1 Introduction The key to survival on rocky wave- and wind-swept seashores for many mussel species is a beard-like array of tethering attachment threads known collectively as the byssus, which is used to secure the mussel to the hard substratum against the lift and drag forces of waves (Yonge, 1962). In addition, the byssus is emerging as an important paradigm for the bio- inspired engineering of water-resistant adhesives (Lee et al., 2006), functionally graded polymers (van Hest and Tirrell, 2001; Waite et al., 2004), and injection molding of liquid crystals (Hassenkam et al., 2004). The utility of such paradigms relies on the depth to which structure–function relationships are understood. A particularly effective strategy for defining structure–function relationships is to investigate homologous structures in closely related species (Hayashi et al., 1999; Brooks et al., 2005). With this report, byssal thread mechanics and the protein chemistry of three species of Mytilus are compared. Of these, the byssus of the California mussel Mytilus californianus (Conrad), stands out as mechanically superior. The threads that make up the byssus of M. californianus are several centimeters long with a diameter of about 200·m, function outside the body of the organism, and are formed from soluble protein precursors secreted by the foot of the mussel. Byssal threads are subdivided into four morphologically and mechanically distinct regions: the stem, the plaque, and the proximal and the distal portions of the thread (Fig.·1). The stem attaches the thread to the mussel tissue, and the plaque contains the adhesive that connects the thread to the hard substratum. The thread connecting the stem to the plaque is further divided into two mechanically distinct regions. The proximal end (closest to the organism) is extensible up to 200% of its original length, has a low initial stiffness, and a corrugated appearance. The distal portion of the thread, in contrast, is characterized by a high initial stiffness followed by a yield point at about 15% strain and a noticeable stress softening (Bell and Gosline, 1996). For most engineered polymeric materials, yield is not reversible and leads to permanent deformation rendering the material functionally useless. However, in distal threads, damage due to yield is reversible in a time-dependent, self-healing manner with threads recovering 25% of the lost modulus and strain energy in 10·min following a cycle to 35% strain (Carrington and Gosline, 2004). Thread formation begins when the mussel foot touches down on a surface it finds suitable for attachment (Waite, 1992). Once The marine mussel Mytilus californianus (Conrad) inhabits the most wave-exposed regions of the rocky intertidal by dint of its extraordinary tenacity. Tenacity is mediated in large part by the byssus, a fibrous holdfast structure. M. californianus byssal threads, which are mechanically superior to the byssal threads of other mytilids, are composed almost entirely of a consortium of three modular proteins known as the preCols. In this study, the complete primary sequence of preCols from M. californianus was deduced and compared to that of two related species with mechanically inferior byssal threads, M. edulis (Linnaeus) and M. galloprovincialis (Lamarck) in order to explore structure–function relationships. The preCols from M. californianus are more divergent from the other two species than they are from one another. However, the degree of divergence is not uniform among the various domains of the preCols, allowing us to speculate on their mechanical role. For instance, the extra spider silk- like runs of alanine-rich sequence in the flanking domains of M. californianus may increase crystalline order, enhancing strength and stiffness. Histidine-rich domains at the termini, in contrast, are highly conserved between species, suggesting a mechanical role common to all three. Mechanical testing of pH-treated and chemically derivatized distal threads strongly suggests that histidine side chains are ligands in reversible, metal-mediated cross- links in situ. By combining the mechanical and sequence data, yield and self-healing in the distal region of threads have been modeled to emphasize the intricate interplay of enthalpic and entropic effects during tensile load and recovery. Key words: mussel, byssus, self-healing, histidine–metal, collagen. Summary The Journal of Experimental Biology 210, 000-000 Published by The Company of Biologists 2007 doi:10.1242/jeb.009753 Holdfast heroics: comparing the molecular and mechanical properties of Mytilus californianus byssal threads Matthew J. Harrington* and J. Herbert Waite Department of Molecular, Cellular, and Developmental Biology, University of California at Santa Barbara (UCSB), Santa Barbara, CA 93106, USA *Author for correspondence (e-mail: harringt@lifesci.ucsb.edu) Accepted 2 October 2007 Page nos Page total Colour pages: Facing pages: Issue Ms order