Daniel E. Morse

Molecular mechanistic origin of the toughness of natural adhesives, fibres and composites

Natural materials are renowned for their strength and toughness,,,,. Spider dragline silk has a breakage energy per unit weight two orders of magnitude greater than high tensile steel,, and is representative of many other strong natural fibres,,. The abalone shell, a composite of calcium carbonate plates sandwiched between organic material, is 3,000 times more fracture resistant than a single crystal of the pure mineral,. The organic component, comprising just a few per cent of the composite by weight, is thought to hold the key to nacres fracture toughness,. Ceramics laminated with organic material are more fracture resistant than non-laminated ceramics,, but synthetic materials made of interlocking ceramic tablets bound by a few weight per cent of ordinary adhesives do not have a toughness comparable to nacre. We believe that the key to nacres fracture resistance resides in the polymer adhesive, and here we reveal the properties of this adhesive by using the atomic force microscope to stretch the organic molecules exposed on the surface of freshly cleaved nacre. The adhesive fibres elongate in a stepwise manner as folded domains or loops are pulled open. The elongation events occur for forces of a few hundred piconewtons, which are smaller than the forces of over a nanonewton required to break the polymer backbone in the threads. We suggest that this ‘modular’ elongation mechanism might prove to be quite general for conveying toughness to natural fibres and adhesives, and we predict that it might be found also in dragline silk.

Biomimetic synthesis of ordered silica structures mediated by block copolypeptides

In biological systems such as diatoms and sponges, the formation of solid silica structures with precisely controlled morphologies is directed by proteins and polysaccharides and occurs in water at neutral pH and ambient temperature. Laboratory methods, in contrast, have to rely on extreme pH conditions and/or surfactants to induce the condensation of silica precursors into specific morphologies or patterned structures. This contrast in processing conditions and the growing demand for benign synthesis methods that minimize adverse environmental effects have spurred much interest in biomimetic approaches in materials science. The recent demonstration that silicatein—a protein found in the silica spicules of the sponge Tethya aurantia—can hydrolyse and condense the precursor molecule tetraethoxysilane to form silica structures with controlled shapes at ambient conditions seems particularly promising in this context. Here we describe synthetic cysteine-lysine block copolypeptides that mimic the properties of silicatein: the copolypeptides self-assemble into structured aggregates that hydrolyse tetraethoxysilane while simultaneously directing the formation of ordered silica morphologies. We find that oxidation of the cysteine sulphydryl groups, which is known to affect the assembly of the block copolypeptide, allows us to produce different structures: hard silica spheres and well-defined columns of amorphous silica are produced using the fully reduced and the oxidized forms of the copolymer, respectively.

Bone indentation recovery time correlates with bond reforming time

Despite centuries of work, dating back to Galileo, the molecular basis of bones toughness and strength remains largely a mystery. A great deal is known about bone microsctructure and the microcracks that are precursors to its fracture, but little is known about the basic mechanism for dissipating the energy of an impact to keep the bone from fracturing. Bone is a nanocomposite of hydroxyapatite crystals and an organic matrix. Because rigid crystals such as the hydroxyapatite crystals cannot dissipate much energy, the organic matrix, which is mainly collagen, must be involved. A reduction in the number of collagen cross links has been associated with reduced bone strength and collagen is molecularly elongated (‘pulled’) when bovine tendon is strained. Using an atomic force microscope, a molecular mechanistic origin for the remarkable toughness of another biocomposite material, abalone nacre, has been found. Here we report that bone, like abalone nacre, contains polymers with ‘sacrificial bonds’ that both protect the polymer backbone and dissipate energy. The time needed for these sacrificial bonds to reform after pulling correlates with the time needed for bone to recover its toughness as measured by atomic force microscope indentation testing. We suggest that the sacrificial bonds found within or between collagen molecules may be partially responsible for the toughness of bone.

γ-Aminobutyric Acid, a Neurotransmitter, Induces Planktonic Abalone Larvae to Settle and Begin Metamorphosis

γ-Aminobutyric acid (a simple amino acid and potent neurotransmitter in human brain and other tissues of higher animals) and certain of its congeners rapidly and synchronously induce planktonic larvae of the red abalone, Haliotis rufescens, to settle and commence behavioral and developmental metamorphosis. These naturally occurring inducers of algal origin apparently are responsible, in part, for the substrate-specific recruitment, induction of settling, and the onset of metamorphosis of abalone and other planktonic larvae upon specific algae which provide naturally favorable habitats for the young of these species in coastal waters. These observations provide a convenient experimental model for further analysis of the basic molecular mechanisms by which environmental and endogenous factors control the recruitment and development of planktonic larvae. Halogenated organic pesticides significantly interfere with larval settling, as quantified in a new bioassay based upon these findings.

Molecular Cloning and Characterization of Lustrin A, a Matrix Protein from Shell and Pearl Nacre of Haliotis rufescens*

A specialized extracellular matrix of proteins and polysaccharides controls the morphology and packing of calcium carbonate crystals and becomes occluded within the mineralized composite during formation of the molluscan shell and pearl. We have cloned and characterized the cDNA coding for Lustrin A, a newly described matrix protein from the nacreous layer of the shell and pearl produced by the abalone, Haliotis rufescens, a marine gastropod mollusc. The full-length cDNA is 4,439 base pairs (bp) long and contains an open reading frame coding for 1,428 amino acids. The deduced amino acid sequence reveals a highly modular structure with a high proportion of Ser (16%), Pro (14%), Gly (13%), and Cys (9%). The protein contains ten highly conserved cysteine-rich domains interspersed by eight proline-rich domains; a glycine- and serine-rich domain lies between the two cysteine-rich domains nearest the C terminus, and these are followed by a basic domain and a C-terminal domain that is highly similar to known protease inhibitors. The glycine- and serine-rich domain and at least one of the proline-rich domains show sequence similarity to proteins of two extracellular matrix superfamilies (one of which also is involved in the mineralized matrixes of bone, dentin, and avian eggshell). The arrangement of alternating cysteine-rich domains and proline-rich domains is strikingly similar to that found in frustulins, the proteins that are integral to the silicified cell wall of diatoms. Its modular structure suggests that Lustrin A is a multifunctional protein, whereas the occurrence of related sequences suggest it is a member of a multiprotein family.

Recruitment and metamorphosis of Haliotis larvae induced by molecules uniquely available at the surfaces of crustose red algae

Crustose red algae induce substratum-specific settlement, attachment and metamorphosis of the planktonic larvae of Haliotis rufescens Swainson (gastropod mollusc), upon direct contact by the larvae with any of a number of algal species tested. Larvae are not induced by contact with intact foliose red, brown or green macroalgae. Geniculate red algae are only slightly active. Larval settlement and metamorphosis are shown to be triggered by a class of chemical inducers associated with macromolecules and found in extracts of all species of crustose, geniculate, and foliose red algae tested; these inducers are not found in extracts of brown or green macroalgae. The substratum specificity of larval settlement and metamorphosis is shown to result from the unique availability of these inducers at the surfaces of the crustose red algae. Using a newly-developed improved method of purification based upon size-separation by gel-filtration, followed by ion-exchange chromatography over a diethylaminoethyl (DEAE)-acrylamide matrix, the principal inducer of Haliotis larval settlement and metamorphosis has been resolved from the red algal phycobiliproteins. Sensitivity of this inducer to reduction in molecular weight by digestion with trypsin demonstrates that this inducer is associated with protein.

Control of larval metamorphosis and recruitment in sympatric agariciid corals

Abstract The larvae of three sympatric shallow-water agariciid corals, Agaricia tenuifolia Dana, A. agaricites humilis Verrill, and A. agaricites dana Milne Edwards et Haime, are shown to be induced to metamorphose by crustose coralline red algae (CCA). These corals display different degrees of stringency and specificity in their requirements for CCA to induce metamorphosis, and different responses to light in the control of the distribution of newly metamorphosed individuals. The morphogenetic inducer from CCA has been fractionated by ultrafiltration, and shown to be a water-insoluble, ether-insoluble, and acetone-insoluble unstable biochemical. This inducer of agariciid metamorphosis is different from the water-soluble peptide inducer of gastropod metamorphosis previously isolated from CCA. Transduction of the morphogenetic signal in the agariciid larvae also is apparently controlled by an internal pathway that is different from the signal-transduction pathway found to control metamorphosis in several other species. Analysis of the distribution of recruits of two of the agariciid species indicates that larval requirements and specificities for metamorphosis may contribute significantly to determine the distribution of recruits in the natural environment. Our evidence suggests that differences in larval requirements for metamorphosis thus may contribute to the maintenance of niche diversification among the sympatric shallow-water agariciids. The competence and CCA requirement of the larvae of these species persist for at least 1–2 wk in the plankton, thereby promoting dispersal and site-specific metamorphosis. For these sympatric shallow-water agariciid corals, then, larval metamorphosis and recruitment are not wholly stochastic lottery-like processes, but instead appear to be determined, in part, by larval recognition of, and responses to, environmental and biochemical factors that can be experimentally resolved and identified.

Silicon biotechnology: harnessing biological silica production to construct new materials

Silicon, the basis of semiconductors and many advanced materials, is an essential element for higher plants and animals, yet its biology is poorly understood. Many invertebrates produce exquisitely controlled silica structures with a nanoscale precision exceeding present human ability. Biotechnology is starting to reveal the proteins, genes and molecular mechanisms that control this synthesis in marine organisms that produce large amounts of silica. Discovering the mechanisms governing biosilicification offers the prospect of developing environmentally benign routes to synthesize new silicon-based materials and to resolve the biological use of silicon in higher organisms.

Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida.

Almost all mineralized tissues contain proteins that are unusually acidic. As they are also often intimately associated with the mineral phase, they are thought to fulfill important functions in controlling mineral formation. Relatively little is known about these important proteins, because their acidic nature causes technical difficulties during purification and characterization procedures. Much effort has been made to overcome these problems, particularly in the study of mollusk‐shell formation. To date about 16 proteins from mollusk‐shell organic matrices have been sequenced, but only two are unusually rich in aspartic and glutamic acids. Here we screened a cDNA library made from the mRNA of the shell‐forming cells of a bivalve, Atrina rigida, using probes for short Asp‐containing repeat sequences, and identified ten different proteins. Using more specific probes designed from one subgroup of conserved sequences, we obtained the full sequences of a family of seven aspartic acid‐rich proteins, which we named “Asprich”; a subfamily of the unusually acidic shell‐matrix proteins. Polyclonal antibodies raised against a synthetic peptide of the conserved acidic1 domain of these proteins reacted specifically with the matrix components of the calcitic prismatic layer, but not with those of the aragonitic nacreous layer. Thus the Asprich proteins are constituents of the prismatic layer shell matrix. We can identify different domains within these sequences, including a signal peptide characteristic of proteins destined for extracellular secretion, a conserved domain rich in aspartic acid that contains a sequence very similar to the calcium‐binding domain of Calsequestrin, and another domain rich in aspartic acid, that varies between the seven sequences. We also identified a domain with DEAD repeats that may have Mg‐binding capabilities. Although we do not know, as yet, the function of these proteins, their generally conserved sequences do indicate that they might well fulfill basic functions in shell formation.

The tube cement of Phragmatopoma californica: a solid foam

SUMMARY Phragmatopoma californica is a marine polychaete that builds protective tubes by joining bits of shell and sand grains with a secreted proteinaceous cement. The cement forms a solid foam (closed cells) via covalent crosslinking, as revealed by electron and laser scanning confocal microscopy. The cement contains extractable calcium and magnesium, and non-extractable phosphorus. Amino acid analysis demonstrated that the phosphorus is in the form of phosphoserine and that >90% of serine in the cement (i.e. 28 mol% of residues) is phosphorylated. In addition to previously identified basic proteins, the cement contains a highly acidic polyphosphoserine protein as a major component. We propose a model for the structure and bonding mechanism of the cement that has the following major features: (1) within the secretory pathway of cement gland cells, the electrostatic association of the oppositely charged proteins and divalent cations (Ca2+ and Mg2+) condense the cement proteins into dehydrated secretory granules; (2) the condensation of the cement leads to the separation of the solution into two aqueous phases (complex coacervation) that creates the closed cell foam structure of the cement; (3) rehydration of the condensed cement granules after deposition onto tube particles contributes to the displacement of water from the mineral substrate to facilitate underwater adhesion; and (4) after secretion, covalent cross-linking through oxidative coupling of DOPA gradually solidifies the continuous phase of the cement to set the porous structure.