Complete Sequences of the Velvet Worm Slime Proteins Reveal that Slime Formation is Enabled by Disulfide Bonds and Intrinsically Disordered Regions

Abstract

The slime of velvet worms (Onychophora) is a strong and fully biodegradable protein material, which upon ejection undergoes a fast liquid-to-solid transition to ensnare prey. However, the molecular mechanisms of slime self-assembly are still not well understood, notably because the primary structures of slime proteins are yet unknown. Combining transcriptomic and proteomic studies, the authors have obtained the complete primary sequences of slime proteins and identified key features for slime self-assembly. The high molecular weight slime proteins contain cysteine residues at the N-and C-termini that mediate the formation of multi-protein complexes via disulfide bonding. Low complexity domains in the N-termini are also identified and their propensity for liquid-liquid phase separation is established, which may play a central role in slime biofabrication. Using solid-state nuclear magnetic resonance, rigid and flexible domains of the slime proteins are mapped to specific peptide domains. The complete sequencing of major slime proteins is an important step toward sustainable fabrication of polymers inspired by the velvet worm slime.

Biological Materials Processing: Time-Tested Tricks for Sustainable Fiber Fabrication

“…The extracted slime can be drawn into fibers in vitro under ambient conditions by mechanically shearing the slime (e.g., with a pipet tip) followed by tensile drawing. 15,51,265,266 While these drawn fibers are quite stable in air, it was discovered that the fibers of the Australian species Euperipatoides rowelli can be resolubilized in distilled water and that new fibers can be regenerated from the solution, providing evidence of a fully circular fiber formation process. 15 It is unclear if this intrinsic recyclability is a universal feature of slimes from all velvet worm species or specific to E. rowelli and closely related species, especially considering that the two major suborders of onychophorans split evolutionarily nearly 400 million years ago.…”

“…reported the full sequences of two high molecular proteins named ES_P1 and ES_P2 with sequences that possess some notable similarities to ER_P1, including the prevalence of proline (some of which is hydroxylated) and an abundance of charged amino acids. 51 However, there were several previously unidentified features of the proteins including glycine and serine-rich sequences in the N-terminal domain with homology to low complexity proteins known to undergo LLPS, as well as the identification of cysteine residues at both the N-and Ctermini. Both features were implicated in the aggregation and assembly of the proteins in vitro, 51 suggesting that the N-and C-terminal domains of these high molecular weight proteins may be crucial for fiber formation, similar to spider silk spidroins and mussel byssus preCols.…”

“…51 However, there were several previously unidentified features of the proteins including glycine and serine-rich sequences in the N-terminal domain with homology to low complexity proteins known to undergo LLPS, as well as the identification of cysteine residues at both the N-and Ctermini. Both features were implicated in the aggregation and assembly of the proteins in vitro, 51 suggesting that the N-and C-terminal domains of these high molecular weight proteins may be crucial for fiber formation, similar to spider silk spidroins and mussel byssus preCols. There is spectroscopic and X-ray based evidence that slime proteins from E. rowelli exhibit a mixture of ordered and disordered protein secondary structure, with a prevalent β-sheet backbone structure, which is organized into extended crystallites (Figure 15).…”

“…indicated that cysteine-based disulfide cross-linking mediated by residues in the N-and C-terminal domains of the proteins resulted in multiprotein complexation, analogous to the role of multimerizing terminal domains in spider silk processing. 51 Additionally, it was demonstrated that recombinantly expressed proteins based on the sequence of the low complexity N-terminal domains could undergo LLPS and thus may play a role in inducing nanoglobule formation and hence, fiber formation. 51 In any case, there are still many open questions as to what drives the formation of these nanoglobules and what their role might be in the slime.…”

“…51 Additionally, it was demonstrated that recombinantly expressed proteins based on the sequence of the low complexity N-terminal domains could undergo LLPS and thus may play a role in inducing nanoglobule formation and hence, fiber formation. 51 In any case, there are still many open questions as to what drives the formation of these nanoglobules and what their role might be in the slime.…”