Article pubs.acs.org/Biomac
Hierarchical Assembly of Tough Bioelastomeric Egg Capsules is Mediated by a Bundling Protein Jun Jie Loke,†,‡ Akshita Kumar,‡,§ Shawn Hoon,∥ Chandra Verma,§,⊥ and Ali Miserez*,†,‡,§ †
School of Materials Science and Engineering, Nanyang Technological University (NTU), Singapore 639798, Singapore Centre for Biomimetic Sensor Science (CBSS), NTU, Singapore 637553, Singapore § School of Biological Sciences, NTU, Singapore 637551, Singapore ∥ Molecular Engineering Lab, Biomedical Sciences Institute, Agency for Science Technology and Research (A*STAR), Singapore 138673, Singapore ⊥ Bioinformatics Institute, A*STAR, 30 Biopolis Street, Singapore 138671, Singapore ‡
S Supporting Information *
ABSTRACT: Marine snail egg capsules are shock-absorbing bioelastomers made from precursor “egg case proteins” (ECPs) that initially lack long-range order. During capsule formation, these proteins self-assemble into coiled-coil filaments that subsequently align into microscopic layers, a multiscale process which is crucial to the capsules’ shockabsorbing properties. In this study, we show that the selfassembly of ECPs into their functional capsule material is mediated by a bundling protein that facilitates the aggregation of coiled-coil building blocks and their gelation into a prefinal capsule prior to final stabilization. This low molecular weight bundling protein, termed Pugilina cochlidium Bundling Protein (PcBP), led to gelation of native extracts from gravid snails, whereas crude extracts lacking PcBP did not gelate and remained as a protein solution. Refolding and reconcentration of recombinant PcBP induced bundling and aggregation of ECPs, as evidenced by ECPs oligomerization. We propose that the secretion of PcBP in vivo is a time-specific event during the embryo encapsulation process prior to cross-linking in the ventral pedal gland (VPG). Using molecular dynamics (MD) simulations, we further propose plausible disulfide binding sites stabilizing two PcBP monomers, as well as a polarized surface charge distribution, which we suggest plays an important role in the bundling mechanism. Overall, this study shows that controlled bundling is a key step during the extra-cellular self-assembly of egg capsules, which has previously been overlooked.
1. INTRODUCTION Marine snails of the Melongenidae family (whelk) are oviparous animals that encapsulate their embryos within proteinaceous egg capsules, which the females lay on the ocean bed of the intertidal zone (Figure 1A).1 The egg capsules must be able to protect the developing embryos against harsh environmental stresses, including predatory attacks, wave forces, or hydration/ dehydration cycles, while at the same time allowing nutrients and gaseous exchange. To fulfill its biological function in such a hostile environment, the egg capsule material is made of a mechanically robust bioelastomer, which is considered a stimulating biological material for bioinspired shock-absorbing bioelastomers and gels. Under uniaxial tension, the capsular material exhibits a complex strain-dependent response (Figure 1C).2−5 At low strains up to ca. 5%, the capsule is linear elastic with a comparatively high elastic modulus in the range 50−70 MPa depending on the species and testing conditions. The intermediate strain regime (5% to 70% engineering strain) corresponds to a pseudo-yielding regime whereby stress increases only moderately with strain. As the strain further © 2017 American Chemical Society
increases, the material strain-stiffens until failure occurs at strain values of about 170%. However, if the material is unloaded prior to failure, it almost instantaneously returns to its original length with a prominent stress−strain hysteresis, resulting in notable mechanical absorption capacity. This combination of properties confers to the capsular material several advantages in comparison to biocompatible elastomers and hydrogels used in tissue engineering or as matrices for 3D stem cell culture.6 In situ tensile testing, in conjunction with wide-angle X-ray scattering (WAXS) as well as Raman spectroscopy, have established that mature capsules comprise of α-helical coiledcoil building blocks, and that large-scale deformation occurs via the transition of coiled-coils into extended β-sheets, with the reversible coiled-coil → extended β-sheet transition responsible for molecular-scale energy absorption,3,4 a mechanism that appears to be widespread in coiled-coil based biological fibers (Figure 1B).7 Moreover, small-angle X-ray scattering (SAXS) Received: December 6, 2016 Revised: February 14, 2017 Published: February 14, 2017 931
DOI: 10.1021/acs.biomac.6b01810 Biomacromolecules 2017, 18, 931−942
Article
Biomacromolecules
a high concentration (8 M) of urea. While the formation of heterodimeric coiled-coil constitutes an important initial milestone toward biomimicry of egg capsule-like materials, there are some limitations with regard to larger-scale assembly. The obtained nanofilaments were limited to less than 10 nm diameter, which may be related to the absence of the R/ KLLEGE peptide, a highly conserved sequence adjacent to the central coiled-coil domain in IFs that enables their lateral alignment and assembly into higher order structures.12 Furthermore, the self-assembly method is questionable from a biological standpoint, since native capsules are readily synthesized in vivo by a process that is unlikely to involve gradual renaturation of the ECPs as done during the stepwise dialysis protocol. Consequently, we previously postulated that egg capsule assembly might be facilitated by the presence of chaperon or “bundling” proteins that could help the larger scale fibrillization process and the ensuing gelation prior to the final cross-linking step. This hypothesis was additionally hinted by our previous observations of the presence of a faint, low-molecular weight protein (
