Role of protein aggregate structure on the strength and underwater performance of barnacle-inspired adhesives.

Nature employs protein aggregates when strong materials are needed to adhere surfaces in extreme environments, allowing organisms to survive conditions ranging from harsh intertidal coasts to open oceans. Amyloids and amyloid-like materials are prevalent and amongst the most densely bonded aggregate structures, though how they contribute to wet adhesion is not well understood. In this work, waterborne protein solutions of individual whey proteins are cured in place using varied temperature to produce model adhesives enriched in amyloid or non-amyloid aggregates. Dry adhesive strengths range from 0.2-1.5 MPa, while wet adhesive strengths range from 0-0.5 MPa across the tested proteins and processing conditions, highlighting that both proper protein selection and controlled aggregation extent are necessary for successful underwater performance. For bovine serum albumin, the amyloid-enriched adhesive was able to retain ca. 500 kPa bond strength underwater throughout extended immersion and thermal degradation testing, while the non-amyloid adhesive weakened by up to 80%. As freestanding gels, higher temperature processing improved underwater stability for all the protein materials, with amyloid-rich structures remaining mostly water-insoluble after 30 days submerged in water. Protein-based adhesives with a controlled aggregate structure shed light on the ability of amyloid-containing materials to remain adhered underwater, a necessary trait for the survival of many organisms.

Extremophilic behavior of catalytic amyloids sustained by backbone structuring.

Enzyme function relies on the placement of chemistry defined by solvent and self-associative hydrogen bonding displayed by the protein backbone. Amyloids, long-range multi-peptide and -protein materials, can mimic enzyme functions while having a high proportion of stable self-associative backbone hydrogen bonds. Though catalytic amyloid structures have exhibited a degree of temperature and solvent stability, defining their full extremophilic properties and the molecular basis for such extreme activity has yet to be realized. Here we demonstrate that, like thermophilic enzymes, catalytic amyloid activity persists across high temperatures with an optimum activity at 81 °C where they are 30-fold more active than at room temperature. Unlike thermophilic enzymes, catalytic amyloids retain both activity and structure well above 100 °C as well as in the presence of co-solvents. Changes in backbone vibrational states are resolved in situ using non-linear 2D infrared spectroscopy (2DIR) to reveal that activity is sustained by reorganized backbone hydrogen bonds in extreme environments, evidenced by an emergent vibrational mode centered at 1612 cm-1. Restructuring also occurs in organic solvents, and facilitates complete retention of hydrolysis activity in co-solvents of lesser polarity. We support these findings with molecular modeling, where the displacement of water by co-solvents leads to shorter, less competitive, bonding lifetimes that further stabilize self-associative backbone interactions. Our work defines amyloid properties that counter classical proteins, where extreme environments induce mechanisms of restructuring to support enzyme-like functions necessary for synthetic applications.