Functional gradient materials are used in Nature to mitigate contact damage between two dissimilar materials. A candidate to study such materials are Humboldt squid beaks (mechanical gradient) which are large in size, acellular and sheet-like, allowing for easy handling. Of particular interest with the squid beak is the use of a hydration gradient to form the beak mechanical gradient. Understanding how this hydration gradient is created and results in a mechanical gradient can potentially provide a strategy to reduce contact damage in multi-component assemblies as well as providing a robust and environmentally-friendly manufacturing alternative to energy-intensive composites such as fiberglass.

Studying squid beak's hydration gradient essentially means scrutinizing the beak proteins and their post-secretion processing since the gradual distribution of beak proteins is proposed to be responsible for the hydration gradient.

Here, full sequences for two families of squid beak proteins are presented and their functions discussed. These were obtained with fast turnaround by employing traditional proteomics techniques together with RNA-Seq, a recent technique based on Next Generation Sequencing.

Chitin-binding beak proteins (DgCBP) which are present throughout the beak make up one of these families and were shown to bind reversibly to chitin using a chitin-binding assay. Thus it is proposed that DgCBPs may be responsible for binding chitin together reversibly during the initial stage of beak formation.

The second group consists of His-rich beak proteins (DgHBP) which are proteins with hydrophobic C-terminal tandem repeats that include His residues arranged in GHG motifs. DgHBPs are likely to be precursors to the polymeric cross-links that are beak pigments as the GHG motifs in the tandem repeats can be cross-linked by catechols to form His-catecholic adducts, similar to those previously isolated from digestion of beak pigments.

In addition, the possibility of DgHBPs coacervates was probed in experiments using synthetic analogs of DgHBPs. Coacervation had been observed in other marine biomaterials and offers an avenue for efficient transport of DgHBPs through the dense chitin network in the squid beak. Results from these experiments suggest that DgHBPs likely formed coacervates when exposed to seawater and are ultimately cross-linked into insoluble complexes by catechols ultimately.