily supports the transformation into insoluble silk fiber. Our study also demonstrated that the molecular structure of the repetitive domain is not affected by pH, indicating that the prefibrillar form of the repetitive domain is not influenced by pH.

This study provides a better understanding of the initial mechanism of spider silk protein self-assembly and the high solubility of spidroin in the major ampullate gland. Future designs to produce strong artificial spider silk should consider a combination of a large number of repetitive and non-repetitive domains CTD and NTD of spidroin as well as a combination of soft glycine-rich region and hard polyalanine region segments in the repetitive domains to form strong and desirable intramolecular and intermolecular interactions.

The monomer, dimer, trimer, hexamer, and mer genes, which correspond to 1, 2, 3, 6, and 15 contiguous copies of this repeat, respectively, were cloned into the pET30a vector. This vector was modified with a linker containing the restriction sites Nhe I and Spe I based on previously published papers 61 , The repetitive domain genes of each construct were confirmed using DNA sequencing.

The monomer, dimer, trimer, hexamer, and mer were overexpressed using E. Next, the pre-culture was transferred to the main culture. The spectra shown were subtracted from the background and averaged over 10 consecutive scans.

Baseline corrections were performed by subtracting the spectra of D 2 O. Each oligomer of the repetitive domain monomer, dimer, trimer, hexamer, and mer was overexpressed in E. Doubly 13 C, 15 N labeled repetitive domains of spidroin were purified in the same way as purification of unlabeled repetitive domain. The pulse sequence for 1 H-detected 15 NT 2 relaxation recordings led to a series of 2D 1 H— 15 N spectra that were correlated with different 15 NT 2 relaxation delays The acquisition times were Backbone 15 NT 2 values were determined by fitting the peak intensities using a single-exponential decay:.

The pulse sequence for 1 H-detected 15 NT 2 relaxation recordings led to a series of 2D 1 H— 15 N spectra correlated with different 15 NT 2 relaxation delays Backbone 15 NT 1 values were determined by fitting the peak intensities using a single-exponential decay:. The steady-state NOE values were determined from the ratios of the average intensities of the peaks with and without proton saturation, and the standard deviation of the NOE values was determined from the background noise level using the following formula 67 :.

The amide 15 N relaxation was analyzed using a reduced spectral density mapping approach The spectral densities of J 0 from the two spectrometer frequencies were calculated from the following expression:. Calculations were performed using Qtiplot. Each spectrum was recorded using and complex points for the direct and indirect domain, respectively, and the number of scans was equal to 4.

All other data are available from the authors upon reasonable request. Altman, G. Silk-based biomaterials. Biomaterials 24 , — Gaines, W. Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes.

Insect Mol. Sponner, A. The conserved C-termini contribute to the properties of spider silk fibroins. Simmons, A. Solid-state 13 C NMR of Nephila clavipes dragline silk establishes structure and identity of crystalline regions. Macromolecules 27 , — Local structure in spider dragline silk investigated by two-dimensional spin-diffusion nuclear magnetic resonance. Macromolecules 29 , — The molecular structure of spider dragline silk: folding and orientation of the protein backbone.

Natl Acad. USA 99 , — Jenkins, J. Characterizing the secondary protein structure of black widow dragline silk using solid-state NMR and X-ray diffraction. Biomacromolecules 14 , — Gosline, J. Spider silk as rubber. Nature , — High resolution magic angle spinning NMR investigation of silk protein structure within major ampullate glands of orb weaving spiders.

Soft Matter 8 , — Askarieh, G. Self-assembly of spider silk proteins is controlled by a pH-sensitive relay. Hagn, F. A conserved spider silk domain acts as a molecular switch that controls fibre assembly. Jaudzems, K. Ittah, S. The coding sequence of AgSp2 is approximately We sequenced and assembled the complete AgSp2 and partial AgSp1 from the toad-like bolas spider species M. AgSp1 and AgSp2 are members of the spider silk spidroin family Collin et al.

AgSp2 was manually assembled from six long reads Supplementary File S1 and is encoded by nearly 63 kb, dwarfing the previous spidroin record holder from A. Interestingly, the AgSp2 ortholog in A. Similar to AgSp2 of A. The intron in M. Unlike A. As the full length of AgSp1 from M. The gene is at least somewhat larger than this, as there are repeats not included in the count total within the N- and C-terminal encoding reads, and, based on unique repeat structure of the terminal-adjacent repeats, there are additional reads needed to bridge these terminal reads to the remainder of the central repeats.

Mastophora phrynosoma repeat motifs are longer and consist of sub-repeats that are each similar to entire repeats of A. Variations from sub-repeat to sub-repeat result in a longer overall repeating unit, however M. Each sub-repeat or repeat is separated into four subgroups and a tail region as in Stellwagen and Renberg The number of amino acid residues within the subgroups is highly conserved across species, and this pattern in maintained in M.

The tail region is highly variable, and there are several different tail organizations across sub-repeats of M. Yellow highlight indicates conserved residues across both species and genes. Both AgSp1 and AgSp2 had variable bases that allowed proper alignment of the long repetitive regions. They varied, however, in the frequency of synonymous changes to the translated amino acids.

While AgSp2 repeat variants were differentiated by a single base change Fig. These included three synonymous substitutions at histidine and valine residues, and three non-synonymous substitutions convert serine, glutamine, and leucine to arginine, arginine, and phenylalanine residues, respectively. Consistent with previous reports for AgSp1 and AgSp2 of both orb weavers and cobweb weavers Stellwagen and Renberg , the number of amino acids for much of M.

Mastophora phrynosoma repeats, however, are formed from larger, variable sub-repeats that are each similar to the full repeat units of other species.

Sequence deviations across sub-repeat units, particularly in the tail region, increase the size of the overall repetitive pattern of amino acids. The toad-like bolas spider M. In other closely related orb-weaving species, the main glue proteins are encoded by two extremely large, repetitive genes AgSp1 and AgSp2 Choresh et al.

AgSp2 has a generally similar structure to that of A. The repeats of both glue proteins are similar to those reported in other species. Due to the extreme repetition of the aggregate spidroin genes in the bolas spider, we relied on the presence of variable DNA bases in repeating sequence units of the gene for alignment.

The translational impact of these base variations is minimal for AgSp2, which had only one variant that led to a synonymous substitution. This result speaks to the overwhelmingly conserved structure of the M. We found six variable bases that were well-characterized by reads of repeat variants in the M. Half of these resulted in synonymous substitutions. The other half produced nonsynonymous substitutions that involved transitions of amino acids with either polar Ser, Gln or hydrophobic Leu side chains to those with positively-charged Arg or hydrophobic side chains Phe.

That the charge and polarity of these residues is not maintained in repeat variants suggests that these nonsynonymous substitutions may have functional significance for AgSp1 protein. This is particularly true for residues that vary in post-translational modification, particularly O -glycosylation of serine, which confers stickiness Dreesbach et al. Interestingly, we found that the AgSp2 of M.

It has been hypothesized that glutamine promotes self-aggregation of silk proteins into fibers Geurts et al. However, unlike the aggregate glues of orb weavers that adhere and then stretch to help dissipate energy and retain fast-moving prey, M. Additionally, protein aggregation is highly concentration dependent Poulson et al.

The aqueous solution that surrounds the bulk of the central protein core of a droplet of spider aggregate glue contains low molecular weight compounds and salts, which are known for their role in ambient water absorption and glue protein lubrication Vollrath et al. Furthermore, salt concentration has been shown to be an important factor for both solubility and aggregation for other spidroins Knight and Vollrath ; Eisoldt et al.

While M. Longer protein length may provide the protein linkage necessary for adhesion, while limiting concentrations that would result in detrimental viscous aggregations, inhibiting flow on prey. In order to evaluate this hypothesis, glycoprotein concentrations of M. After size correction, we would expect M. There is likely a delicate balance between protein size and concentration, as well as the concentration of other small molecules within glue droplets that help regulate protein behavior.

Future focus on the role of aggregation in regulating solubility and viscosity would be important for evaluating the suitability of spidroins for medical application Abdelrahman et al.

Repeats across M. The unique pattern of these variations from repeat to repeat allows alignment of long reads that is not otherwise possible. However, likely due to the error rate within long reads and the length of these repetitive sequences, the alignment algorithms we applied were unable to identify and utilize nucleotide variations across repeats for alignment. Furthermore, using short sequence reads mapped to long reads for error correction can lead to mis-mapping.

Any repeats with a few non-typical bases which are common near spidroin termini and can be difficult to discern from sequencing errors become overwhelmed by the large number of reads from the central repeats that otherwise map successfully, resulting in an incorrect consensus Fig.

Nanopore long reads and corresponding alignment consensus Nanopore Consensus , Illumina short reads mapped to the Nanopore Consensus, and the consensus formed from Illumina read mapping Illumina Consensus. An initial consensus sequence Nanopore Consensus, gray box for a section of a terminal-adjacent repeat which typically contain a few base variations compared with the bulk of central repeats from a spidroin of a species that was not part of this study is obtained by aligning Nanopore reads 1—5.

Depth of coverage using Nanopore long reads is not sufficient to resolve all errors with confidence and is therefore often corrected using Illumina data.