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Research Article
Holdfast heroics: comparing the molecular and mechanical properties of Mytilus californianus byssal threads
Matthew J. Harrington, J. Herbert Waite
Journal of Experimental Biology 2007 210: 4307-4318; doi: 10.1242/jeb.009753
Matthew J. Harrington
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J. Herbert Waite
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  • Fig. 1.
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    Fig. 1.

    Schematic of an isolated byssal thread showing important structural features. Morphological and mechanical differences graded from distal to proximal correspond with a gradient in the relative composition of preCol variants D, NG and P.

  • Table 1.

    PCR reactions and primers used to determine the cDNA sequence of M. californianus preCols

    PreCol D5′RACE/DR-1, DF-2/DR-2, DF-3/DR-3
       McDR-15′ TCCGCCACCTACAACTGGACC 3′
       McDF-25′ GCAGCAGCAAACGCAGCAGCAGGAGGAT 3′
       McDR-25′ CTCCGGCTGTTCTGAGGTCTTCAAT 3′
       McDF-3*5′ GTGGTATGGGTAGACGAG 3′
       McDR-3*5′ AACACTTGCAGATTTTATTGATA 3′
    PreCol NG5′RACE/NGR-1, NGF-2/NGR-2dg, NGF-3/NGR-3, NGf-4/NGR-4
       McNGR-15′ AAGTGGGAATCCGCCGCCACCAGTTGC 3′
       McNGF-25′ GCGGCTATCGCCCGCACAGGACTA 3′
       McNGR-2dg5′ YTGWCCWGGDGCWCCWCCDGCATTWCCWGGTTGG 3′
       McNGF-35′ GCAACTGGTGGCGGCGGATTCCCACTT 3′
       McNGR-35′ CCACCAGCCTCTCCTGCTGGTCCTTCT 3′
       McNGF-4*5′ AAGGAGAACTTGGACCAGTCG 3′
       McNGR-4*5′ AGGAACTTGCACTTTTTAT 3′
    PreCol P5′RACE/PR-1, PF-2/PR-2, PF-3/3′RACE
       McPR-15′ GGATCAGGCAGTGGCAGAGCATTTGGTGG 3′
       McPR-2*5′ GAATAACACCTGGTGCTCCT 3′
       McPF-2*5′ GAGGATTCGGTGGACCAGGTAC 3′
       McPF-35′ GGGACCAGGAGGTGAAAGAGGAGGCCAAGG 3′
    • ↵* Primers from M. edulis. McDR-3 and McNGR-4 are based on the 3′ UTR

    • ↵dg Degenerate primer

  • Table 2.

    Percent identity of the preCol variants for M. californianus, M. edulis and M. galloprovincialis by domain

    Identity (%)
    PreColvariantOverallSignalN-HisN-flankCollagenAcidicC-flankC-His
    D
       Mc:Me8285677093956966
       Mc:Mg79856552879572100
       Me:Mg891009283931008976
    NG
       Mc:Me83100807989907170
       Mc:Mg8195838589906862
       Me:Mg89958987971007879
    P
       Mc:Me79100827583936288
       Mc:Mg80100827585866383
       Me:Mg91100849194939193
    • Mc, M. californianus; Me, M. edulis; Mg, M. galloprovincialis

      Identity scores were determined by ClusterW and represent the number of identical residues in the best pairwise match, as determined by the program divided by the number of residues that were matched. This score does not take into account regions that do not overlap due to inserted sequence

  • Fig. 2.
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    Fig. 2.

    Schematics of histidine chemistry concerning protonation, metal coordination and reaction with diethyl pyrocarbonate (DEPC). (I,II) The titration of a proton on the imidazole ring of His has a pKa∼ 6.5. The nitrogen shown losing the proton in the figure is the more likely of the two since it has slightly lower pKa; however, depending on the local environment, either can be deprotonated. As shown in (III), it is possible for the deprotonated nitrogen to contribute a lone pair of electrons as a ligand in a metal coordination bond with a divalent transition metal ion such as Zn2+ or Cu2+. When both nitrogens are protonated (I), the ring is unable to participate as a ligand in metal bonding. Reacting unbound His residues with DEPC (IV) results in a carbethoxylation of the imidazole ring, rendering it unable to participate in coordination metal chemistry. Metal bound His will not react with DEPC. DEPC treatment is specific for His at pH 5.5 and at this pH, there will be few side chains bound to metal. EtOH, ethanol.

  • Fig. 3.
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    Fig. 3.

    Deduced protein sequence of preCol D from M. californianus. Histidine and tyrosine residues in the His-rich domains are in bold type. Breaks in the Gly-X-Y canonical repeat are underlined in the collagen domain and deletions are denoted with an asterisk.

  • Fig. 4.
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    Fig. 4.

    Deduced protein sequence of preCol NG from M. californianus. Histidine and tyrosine residues in the His-rich domains are in bold type. Breaks in the Gly-X-Y canonical repeat are underlined in the collagen domain and deletions are denoted with an asterisk.

  • Fig. 5.
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    Fig. 5.

    Deduced protein sequence of preCol P from M. californianus. Histidine and tyrosine residues in the His-rich domains are in bold type. Breaks in the Gly-X-Y canonical repeat are underlined in the collagen domain and deletions are denoted with an asterisk.

  • Fig. 6.
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    Fig. 6.

    ClustalW alignment of (A) the N-terminal silk domain and (B) histidine-rich domain of preCol D from M. californianus (Mc), M. edulis (Me) and M. galloprovincialis (Mg). Silk alignment reveals that M. californianus has two major inserts consisting of polyalanine runs in the N terminus. His-rich domain alignment reveals that while overall the identity between M. californianus and the other species is low, the regions containing histidine are more conserved. Residues in bold type are conserved between all three species.

  • Fig. 7.
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    Fig. 7.

    (A) Representative stress–strain curve of a single distal thread cycled to incrementally increasing strain values from 10% to 70%. Yield, as seen in cycles to 20, 30 and 40%, is followed by a loss in stiffness (stress softening) in the subsequent cycle. (B) Percentage loss in modulus plotted as a function of the strain that caused the loss. Values are means ± s.e.m.; N=10. Most stress softening is occurring between 10% and 40% strain.

  • Fig. 8.
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    Fig. 8.

    Initial modulus of distal byssal threads plotted against the pH at which they were incubated. Red dotted line and circle denote the midway point of the Young's modulus and the corresponding pH of 6.6. The titration of a nitrogen proton on the histidine imidazole ring (pKa=6.6) is plotted in light gray (A/[A+AH]) to illustrate that the modulus curve closely follows the characteristic sigmoidal shape, suggesting that histidine protonation may influence thread mechanics. The blue inverted triangle represents the initial stiffness of threads treated at pH 5.5 and brought back to pH 8 and tested, indicating that pH-induced softening is reversible. The pink triangle represents the initial stiffness of threads treated in DEPC at pH 5.5, brought back to pH 8, and then tested. DEPC treatment further supports the role of His in the mechanical properties of threads. Values are means ± s.e.m.; N ranged from 6 to 9 threads for each treatment.

  • Fig. 9.
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    Fig. 9.

    (A) Young's modulus plotted against the previous strain cycle value for each pH treatment. Threads treated at pH 5 and below show a pH-induced reduction in modulus that becomes less prominent as the thread is cycled to higher strain values. (B) Reduction in modulus (ΔE) between strain cycles in Fig. 9A plotted against the strain that caused it for each of the pH treatments. Asterisks indicate where ΔE at pH 5 is significantly different from ΔE at pH 8 (P<0.01). Values are means ± s.e.m.; N ranged from 6 to 10 threads for each treatment.

  • Fig. 10.
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    Fig. 10.

    Molecular model of yield and self-healing in the distal byssal thread. Our data suggests that His–metal coordinate cross-links in the termini of the preCols are bonds that are reversibly sacrificed in yield and self-healing. A difference in healing rate between species, despite high homology of the His-rich domains, suggests that there is a separate entropic elastomeric component driving recovery. Since preCol D and preCol NG are co-localized in the distal thread, and since preCols are believed to align in register, we propose that the flanking domain of NG is playing this role. In this model, the stiffness of the amorphous Gly-rich flanking domain of preCol NG is roughly the same as the His-rich domain of preCol D, and the silk domain and NG His-rich domain are somewhat stiffer. During yield, the His–metal cross-links of preCol D begin to rupture as the Gly-rich domain begins to unravel, whereas the stiffer silk domain, collagen domains, and NG His-rich domain stay folded. When the tensile force is released, there is a time-dependent entropic drive for the Gly-rich domain of preCol NG to recover its initial length. In doing so, the histidine residues are brought back within proximity of one another, allowing the metal coordination bonds to reform and initial mechanical properties to be recovered. The covalent bonds between the ends of preCols in series have been proposed to be diDOPA cross-links.

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Research Article
Holdfast heroics: comparing the molecular and mechanical properties of Mytilus californianus byssal threads
Matthew J. Harrington, J. Herbert Waite
Journal of Experimental Biology 2007 210: 4307-4318; doi: 10.1242/jeb.009753
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Research Article
Holdfast heroics: comparing the molecular and mechanical properties of Mytilus californianus byssal threads
Matthew J. Harrington, J. Herbert Waite
Journal of Experimental Biology 2007 210: 4307-4318; doi: 10.1242/jeb.009753

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