Suckerin proteins, recently discovered in the sucker ring teeth of squids, represent a family of promising structural biomacromolecules that can form supramolecular networks stabilized by nanoconfined β-sheets. Exploiting this feature as well as their specific amino acid composition, we demonstrate that artificial suckerin-19 (S-19) can be engineered into nanocarriers for efficient drug delivery and gene transfection in vitro and in vivo. First, we demonstrate that S-19 self-assembles into β-sheet stabilized nanoparticles with controlled particle sizes of 100-200 nm that are able to encapsulate hydrophobic drugs for pH-dependent release in vitro, and that can effectively inhibit tumor growth in vivo. We also show that S-19 can complex and stabilize plasmid DNA, with the complexes stabilized by hydrophobic interactions of the β-sheet domains as opposed to electrostatic interactions commonly achieved with cationic polymers, thus lowering cytotoxicity. The elevated Histidine content of S-19 appears critical to trigger endosomal escape by the proton sponge effect, thereby ensuring efficient gene transfection both in vitro and in vivo. Our study demonstrates that S-19 represents a promising functional protein nanocarrier that could be used for various drug and gene delivery applications.

Biomineralization, the process by which mineralized tissues grow and harden via biogenic mineral deposition, is a relatively lengthy process in many mineral-producing organisms, resulting in challenges to study the growth and biomineralization of complex hard mineralized tissues. Arthropods are ideal model organisms to study biomineralization because they regularly molt their exoskeletons and grow new ones in a relatively fast timescale, providing opportunities to track mineralization of entire tissues. Here, we monitored the biomineralization of the mantis shrimp dactyl club—a model bioapatite-based mineralized structure with exceptional mechanical properties—immediately after ecdysis until the formation of the fully functional club and unveil an unusual development mechanism. A flexible membrane initially folded within the club cavity expands to form the new club’s envelope. Mineralization proceeds inwards by mineral deposition from this membrane, which contains proteins regulating mineralization. Building a transcriptome of the club tissue and probing it with proteomic data, we identified and sequenced Club Mineralization Protein 1 (CMP-1), an abundant mildly phosphorylated protein from the flexible membrane suggested to be involved in calcium phosphate mineralization of the club, as indicated by in vitro studies using recombinant CMP-1. This work provides a comprehensive picture of the development of a complex hard tissue, from the secretion of its organic macromolecular template to the formation of the fully functional club.

The catecholic Dopa side-chain indeed exhibits versatile physicochemical features that makes it well-suited to adsorb and adhere to immersed substrates, including the ability to engage into numerous types of intermolecular interactions such as coordination bonding, [2,5] hydrogen bonding, [6] and hydrophobic interactions. [7] While there has been undeniable success in synthesizing catechol-containing polymers for enhanced water-resistant adhesives and coatings, [8,9] recent investigations have indicated that this Dopa "paradigm" of mussel adhesion may be subtler than previously proposed. Notably, both nano-scale and macroscopic adhesion studies of recombinant MFPs and MFP-derived peptides from the adhesive plaque-the section of the byssus in direct contact with substrates-have indicated that comparable levels of adhesion can be achieved even when tyrosine residues (Tyr) are not post-translated into Dopa. In particular for the Asian green mussel (Perna viridis), Bilotto et al. [10] reported surface force apparatus (SFA) adhesion measurements of the foot protein 5 (Pvfp-5β) [11] containing either Tyr or with most Tyr substituted to Dopa, and did not find statistically-significant differences of adhesion strengths and energies between the two variants. Likewise, Ou et al. [12] -also using Pvfp-5β as a model mussel adhesive protein-measured equivalent macroscopic adhesive strength values for Pvfp-5β when Tyr was enzymatically modified to Dopa. These results can be reconciled by the study of Maier et al. [13] together with molecular dynamic (MD) simulations by Ou et al. [12] The former reported that hydrated ions on oxide surfaces are evicted by Lys residues, enabling adjacent Dopa residues to form bidentate H-bonding with the oxide surface unimpeded by these hydrated ions, whereas the latter predicted that the reverse stepwise process, namely initial eviction of surface ions by aromatic residues followed by electrostatic binding of positively-charged residues, was also possible. Importantly, MD simulations revealed that Tyr is equally efficient at sweeping the surface of hydrated ions, thus eliminating their screening effect and enabling adjacent Lys residues to bind to the surface via electrostatic interactions. In addition, The underwater adhesive prowess of aquatic mussels has been largely attributed to the abundant post-translationally modified amino acid l-3,4-dihydroxyphenylalanine (Dopa) in mussel foot proteins (MFPs) that make up their adhesive threads. More recently, it has been suggested that during thread fabrication, MFPs form intermediate fluidic phases such as liquid crystals or coacervates regulated by a liquid-liquid phase separation (LLPS) process. Here, it is shown that Dopa plays another central role during mussel fiber formation, by enabling LLPS of Pvfp-5β, a main MFP of the green mussel Perna viridis. Using residue-specific substitution of Tyrosine (Tyr) for Dopa during recombinant expression, Dopa-substituted Pvfp-5β is shown to exhibit LLPS under seawater-like conditions, whereas the Tyr-only ...

Nature has evolved several molecular strategies to ensure adhesion in aqueous environments, where artificial adhesives typically fail. One recentlyunveiled molecular design for wet-resistant adhesion is the cohesive cross-β structure characteristic of amyloids, complementing the well-established surface-binding strategy of mussel adhesive proteins based on 3,4-l-dihydroxyphenylalanine (Dopa). Structural proteins that self-assemble into cross β-sheet networks are the suckerins discovered in the sucker ring teeth of squids. Here, light is shed on the wet adhesion of cross-β motifs by producing recombinant suckerin-12, naturally lacking Dopa, and investigating its wet adhesion properties. Surprisingly, the adhesion forces measured on mica reach 70 mN m −1 , exceeding those measured for all mussel adhesive proteins to date. The pressure-sensitive adhesion of artificial suckerins is largely governed by their cross-β motif, as evidenced using control experiments with disrupted cross-β domains that result in complete loss of adhesion. Dopa is also incorporated in suckerin-12 using a residue-specific incorporation strategy that replaces tyrosine with Dopa during expression in Escherichia coli. Although the replacement does not increase the long-term adhesion, it contributes to the initial rapid contact and enhances the adsorption onto model oxide substrates. The findings suggest that suckerins with supramolecular cross-β motifs are promising biopolymers for wet-resistant adhesion.

A novel thermostable ω-transaminase from Thermomicrobium roseum showing broad substrate specificity and high enantioselectivity was identified, expressed and biochemically characterized and it could produce chiral amines at high temperature.