Rotational Echo Double Resonance Detection of Cross-links Formed in Mussel Byssus under High-Flow Stress

Abstract

13 C{2 H} rotational echo double resonance NMR has been used to provide the first evidence for the formation of quinone-derived cross-links in mussel byssal plaques. Labeling of byssus was achieved by allowing mussels to filter feed from seawater containing L-[phenol-4-13 C]tyrosine and L-[ring-d 4 ]tyrosine for 2 days. Plaques and threads were harvested from two groups of mussels over a period of 28 days. One group was maintained in stationary water while the other was exposed to turbulent flow at 20 cm/s. The flow-stressed byssal plaques exhibited significantly enhanced levels of 5, 5-di-dihydroxyphenylalanine cross-links. The average concentration of di-dihydroxyphenylalanine cross-links in byssal plaques is 1 per 1800 total protein amino acid residues.The attachment strategies of marine organisms that rely on DOPA-containing 1 adhesive proteins have recently come under much scrutiny. A number of these organisms, including tubebuilding polychaetes, black corals, ascidians, and mussels, cure their proteinaceous adhesives by a process called "quinone tanning" (1). Despite frequent references to in vitro studies postulating that quinone tanning involves nucleophilic addition of amine groups to quinones (2), nothing of substance is known about the actual cross-linking chemistry in these organisms. Indeed, previous efforts from our own laboratories seemed to rule out the existence of lysine-aromatic or aromaticaromatic coupling products in the mussel byssus (3, 4). In 1994, Dolmer and Svane (5) reported that individual threads in a mussel byssus behaved as "smart materials" in flow: by doubling the flow of seawater, the tensile strength of the threads could be doubled. This suggested that there were different degrees of quinone-tanning in a given material that relied in some way on maturation by imposed stresses. This paper reports on the continuation of work on the biosynthetic labeling of byssus with 13 C-and 2 H-containing analogs of tyrosine and the in situ analysis of this label-rich material by rotational echo double resonance (REDOR) NMR (6). We have repeated the byssus labeling under high flow conditions and compared directly REDOR spectra of byssal plaques collected under stressed (flow) and unstressed (stationary or minimal flow) conditions. The difference between these REDOR spectra reveal the preferential routing of tyrosine labels to diphenolics. The 13 C{ 2 H} REDOR dephasing is only consistent with the formation of 5, 5Ј-di-DOPA covalent crosslinks stabilizing the byssal plaques formed under stress. EXPERIMENTAL PROCEDURESMussel Labeling-170 Adult mussels (Mytilus edulis) were collected in the vicinity of Roosevelt Inlet at Lewes, Delaware. The mussels ranged in size from 3 to 10 cm. They were tethered to acrylic plates as described previously (4). The plates were put into two 140-liter marine aquarium tanks with salt water (no flow) at 14°C. The tanks were aerated. Labels H 4 ]tyrosine. The mussels were incubated with the labels for 48 h, after which control samples of plaques and threads were harv...

Cement Proteins of the Tube-building Polychaete Phragmatopoma californica

“…Three assumptions were made in this calculation: (i) that the glycine in cement is contributed only by Pc-1 (Ͼ45 mol %) and Pc-2 (Ͼ29 mol %), (ii) that histidine in cement is derived from Pc-2, and (iii) that Pc-1 and Pc-2 each contribute a third to the composite whole. The accuracy of this approach is compromised mostly by the considerable amounts of tyrosine in the Pcs known to be lost first to Dopa, then to Dopaquinone, and finally to cross-links and/or oxidation products that ultimately involve collateral losses of other amino acids (15)(16)(17) as well. Given the Ser/ Ser(P) content in cement at 28.5 mol %, the Ser contributions of Pc-1 and Pc-2 are 0.6/3 and 3.7/3 Ser/Ser(P), respectively, or about 1.5 parts out of 28.5.…”

“…If future studies determine that the cysteinyldopa cross-links in cement are intermolecular, then this would fulfill the setting requirement. In mussel byssal adhesive plaques, diDopa cross-links prevail (15), but whether they also occur in Phragmatopoma cement is not known. A role for phosphoserines in setting is suggested by the high levels of Mg/Ca 2ϩ in the cement and cement gland (6,7,33) and cement softening by EDTA treatment.…”

Elastomeric gradients: a hedge against stress concentration in marine holdfasts?

“…DOPA residues not engaged in chelate complexes are prone to oxidation in seawater. In a study of byssus 'stressed' by conditions of high flow and aeration, the primary product of oxidation was found by solid-state NMR with 2 H-and 13 Ctyrosine-labelled byssus to be 5,5Ј diDOPA (McDowell et al 1999). diDOPA was also formed during in vitro oxidation of DOPA-containing byssal precursor proteins (Burzio & Waite 2000).…”

Adhesive strength and curing rate of marine mussel protein extracts on porcine small intestinal submucosa

“…Although several recent studies have increased the scientific community's understanding of how blue mussels cure their adhesive precursors [26][27][28][29][30][42][43][44][45][46][47][48][49][50][51], the mechanism is not yet completely understood. It appears that the precursor proteins are cross-linked by a complex interaction of dihydroxyphenylalanine (DOPA) motifs in the protein, facilitated by enzymes and oxidative reagents such as metal ions.…”