Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376 MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230 MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers.

The relationship between microstructure and mechanical properties has been investigated in Argiope trifasciata dragline silk fibers (major ampullate silk, MAS) by X-ray diffraction, Raman spectroscopy and tensile testing. We have analyzed three fractions of the material, i.e. amorphous, highly oriented nanocrystals and weakly oriented material, for different values of the macroscopic alignment parameter a, calculated as the relative difference between the length of the fiber and its length when supercontracted. Two distinct regimes have been identified: for low values of the alignment parameter a, microstructural changes are dominated by the reorientation of the nanocrystals; however, at high values (a > 0.5) of the alignment parameter, an increase in the fraction of the crystalline phase is revealed. The two regimes are also reflected in the mechanical behaviour, which can be explained by microstructural changes. This finding of the two distinct regimes in the microstructural evolution, which separates the reorientation and the increase in the crystalline phase, will be valuable to develop and validate molecular models of natural and artificial silk fibers, as well as to deepen our present knowledge of the origin of the outstanding properties of MAS fibers. In addition, we have analyzed the characteristics of the crystal lattice, and discussed the relationship between the percentage of short sidechain residues and the unit cell dimensions in different silks.

The mechanical behavior and microstructure of minor ampullate gland silk (miS) of two orb-web spinning species, Argiope trifasciata and Nephila inaurata, were extensively characterized, enabling detailed comparison with other silks. The similarities and differences exhibited by miS when compared with the intensively studied major ampullate gland silk (MAS) and silkworm (Bombyx mori) silk offer a genuine opportunity for testing some of the hypotheses proposed to correlate microstructure and tensile properties in silk. In this work, we show that miSs of different species show similar properties, even when fibers spun by spiders that diverged over 100 million years are compared. The tensile properties of miS are comparable to those of MAS when tested in air, significantly in terms of work to fracture, but differ considerably when tested in water. In particular, miS does not show a supercontraction effect and an associated ground state. In this regard, the behavior of miS in water is similar to that of B. mori silk, and it is shown that the initial elastic modulus of both fibers can be explained using a common model. Intriguingly, the microstructural parameters measured in miS are comparable to those of MAS and considerably different from those found in B. mori. This fact suggests that some critical microstructural information is still missing in our description of silks, and our results suggest that the hydrophilicity of the lateral groups or the large scale organization of the sequences might be routes worth exploring.

The extraordinary mechanical performance of spider dragline silk is explained by its highly ordered microstructure and results from the sequences of its constituent proteins. This optimized microstructural organization simultaneously achieves high tensile strength and strain at breaking by taking advantage of weak molecular interactions. However, elucidating how the original design evolved over the 400 million year history of spider silk, and identifying the basic relationships between microstructural details and performance have proven difficult tasks. Here we show that the analysis of maximum supercontracted single spider silk fibers using X ray diffraction shows a complex picture of silk evolution where some key microstructural features are conserved phylogenetically while others show substantial variation even among closely related species. This new understanding helps elucidate which microstructural features need to be copied in order to produce the next generation of biomimetic silk fibers.

Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine β-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the –GGX– and –GPG– motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract.