Pseudoelastic behaviour of a natural material is achieved via reversible changes in protein backbone conformation

Abstract

The egg capsules of marine prosobranch gastropods, commonly know as whelks, function as a protective encapsulant for whelk embryos in wave-swept marine environments. The proteinaceous sheets comprising the wall of whelk egg capsules (WEC) exhibit long-range reversible extensibility with a hysteresis of up to 50 per cent, previously suggested to result from reversible changes in the structure of the constituent protein building blocks. Here, we further investigate the structural changes of the WEC biopolymer at various hierarchical levels using several different time-resolved in situ approaches. We find strong evidence in these biological polymers for a strain-induced reversible transition from an ordered conformational phase to a largely disordered one that leads to the characteristic reversible hysteretic behaviour, which is reminiscent of the pseudoelastic behaviour in some metallic alloys. On the basis of these results, we generate a simple numerical model incorporating a worm-like chain equation to explain the phase transition behaviour of the WEC at the molecular level.

Figure 1.

WEC mechanical performance. (a) Strand of egg capsules laid by B. canaliculatus (illustration top-right inset). For mechanical testing, individual egg capsules were removed from the strand, and strips were excised as indicated in the bottom-right inset. (b) Typical tensile stress–true strain curve for a strip of WEC material cycled four times with no rest between cycles. Arrows indicate loading and unloading, and the yield region of the curve is highlighted in grey.

Figure 2.

WAXD analysis of WEC during in situ tensile testing. Typical diffraction patterns of hydrated WEC tissue at (a) rest and (b) stretched to 69% true strain.

Figure 3.

SAXS reflections of WEC at rest and in the post-yield region. Small angle scattering patterns of WEC tissue at (a) 0% and (b) 40% true strain. (c) The intensity profiles from the areas indicated in (a) and (b) are plotted and represent two entirely different structural phases. The order of each peak is indicated and the D-spacings are calculated.

Figure 4.

In situ confocal Raman spectroscopic characterization of protein conformation in WEC. (a) Visual models of the peptide backbones in an α-helical versus β-strand conformation. A detailed structure of the β-strand conformation is shown, including side chains, to clarify the position of the dihedral bonds. (b) Rough illustration of a typical Ramachandran plot in which the Φ and Ψ dihedral angles are plotted on opposite axes. The dihedral angle combinations representative of α-helical (green) and extended β* (red) are outlined. The blue region represents partially allowed, but less energetically favourable conformations. White regions represent conformations that are not allowed for the majority of amino acid residues owing largely to steric conflicts. (c) Isotropic Raman spectra from the WEC tissue at several different strain values. Prominent Raman bands (amides I and III) undergo transitions during strain that are consistent with a shift from α-helical to more extended conformations (β*). (d) Raman depth scan of WEC layers at rest and at 69% true strain. Intensity profiles from polarized Raman imaging represent the ratio between the area under the β* peak and α peak of the amide III band. Conversion from α to β* occurs only in layers where protein fibres are oriented in the direction of tension.

Figure 5.

A summary of Raman, XRD and SAXS measurements during in situ tensile deformation of WEC tissue. The left and right columns of graphs represent the same data points plotted versus relative stress and true strain, respectively. Relative stress is defined such that the yield stress for each of the separate measurements is normalized to 1, in order to allow the measurements of the different techniques to be compared. The shapes of the data points correspond to a specific stress–strain curve in the first row used for normalization. The colour of the data point in the other rows indicates whether it corresponds to the α- (white) or β* (black)-phase. Raman and WAXD peak intensity values corresponding to the α- and β*-phases, respectively, are background corrected, but otherwise represent the raw data. The grey area in the left column is the yield region where both phases coexist, while the white regions on the left and the right correspond to the regions of the pure phases of α and β*, respectively. The dotted line in the lower right panel is a linear fit to the data in the β*-phase.

Figure 6.

Model for the phase coexistence in WEC fibres. (a) Simple schematic of conformational changes occurring in the WEC biopolymer during mechanical deformation. Unfolding of α-helical domains into a worm-like chain (WLC) extended conformation (β*) begins occurring at the onset of yield (position C) as a sudden conversion of discrete domains. During yield (position D) more and more α-domains are unfolded until all domains are in the WLC phase (position E). With further straining, the soft β* (WLC) domains continue to extend (position F). (b) Elastic energy of extended α-helices (green) and WLCs (red) according to equations (3.1) and (3.2) and with the parameters given in the text. The black dotted line corresponds to the common tangent between the energy curves. (c) The stress in the fibre can be obtained by taking the derivative of the elastic energy with respect to strain. The model predicts that for intermediate strains, the stress will remain constant (at the value given by the slope of the common tangent in (b)), allowing for a sudden transformation of individual α domains into β* domains, which occurs gradually over a true strain of approximately 40%. The green and red dots are measured values for the stress and strain in the two phases derived directly from the SAXS data (figure 3).