pH-sulfate synergy regulates processing and mechanics of mussel byssus protein condensates

Fluid protein condensates are used as precursor phases for fabricating extracellular protein-based materials including elastin, spider silk, and mussel byssus. The byssus, utilized by mussels for anchoring in marine environments, consists of tough, self-healing adhesive fibers. Byssus formation involves the secretion of protein condensate droplets under acidic conditions that subsequently solidify under basic seawater conditions. We currently have a poor understanding of the physicochemical triggers and molecular-level interactions at play, in particular the role of pH and sulfate anions previously identified during native fabrication. Here, we investigated the pH and sulfate-dependent structural and mechanical response of condensates made from a recombinant byssus protein (mfp-1) using optical tweezers microrheology, FRAP, confocal Raman spectroscopy, NMR, and cryo-EM. We found that the protein concentration in condensates increased, and the viscoelastic response became more rigid under basic conditions in the presence of sulfate ions compared with chloride ions, consistent with spectroscopic analysis indicating different molecular interactions under these different chemical conditions. These studies highlight the crucial interplay between sulfate anions and pH in tuning condensate viscoelasticity via control of intermolecular interactions, providing insights into the natural byssus formation process with relevance for bio-inspired materials processing of sustainable plastics and materials for tissue engineering.