James C Weaver

Publications of James C Weaver

• Four-Stranded Coiled-Coil Elastic Protein in the Byssus of the Giant Clam, Tridacna maxima.

  Authors: Ali Miserez, Youli Li, Joel Cagnon, James C Weaver, J Herbert Waite

  Biomacromolecules. 12/2011; 13(2):332-41.

  An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle ofAn elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.

• Microindentation for in vivo measurement of bone tissue mechanical properties in humans.

  Authors: Adolfo Diez-Perez, Roberto Güerri, Xavier Nogues, Enric Cáceres, Maria Jesus Peña, Leonardo Mellibovsky, Connor Randall, Daniel Bridges, James C Weaver, Alexander Proctor, Davis Brimer, Kurt J Koester, Robert O Ritchie, Paul K Hansma

  Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 02/2010; 25(8):1877-85.

  Bone tissue mechanical properties are deemed a key component of bone strength, but their assessment requires invasive procedures. Here we validate a new instrument, a reference point indentationBone tissue mechanical properties are deemed a key component of bone strength, but their assessment requires invasive procedures. Here we validate a new instrument, a reference point indentation (RPI) instrument, for measuring these tissue properties in vivo. The RPI instrument performs bone microindentation testing (BMT) by inserting a probe assembly through the skin covering the tibia and, after displacing periosteum, applying 20 indentation cycles at 2 Hz each with a maximum force of 11 N. We assessed 27 women with osteoporosis-related fractures and 8 controls of comparable ages. Measured total indentation distance (46.0 +/- 14 versus 31.7 +/- 3.3 microm, p = .008) and indentation distance increase (18.1 +/- 5.6 versus 12.3 +/- 2.9 microm, p = .008) were significantly greater in fracture patients than in controls. Areas under the receiver operating characteristic (ROC) curve for the two measurements were 93.1% (95% confidence interval [CI] 83.1-100) and 90.3% (95% CI 73.2-100), respectively. Interobserver coefficient of variation ranged from 8.7% to 15.5%, and the procedure was well tolerated. In a separate study of cadaveric human bone samples (n = 5), crack growth toughness and indentation distance increase correlated (r = -0.9036, p = .018), and scanning electron microscope images of cracks induced by indentation and by experimental fractures were similar. We conclude that BMT, by inducing microscopic fractures, directly measures bone mechanical properties at the tissue level. The technique is feasible for use in clinics with good reproducibility. It discriminates precisely between patients with and without fragility fracture and may provide clinicians and researchers with a direct in vivo measurement of bone tissue resistance to fracture.

• Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.

  Authors: Michi Izumi, Alison M Sweeney, Daniel DeMartini, James C Weaver, Meghan L. Powers, Andrea Tao, Tania V. Silvas, Ryan M Kramer, Wendy J Crookes-Goodson, Lydia M Mäthger, Rajesh R Naik, Roger T Hanlon, Daniel E Morse

  Journal of the Royal Society, Interface / the Royal Society. 09/2009;

  Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static andMany cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in which the dermis possesses a dynamic iridescent component with reflective, coloured structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (ACh). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses ACh- and calcium-induced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous ACh. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo.

• Hierarchical assembly of the siliceous skeletal lattice of the hexactinellid sponge Euplectella aspergillum.

  Authors: James C Weaver, Joanna Aizenberg, Georg E Fantner, David Kisailus, Alexander Woesz, Peter Allen, Kirk Fields, Michael J Porter, Frank W Zok, Paul K Hansma, Peter Fratzl, Daniel E Morse

  Journal of structural biology. 05/2007; 158(1):93-106.

  Despite its inherent mechanical fragility, silica is widely used as a skeletal material in a great diversity of organisms ranging from diatoms and radiolaria to sponges and higher plants. In additionDespite its inherent mechanical fragility, silica is widely used as a skeletal material in a great diversity of organisms ranging from diatoms and radiolaria to sponges and higher plants. In addition to their micro- and nanoscale structural regularity, many of these hard tissues form complex hierarchically ordered composites. One such example is found in the siliceous skeletal system of the Western Pacific hexactinellid sponge, Euplectella aspergillum. In this species, the skeleton comprises an elaborate cylindrical lattice-like structure with at least six hierarchical levels spanning the length scale from nanometers to centimeters. The basic building blocks are laminated skeletal elements (spicules) that consist of a central proteinaceous axial filament surrounded by alternating concentric domains of consolidated silica nanoparticles and organic interlayers. Two intersecting grids of non-planar cruciform spicules define a locally quadrate, globally cylindrical skeletal lattice that provides the framework onto which other skeletal constituents are deposited. The grids are supported by bundles of spicules that form vertical, horizontal and diagonally ordered struts. The overall cylindrical lattice is capped at its upper end by a terminal sieve plate and rooted into the sea floor at its base by a flexible cluster of barbed fibrillar anchor spicules. External diagonally oriented spiral ridges that extend perpendicular to the surface further strengthen the lattice. A secondarily deposited laminated silica matrix that cements the structure together additionally reinforces the resulting skeletal mass. The mechanical consequences of each of these various levels of structural complexity are discussed.

• In vitro study of magnesium-calcite biomineralization in the skeletal materials of the seastar Pisaster giganteus.

  Authors: Subramanyam Gayathri, Rajamani Lakshminarayanan, James C Weaver, Daniel E Morse, R Manjunatha Kini, Suresh Valiyaveettil

  Chemistry (Weinheim an der Bergstrasse, Germany). 02/2007; 13(11):3262-8.

  The mechanisms of formation of biogenic magnesium-rich calcite remain an enigma. Here we present ultrastructural and compositional details of ossicles from the seastar Pisaster giganteusThe mechanisms of formation of biogenic magnesium-rich calcite remain an enigma. Here we present ultrastructural and compositional details of ossicles from the seastar Pisaster giganteus (Echinodermata, Asteroidea). Powder X-ray diffraction, infrared spectroscopy and elemental analyses confirm that the ossicles are composed of magnesium-rich calcite, whilst also containing about 0.01 % (w/w) of soluble organic matrix (SOM) as an intracrystalline component. Amino acid analysis and N-terminal sequencing revealed that this mixture of intracrystalline macromolecules consists predominantly of glycine-rich polypeptides. In vitro calcium carbonate precipitation experiments indicate that the SOM accelerates the conversion of amorphous calcium carbonate (ACC) into its final crystalline product. From this observation and from the discovery of ACC in other closely related taxa, it is suggested that substitution of magnesium into the calcite lattice through a transient precursor phase may be a universal phenomenon prevalent across the phylum echinodermata.

• Kinetically controlled catalytic formation of zinc oxide thin films at low temperature.

  Authors: David Kisailus, Birgit Schwenzer, John Gomm, James C Weaver, Daniel E Morse

  Journal of the American Chemical Society. 09/2006; 128(31):10276-80.

  We developed a unique method to produce ZnO thin films by kinetically controlled catalytic hydrolysis of a molecular precursor at low temperature, operating in conjunction with the vectorial controlWe developed a unique method to produce ZnO thin films by kinetically controlled catalytic hydrolysis of a molecular precursor at low temperature, operating in conjunction with the vectorial control of crystal growth. Using a system in which the diffusion of a volatile catalyst into a solution of molecular precursor of the metal oxide limits the rate of hydrolysis and establishes a gradient of catalyst concentration, we investigated the nucleation of textured nanoparticles at the gas-liquid interface and characterized their subsequent growth. Use of this slow diffusion method combined with prediction of molecular species using a partial charge model enables a higher level of organizational control than obtained in other low-temperature synthesis methods, without the use of organic molecules. Various metal oxides and their morphologies and chemical compositions can be tailored for specific applications using this relatively simple approach.

• Self-assembled bifunctional surface mimics an enzymatic and templating protein for the synthesis of a metal oxide semiconductor.

  Authors: David Kisailus, Quyen Truong, Yosuke Amemiya, James C Weaver, Daniel E Morse

  Proceedings of the National Academy of Sciences of the United States of America. 05/2006; 103(15):5652-7.

  The recent discovery and characterization of silicatein, a mineral-synthesizing enzyme that assembles to form the filamentous organic core of the glassy skeletal elements (spicules) of a marineThe recent discovery and characterization of silicatein, a mineral-synthesizing enzyme that assembles to form the filamentous organic core of the glassy skeletal elements (spicules) of a marine sponge, has led to the development of new low-temperature synthetic routes to metastable semiconducting metal oxides. These protein filaments were shown in vitro to catalyze the hydrolysis and structurally direct the polycondensation of metal oxides at neutral pH and low temperature. Based on the confirmation of the catalytic mechanism and the essential participation of specific serine and histidine residues (presenting a nucleophilic hydroxyl and a nucleophilicity-enhancing hydrogen-bonding imidazole nitrogen) in silicatein's catalytic active site, we therefore sought to develop a synthetic mimic that provides both catalysis and the surface determinants necessary to template and structurally direct heterogeneous nucleation through condensation. Using lithographically patterned poly(dimethylsiloxane) stamps, bifunctional self-assembled monolayer surfaces containing the essential catalytic and templating elements were fabricated by using alkane thiols microcontact-printed on gold substrates. The interface between chemically distinct self-assembled monolayer domains provided the necessary juxtaposition of nucleophilic (hydroxyl) and hydrogen-bonding (imidazole) agents to catalyze the hydrolysis of a gallium oxide precursor and template the condensed product to form gallium oxohydroxide (GaOOH) and the defect spinel, gamma-gallium oxide (gamma-Ga(2)O(3)). Using this approach, the production of patterned substrates for catalytic synthesis and templating of semiconductors for device applications can be envisioned.

• Developing macroporous bicontinuous materials as scaffolds for tissue engineering.

  Authors: Monique Martina, Gayathri Subramanyam, James C Weaver, Dietmar W Hutmacher, Daniel E Morse, Suresh Valiyaveettil

  Biomaterials. 11/2005; 26(28):5609-16.

  Calcareous skeletal elements (ossicles) isolated from the seastar, Pisaster giganteus, were characterized and tested as potential biocompatible substrates for cellular attachment. These ossicles haveCalcareous skeletal elements (ossicles) isolated from the seastar, Pisaster giganteus, were characterized and tested as potential biocompatible substrates for cellular attachment. These ossicles have a remarkably robust open-framework architecture with an interconnected network of ca. 10 microm diameter pores. Scanning electron and confocal microscopy was used to characterize the cell-substrate interaction. Cell culturing experiments revealed that the cells firmly attach to the ossicle surface, forming cell aggregates of several layers thick. The anchored cells extended to form 'bridges' between the openings in the bicontinuous framework and the degree of coverage increased as culture time progressed. Osteoblasts grown on the ossicles were found to be viable up to 32 days after initial seeding, as proven by assaying with AlamarBlue and FDA/PI staining indicating the ossicle's potential as an alternative highly effective tissue scaffold. Given the limitation in availability of this natural material, the results presented here should be seen as offering guidelines for future development of synthetic materials with physical and chemical properties strongly conducive to bone repair and restoration.

• Sacrificial bonds and hidden length dissipate energy as mineralized fibrils separate during bone fracture.

  Authors: Georg E Fantner, Tue Hassenkam, Johannes H Kindt, James C Weaver, Henrik Birkedal, Leonid Pechenik, Jacqueline A Cutroni, Geraldo A G Cidade, Galen D Stucky, Daniel E Morse, Paul K Hansma

  Nature materials. 09/2005; 4(8):612-6.

  Properties of the organic matrix of bone as well as its function in the microstructure could be the key to the remarkable mechanical properties of bone. Previously, it was found that on the molecularProperties of the organic matrix of bone as well as its function in the microstructure could be the key to the remarkable mechanical properties of bone. Previously, it was found that on the molecular level, calcium-mediated sacrificial bonds increased stiffness and enhanced energy dissipation in bone constituent molecules. Here we present evidence for how this sacrificial bond and hidden length mechanism contributes to the mechanical properties of the bone composite, by investigating the nanoscale arrangement of the bone constituents and their interactions. We find evidence that bone consists of mineralized collagen fibrils and a non-fibrillar organic matrix, which acts as a 'glue' that holds the mineralized fibrils together. We believe that this glue may resist the separation of mineralized collagen fibrils. As in the case of the sacrificial bonds in single molecules, the effectiveness of this mechanism increases with the presence of Ca2+ ions.

• Skeleton of Euplectella sp.: structural hierarchy from the nanoscale to the macroscale.

  Authors: Joanna Aizenberg, James C Weaver, Monica S Thanawala, Vikram C Sundar, Daniel E Morse, Peter Fratzl

  Science (New York, N.Y.). 08/2005; 309(5732):275-8.

  Structural materials in nature exhibit remarkable designs with building blocks, often hierarchically arranged from the nanometer to the macroscopic length scales. We report on the structuralStructural materials in nature exhibit remarkable designs with building blocks, often hierarchically arranged from the nanometer to the macroscopic length scales. We report on the structural properties of biosilica observed in the hexactinellid sponge Euplectella sp. Consolidated, nanometer-scaled silica spheres are arranged in well-defined microscopic concentric rings glued together by organic matrix to form laminated spicules. The assembly of these spicules into bundles, effected by the laminated silica-based cement, results in the formation of a macroscopic cylindrical square-lattice cagelike structure reinforced by diagonal ridges. The ensuing design overcomes the brittleness of its constituent material, glass, and shows outstanding mechanical rigidity and stability. The mechanical benefits of each of seven identified hierarchical levels and their comparison with common mechanical engineering strategies are discussed.

• The tube cement of Phragmatopoma californica: a solid foam.

  Authors: Russell J Stewart, James C Weaver, Daniel E Morse, J Herbert Waite

  The Journal of experimental biology. 01/2005; 207(Pt 26):4727-34.

  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 (closedPhragmatopoma 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.

• Influence of the degradation of the organic matrix on the microscopic fracture behavior of trabecular bone.

  Authors: Georg E Fantner, Henrik Birkedal, Johannes H Kindt, Tue Hassenkam, James C Weaver, Jacquelin A Cutroni, Bonnie L Bosma, Lukmaan Bawazer, Marquesa M Finch, Geraldo A G Cidade, Daniel E Morse, Galen D Stucky, Paul K Hansma

  Bone. 12/2004; 35(5):1013-22.

  In recent years, the important role of the organic matrix for the mechanical properties of bone has become increasingly apparent. It is therefore of great interest to understand the interactionsIn recent years, the important role of the organic matrix for the mechanical properties of bone has become increasingly apparent. It is therefore of great interest to understand the interactions between the organic and inorganic constituents of bone and learn the mechanisms by which the organic matrix contributes to the remarkable properties of this complex biomaterial. In this paper, we present a multifaceted view of the changes of bone's properties due to heat-induced degradation of the organic matrix. We compare the microscopic fracture behavior (scanning electron microscopy; SEM), the topography of the surfaces (atomic force microscopy; AFM), the condition of bone constituents [X-ray diffraction (XRD), thermogravimetric analysis (TGA), and gel electrophoresis], and the macromechanical properties of healthy bovine trabecular bone with trabecular bone that has a heat-degraded organic matrix. We show that heat treatment changes the microfracture behavior of trabecular bone. The primary failure mode of untreated trabecular bone is fibril-guided delamination, with mineralized collagen filaments bridging the gap of the microcrack. In contrast, bone that has been baked at 200 degrees C fractures nondirectionally like a brittle material, with no fibers spanning the microcracks. Finally, bone that has been boiled for 2 h in PBS solution fractures by delamination with many small filaments spanning the microcracks, so that the edges of the microcracks become difficult to distinguish. Of the methods we used, baking most effectively weakens the mechanical strength of bone, creating the most brittle material. Boiled bone is stronger than baked bone, but weaker than untreated bone. Boiled bone is more elastic than untreated bone, which is in turn more elastic than baked bone. These studies clearly emphasize the importance of the organic matrix in affecting the fracture mechanics of bone.

• High-resolution AFM imaging of intact and fractured trabecular bone.

  Authors: Tue Hassenkam, Georg E Fantner, Jacqueline A Cutroni, James C Weaver, Daniel E Morse, Paul K Hansma

  Bone. 08/2004; 35(1):4-10.

  Nanoscale structural analyses of biomineralized materials can frequently help elucidate important structure-function relationships in these complex organic-inorganic composites. Atomic forceNanoscale structural analyses of biomineralized materials can frequently help elucidate important structure-function relationships in these complex organic-inorganic composites. Atomic force microscope (AFM) imaging of the exterior surface of trabecular bone reveals a densely woven structure of collagen fibrils, banded with a 67-nm periodicity, and densely packed mineral plates. The mineral plates on the collagen fibrils overlap and exhibit a large range of plate diameters from 30 to 200 nm. On the collagen fibrils, small nodular features, spaced 20-30 nm, run perpendicular to the fibrils. In some cases, these nodules are also seen on filaments extending between collagen fibrils. We hypothesize that these protrusions are noncollagenous proteins such as proteoglycans and may have collapsed into compact structures when the sample was dried. AFM images of pristine fractured surfaces reveal a dense array of mineral plates. In a few isolated locations, short sections of bare collagen fibrils are visible. In other regions, the existence of the underlying collagen fibrils can be inferred from the linear patterns of the mineral plates. Fractured samples, rinsed to remove mineral plates, reveal separated collagen fibrils on the fractured surfaces. These fibrils are often covered with protrusions similar to those observed on the exterior surfaces but are less organized. In addition, as on the exterior surfaces, there are sometimes small filaments extending between neighboring collagen fibrils. These studies provide important insights into the nanostructured architecture of this complex biocomposite.

• Biological glass fibers: correlation between optical and structural properties.

  Authors: Joanna Aizenberg, Vikram C Sundar, Andrew D Yablon, James C Weaver, Gang Chen

  Proceedings of the National Academy of Sciences of the United States of America. 04/2004; 101(10):3358-63.

  Biological systems have, through the course of time, evolved unique solutions for complex optical problems. These solutions are often achieved through a sophisticated control of fine structuralBiological systems have, through the course of time, evolved unique solutions for complex optical problems. These solutions are often achieved through a sophisticated control of fine structural features. Here we present a detailed study of the optical properties of basalia spicules from the glass sponge Euplectella aspergillum and reconcile them with structural characteristics. We show these biosilica fibers to have a distinctive layered design with specific compositional variations in the glass/organic composite and a corresponding nonuniform refractive index profile with a high-index core and a low-index cladding. The spicules can function as single-mode, few-mode, or multimode fibers, with spines serving as illumination points along the spicule shaft. The presence of a lens-like structure at the end of the fiber increases its light-collecting efficiency. Although free-space coupling experiments emphasize the similarity of these spicules to commercial optical fibers, the absence of any birefringence, the presence of technologically inaccessible dopants in the fibers, and their improved mechanical properties highlight the advantages of the low-temperature synthesis used by biology to construct these remarkable structures.

• Nanostructural features of demosponge biosilica.

  Authors: James C Weaver, Lía I Pietrasanta, Niklas Hedin, Bradley F Chmelka, Paul K Hansma, Daniel E Morse

  Journal of structural biology. 01/2004; 144(3):271-81.

  Recent interest in the optical and mechanical properties of silica structures made by living sponges, and the possibility of harnessing these mechanisms for the synthesis of advanced materials andRecent interest in the optical and mechanical properties of silica structures made by living sponges, and the possibility of harnessing these mechanisms for the synthesis of advanced materials and devices, motivate our investigation of the nanoscale structure of these remarkable biomaterials. Scanning electron and atomic force microscopic (SEM and AFM) analyses of the annular substructure of demosponge biosilica spicules reveals that the deposited material is nanoparticulate, with a mean particle diameter of 74+/-13 nm. The nanoparticles are deposited in alternating layers with characteristic etchant reactivities. Further analyses of longitudinally fractured spicules indicate that each deposited layer is approximately monoparticulate in thickness and exhibits extensive long range ordering, revealing an unanticipated level of nanoscale structural complexity. NMR data obtained from differentially heated spicule samples suggest that the etch sensitivity exhibited by these annular domains may be related to variation in the degree of silica condensation, rather than variability in the inclusion of organics. In addition, AFM phase imaging in conjunction with results obtained from HF and alkaline etching experiments suggest that at various stages in spicule biosynthesis, regions of unusually low silica condensation are deposited, indicating a possible interruption in normal spicule formation. While this discovery of nanoparticulate silica aggregation in demosponge skeletal elements is likely to reflect the intrinsic kinetic tendency of silica to form such particles during polycondensation, the heirarchical organization of these nanoparticles is biologically unique.

• Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains.

  Authors: Martina Michenfelder, Germaine Fu, Camille Lawrence, James C Weaver, Brandon A Wustman, Laura Taranto, John Spencer Evans, Daniel E Morse

  Biopolymers. 01/2004; 70(4):522-33.

  Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation,Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation, and phase of the deposited mineral. Previously, atomic force microscopy demonstrated that abalone nacre proteins bind to growing step edges and to specific crystallographic faces of calcite, suggesting that inhibition of calcite growth may be one of the molecular processes required for growth of the less thermodynamically stable aragonite phase. Previous experiments were done with protein mixtures. To elucidate the role of single proteins, we have characterized two proteins isolated from the aragonitic component of nacre of the red abalone, Haliotis rufescens. These proteins, purified by hydrophobic interaction chromatography, are designated AP7 and AP24 (aragonitic protein of molecular weight 7 kDa and 24 kDa, respectively). Degenerate oligonucleotide primers corresponding to N-terminal and internal peptide sequences were used to amplify cDNA clones by a polymerase chain reaction from a mantle cDNA library; the deduced primary amino acid sequences are presented. Preliminary crystal growth experiments demonstrate that protein fractions enriched in AP7 and AP24 produced CaCO(3) crystals with morphology distinct from crystals grown in the presence of the total mixture of soluble aragonite-specific proteins. Peptides corresponding to the first 30 residues of the N-terminal sequences of both AP7 and AP24 were generated. The synthetic peptides frustrate the progression of step edges of a growing calcite surface, indicating that sequence features within the N-termini of AP7 and AP24 include domains that interact with CaCO(3). CD analyses demonstrate that the N-terminal peptide sequences do not possess significant percentages of alpha-helix or beta-strand secondary structure in solution. Instead, in both the presence and absence of Ca(II), the peptides retain unfolded conformations that may facilitate protein-mineral interaction.

• Molecular biology of demosponge axial filaments and their roles in biosilicification.

  Authors: James C Weaver, Daniel E Morse

  Microscopy research and technique. 12/2003; 62(4):356-67.

  For hundreds of years, the skeletal elements of marine and freshwater sponges have intrigued investigators with a diverse array of remarkably complex morphologies. Early studies of demospongeFor hundreds of years, the skeletal elements of marine and freshwater sponges have intrigued investigators with a diverse array of remarkably complex morphologies. Early studies of demosponge monaxonal megascleres revealed the presence of a central organic axial filament running their entire length. Until recently, however, the precise function of these axial filaments was largely unknown. The spicules from the temperate Eastern Pacific demosponge, Tethya aurantia, comprise approximately 75% of the dry weight of this species, facilitating the large-scale isolation and purification of the biosilica-associated proteins. Silicateins, the most abundant proteins comprising the axial filaments of these spicules, prove to be members of a well-known superfamily of proteolytic and hydrolytic enzymes and can be easily collected after silica demineralization with hydrofluoric acid. Consistent with these findings, the intact filaments are more than simple, passive templates; in vitro, they actively catalyze and spatially direct the hydrolysis and polycondensation of silicon alkoxides to yield silica at neutral pH and low temperature. Catalytic activity also is exhibited by the monomeric subunits obtained by disaggregation of the protein filaments and those produced from recombinant DNA templates cloned in bacteria. These proteins also can be used to direct the polymerization of organosilicon polymers (silicones) from the corresponding organically functionalized silicon alkoxides. Based on these observations, the silicateins are currently being used as models for the design of biomimetic agents with unique catalytic and structure-directing properties. The presence of axial filaments in a diversity of spicule types and the evolutionary implications of these findings are also discussed.

• Structure-function studies of the lustrin A polyelectrolyte domains, RKSY and D4.

  Authors: Brandon A Wustman, James C Weaver, Daniel E Morse, John Spencer Evans

  Connective tissue research. 02/2003; 44 Suppl 1:10-5.

  The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins not onlyThe lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins not only exhibit elastomeric behavior within the mineralized matrix, but also adhesion to the aragonite-containing composite layer. One member of the lustrin superfamily, Lustrin A, has been sequenced; the protein is organized into defined, modular sequence domains that are hypothesized to perform separate functions (i.e., force unfolding, mineral adhesion, intermolecular binding) within the Lustrin A protein. Using nuclear magnetic resonance (NMR) and in vitro mineralization assays, we investigated structure-function relationships for two Lustrin A putative mineral binding domains, the 30 AA Arg, Lys, Tyr, Ser-rich (RKSY) and the 24 AA Asp-rich (D4) sequence regions domain of the Lustrin A protein. The results indicate that both sequences adopt open, unfolded structures that represent either extended or random coil states. Using geologic calcite overgrowth assays and scanning electron microscopic analyses, we observe that the RKSY polypeptide does not significantly perturb calcium carbonate growth. However, the D4 domain does influence crystal growth in a concentration-dependent manner. Collectively, our data indicate that D4, and not the RKSY domain, exhibits structure-function activity consistent with a mineral binding region.

• Hierarchical interconnections in the nano-composite material bone: Fibrillar cross-links resist fracture on several length scales

  Authors: Georg E. Fantner, Olexandr Rabinovych, Georg Schitter, Philipp Thurner, Johannes H. Kindt, Marquesa M. Finch, James C. Weaver, Laura S. Golde, Daniel E. Morse, Everett A. Lipman, Ivo W. Rangelow, Paul K. Hansma

  Composites Science and Technology.

  Bone is a complex and very important multi-constituent bio-composite. In this work, we focus on the arrangement of bone constituents from the nanoscopic to the microscopic scale, and investigate theBone is a complex and very important multi-constituent bio-composite. In this work, we focus on the arrangement of bone constituents from the nanoscopic to the microscopic scale, and investigate the influence of their arrangements on the fracture mechanisms of the whole composite. We find that bone, on the nanoscopic scale, consists of mineralized collagen fibrils held together by a non-fibrillar organic matrix, which results in a primary failure mode of delamination between mineralized fibrils. In turn, these mineralized fibrils form one of three types of filaments that span microcracks in fractured bone samples, possibly resisting the propagation of these cracks.

• Nanostructural features of demosponge biosilica

  Authors: James C. Weaver, Lı́a I. Pietrasanta, Niklas Hedin, Bradley F. Chmelka, Paul K. Hansma, Daniel E. Morse

  Journal of Structural Biology.

  Recent interest in the optical and mechanical properties of silica structures made by living sponges, and the possibility of harnessing these mechanisms for the synthesis of advanced materials andRecent interest in the optical and mechanical properties of silica structures made by living sponges, and the possibility of harnessing these mechanisms for the synthesis of advanced materials and devices, motivate our investigation of the nanoscale structure of these remarkable biomaterials. Scanning electron and atomic force microscopic (SEM and AFM) analyses of the annular substructure of demosponge biosilica spicules reveals that the deposited material is nanoparticulate, with a mean particle diameter of 74 ± 13 nm. The nanoparticles are deposited in alternating layers with characteristic etchant reactivities. Further analyses of longitudinally fractured spicules indicate that each deposited layer is approximately monoparticulate in thickness and exhibits extensive long range ordering, revealing an unanticipated level of nanoscale structural complexity. NMR data obtained from differentially heated spicule samples suggest that the etch sensitivity exhibited by these annular domains may be related to variation in the degree of silica condensation, rather than variability in the inclusion of organics. In addition, AFM phase imaging in conjunction with results obtained from HF and alkaline etching experiments suggest that at various stages in spicule biosynthesis, regions of unusually low silica condensation are deposited, indicating a possible interruption in normal spicule formation. While this discovery of nanoparticulate silica aggregation in demosponge skeletal elements is likely to reflect the intrinsic kinetic tendency of silica to form such particles during polycondensation, the heirarchical organization of these nanoparticles is biologically unique.