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• W2004626100 abstract "13C{2H} 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 containingl-[phenol-4-13C]tyrosine andl-[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. 13C{2H} 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 containingl-[phenol-4-13C]tyrosine andl-[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 1The abbreviations DOPAdihydroxyphenylalanineREDORrotational-echo double resonance 1The abbreviations DOPAdihydroxyphenylalanineREDORrotational-echo double resonanceadhesive proteins have recently come under much scrutiny. A number of these organisms, including tube-building polychaetes, black corals, ascidians, and mussels, cure their proteinaceous adhesives by a process called “quinone tanning” (1Waite J.H. Comp. Biochem. Physiol. 1990; 97B: 19-29Google Scholar). Despite frequent references toin vitro studies postulating that quinone tanning involves nucleophilic addition of amine groups to quinones (2Mason H.S. Adv. Enzymol. 1955; 16: 105-184PubMed Google Scholar), 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 aromatic-aromatic coupling products in the mussel byssus (3Holl S.M. Hansen D.C. Waite J.H. Schaefer J. Arch. Biochem. Biophys. 1993; 302: 255-258Crossref PubMed Scopus (44) Google Scholar, 4Klug C.A. Burzio L.A. Waite J.H. Schaefer J. Arch. Biochem. Biophys. 1996; 333: 221-224Crossref PubMed Scopus (24) Google Scholar). In 1994, Dolmer and Svane (5Dolmer P. Svane I. Ophelia. 1994; 40: 63-74Crossref Scopus (51) Google Scholar) 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. dihydroxyphenylalanine rotational-echo double resonance dihydroxyphenylalanine rotational-echo double resonance This paper reports on the continuation of work on the biosynthetic labeling of byssus with 13C- and 2H-containing analogs of tyrosine and the in situ analysis of this label-rich material by rotational echo double resonance (REDOR) NMR (6Gullion T. Schaefer J. J. Magn. Reson. 1989; 81: 196-200Crossref Scopus (1549) Google Scholar). 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. The13C{2H} REDOR dephasing is only consistent with the formation of 5, 5′-di-DOPA covalent cross-links stabilizing the byssal plaques formed under stress. 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 (4Klug C.A. Burzio L.A. Waite J.H. Schaefer J. Arch. Biochem. Biophys. 1996; 333: 221-224Crossref PubMed Scopus (24) Google Scholar). The plates were put into two 140-liter marine aquarium tanks with salt water (no flow) at 14 °C. The tanks were aerated. Labels (13C, 99 atom %; 2H, 98 atom %; Cambridge Isotopes) were added in equal amounts to each tank to make the following concentrations: 49.3 μml-[phenol-4-13C]tyrosine and 48.5 μml-[ring-2H4]tyrosine. The mussels were incubated with the labels for 48 h, after which control samples of plaques and threads were harvested. The plaque harvests used here derive from two mussel groups. The “unstressed” group was maintained in the 140-liter holding tank with essentially stationary flow (4Klug C.A. Burzio L.A. Waite J.H. Schaefer J. Arch. Biochem. Biophys. 1996; 333: 221-224Crossref PubMed Scopus (24) Google Scholar). The “stressed” group was transferred to a flow tank which consisted of a water table that was inclined three degrees from level at one end with an unobstructed laminar flow of 20 cm/s. Low partitions (2–3 cm height) were inserted to allow for the formation of shallow pools in which mussels could be submerged. The water temperature was maintained at 16 °C. Threads and plaques from the stressed mussels were harvested on days 7, 10, 13, 17, 21, and 28. The average thread harvest was 150 mg, and the average plaque harvest was 60 mg. A loss of 13 mussels occurred during the course of the experiment. The same spectrometer was used as described before (4Klug C.A. Burzio L.A. Waite J.H. Schaefer J. Arch. Biochem. Biophys. 1996; 333: 221-224Crossref PubMed Scopus (24) Google Scholar) but with the performance of the 2H channel improved. The dephasing 2H π pulses were shortened to 6.6 μs from 7.1 μs, which increased the observed REDOR dephasing for the same mussel sample by about a factor of two. The relative incorporation of label as a function of time after the 2-day labeling period was similar to that reported earlier (4Klug C.A. Burzio L.A. Waite J.H. Schaefer J. Arch. Biochem. Biophys. 1996; 333: 221-224Crossref PubMed Scopus (24) Google Scholar). Carbon- and deuterium-labeled rings are assumed to be equally probable. There is no scrambling of label. Threads and plaques harvested from 7–17 days after labeling were close to 20% 13C-enriched in tyrosyl residues. Based on a comparison of the intensity of the 155-ppm tyrosyl, oxygenated aromatic carbon peak relative to that of the 175-ppm natural abundance, carbonyl carbon peak (4Klug C.A. Burzio L.A. Waite J.H. Schaefer J. Arch. Biochem. Biophys. 1996; 333: 221-224Crossref PubMed Scopus (24) Google Scholar), the isotopic enrichment for the unstressed mussel plaques is slightly higher than that of the stressed mussel plaque (Fig.1, left and right). Nevertheless, the 145-ppm diphenolic peak (4Klug C.A. Burzio L.A. Waite J.H. Schaefer J. Arch. Biochem. Biophys. 1996; 333: 221-224Crossref PubMed Scopus (24) Google Scholar, 7Schaefer J. Kramer K.J. Garbow J.R. Jacob G.S Stejskal E.O. Hopkins T.L. Spiers R.D. Science. 1987; 235: 1200-1204Crossref PubMed Scopus (206) Google Scholar) of the stressed plaque is increased by 50% relative to the unstressed plaque (Fig. 1,bottom), suggesting creation of increased levels of DOPA. The 175-ppm REDOR difference at the top of Fig. 1 (left) shows 4% dephasing, which we assume arises from random placement of2H-labeled rings near peptide carbonyl carbons. (The dephasings of the α-carbon and aliphatic carbon regions at 50 and 20 ppm, respectively, appear to be somewhat less than 4%. It is conceivable that some of the tyrosyl aromatic carbon label has been transformed to quinone carbonyl carbons, with an expected chemical shift near 180 ppm, and that these carbons are near 2H labels in the covalently attached rings of diquinone or DOPA-quinone moieties. This possibility is under further examination.) The dephasing for the 155-ppm peak is about double the assumed 4% random placement value, which we interpret as indicating local ordering in the inherent positioning of labeled rings in tyrosyl- and DOPA-rich proteins. However, the REDOR dephasing for the increase in intensity of the 145-ppm diphenolic peak of stressed relative to unstressed byssal plaques is close to the 20% level of the isotopic enrichment itself (Fig. 2, ΔΔS/ΔS 0). This level of dephasing cannot be accounted for by the adventitious placements of13C- and 2H-labeled rings but requires the proximity of labels arising from a covalent linkage. Regardless of the relative orientation of the two rings of a 5,5′-di-DOPA cross-link (Fig. 3,inset), the 4-bond 13C-2H distance is between 2.8 and 4.2 Å. Within this distance range,13C{2H} dephasing is complete after 64 rotor cycles with magic-angle spinning at 5 kHz (Fig. 3). Moreover, the 6-bond, 6-Å separation of the distal 2H in 5,5′-di-DOPA will provide additional dephasing, which is possible because of the 1/3 probability of an m = 0 spin state for the proximal2H (8Schmidt A. McKay R.A. Schaefer J. J. Magn. Reson. 1992; 96: 644-650Google Scholar). The combination of the two labels accounts for the observed 20% dephasing. This level of dephasing is not possible for other types of tyrosyl cross-links resulting in a 145-ppm diphenolic aromatic carbon, such as, for example, an isodityrosine linkage, which can have a 5-bond separation for its nearest neighbor13C-2H pair. This is the first successful detection of a covalent cross-link in mussel byssus, or for that matter, in any marine holdfast. It is noteworthy that other dicatechols, 6,6′-di-[1-methylcatechol], as well as related quinone derivatives, were enzymatically synthesized and characterized by Andersen et al. (9Andersen S.O. Jacobsen J.P. Bojesen G. Roepstorff P. Biochim. Biophys. Acta. 1992; 1118: 134-138Crossref PubMed Scopus (36) Google Scholar) who wrote that such compounds “ … are preferentially produced when catechols are oxidized under conditions with excess water, rather high concentrations of catechols, and few other compounds available for reaction with intermediary o-quinones.” Whether this statement describes the microenvironments in which the peptidyl DOPA residues of byssal proteins function is not yet known. Based on spin counts for carbons at 145- and 175-ppm (Fig. 2), we estimate that the increase in cross-link density resulting from higher levels of flow stress is 1 di-DOPA cross-link per 1800 total protein amino acid residues. This estimate is an average for bulk byssal plaque material. Naturally, the cross-link density in specific DOPA proteins can be higher. Given that byssal plaques stressed with a flow of 20 cm/s contained enhanced levels of di-DOPA cross-links, it would be intriguing to determine whether yet higher flows lead to higher cross-link densities. Mussels can survive in flow regimes in excess of 15 m/s (10Bell E.C. Gosline J.M. Mar. Ecol. Prog. Ser. 1997; 159: 197-208Crossref Scopus (148) Google Scholar). We thank Barbara Poliks (SUNY, Binghamton) for calculation of the conformationally dependent distances in 5–5′-di-DOPA." @default.
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• W2004626100 title "Rotational Echo Double Resonance Detection of Cross-links Formed in Mussel Byssus under High-Flow Stress" @default.
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