We would like to mimic the mechanical properties of animal systems for the development of novel materials. Insect cuticle serves as one source of inspiration for the design of these materials. Cuticle is composed of chitin embedded in a protein matrix which may also contain plasticizers, fillers, crosslinkers, and minerals. The specific properties of the cuticle depend on the type, amount and interactions between each component. We are renewing the investigation of the elastic cuticle, resilin. Resilin, a protein-based elastomer first described in the early 1960s, has properties which have been reported to be most like those of ideal rubbers. We have examined resilin isolated from the prealar arms of the cockroach, Periplaneta americana. The results of amino acid analysis are in good agreement with earlier data reported for resilin. A series of tryptic fragments have been isolated and sequenced. These peptides have been used for the design of oligonucleotide probes for the identification of the gene(s) from a teneral cockroach cDNA library. A biopolymer, based on one tryptic fragment, has been designed and synthesized. We are continuing to treat resilin with residue specific proteases in order to map the resilin protein.

A phosphorylated, glycoprotein preparation has been obtained from orb webs of the araneid spider Argiope aurantia. This preparation probably contains proteins from more than one gland type, but resolution of these proteins has not yet been achieved. Nevertheless, a major component appears to be the adhesive glycoprotein(s) from the adhesive spiral. A product of the aggregate glands, this glycoprotein(s) occurs as discrete nodules along the core fibers of the adhesive spiral, within the viscid, aqueous droplets.

The glycoprotein preparation has a high apparent molecular weight (> 200 kDa) and is polydisperse. The only monosaccharide constituent identified by gas-liquid chromatography or in lectin studies is N-acetylgalactosamine and this is at least primarily O-linked to threonine. By electron microscopy, linear, unbranched and apparently flexible filaments are observed. Phosphorylated serine and threonine residues are present in the preparation and glycine, proline and threonine together account for about 57 mole % of the preparation's amino acid content. Thus, in some, but not all, respects, this glycoprotein preparation is reminiscent of a secretory mucin.

Dragline silk has been shown to consist of two proteins, Spidroins 1 and 2, which form this unique fiber. The cDNAs for these two proteins have been sequenced and a structure proposed which accounts for both the tensile strength and elasticity of dragline silk.

The shell (pericarp) of nuts protects the seed from being eaten before it can germinate. It is presumably designed to resist fracture by external forces, and evolution will have optimised its design for this purpose. It appears that the material of larger shells, which will tend to be structurally more brittle, is tougher. However, the fracture mechanics theory for shells in compression is not available, nor the information on the biological selection pressures.

Rhinoceros horn has been used medicinally and as a talisman in many cultures and animals are slaughtered to obtain the horn. With the dwindling populations of rhinos, and the limited number and breeding success of captive rhinos, there is a critical need to learn as much as is possible about their horns to find an adequate substitute. Examination of rhino horn was made using optical microscopy, scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDS), and x-ray diffraction (XRD). The structure of the horn is unusual and consists of two separate phases, one of hair-like filaments, built around a central core in circumferential layers and the other surrounding and filling in the spaces between the filaments as a matrix. Together, these two structures make up a biological composite, structurally similar to metal, ceramic or polymer based composites. The structural morphology, the dimensions of the structures, and the chemistry of the horn are discussed. Comparisons are made between horn, hoof, and hair of rhinos and hoof and hair from horses, their nearest living relatives.

Biomineralization is precisely controlled by complex templating relationships ultimately encoded in the genes. In the formation of the molluscan shell, polyanionic pleated sheet proteins serve as templates for the nucleation and epitaxial growth of calcium carbonate crystalline domains to yield microlaminate composites of exceptional strength and crystal ordering. The strength and fracture-resistance of these composites far exceed those of the minerals themselves, as a result of both the capacity for flexible deformation of the organic matrix layers and the retardation of crack propagation at each mineral-organic interface. The basic principles controlling low temperature biosynthesis of these materials thus are of both fundamental and applied importance. The abalone shell consists of microlaminates with a remarkable regularity of lamina thickness (ca. 0.5 micron), the formation of which defies present understanding. We have found that shells of abalone larvae formed prior to metamorphosis contain only aragonite, whereas the adult shell made after metamorphosis contains both aragonite and calcite. This transition is accompanied by a switch in genetic expression of the template proteins, suggesting that the premetamorphic protein may serve as a template for aragonite nucleation and growth, while template proteins synthesized after metamorphosis may direct crystallization of calcite. These analyses are based on improvements we recently reported for the detection and purification of proteins from the demineralized shell matrix. Genetic cloning experiments now in progress are aimed at discovering additional protein sequences responsible for the programmed control of crystal phase termination, since it is the termination and reinitiation of mineralization that is responsible for the regularity of highly ordered microlaminates produced in nature.

The schistosome eggshell is composed of two kinds of cross-linked proteins. One is glycine rich and the other is extremely tyrosine rich and highly repetitive. The highly conserved sequence of the tyrosine residues in the tyrosine rich protein suggests that it may play a role as an electron transport chain during eggshell cross-linking. Studies using model synthetic peptides suggest that the tyrosine rich protein may adopt a left-handed structure, possibly a left-handed alpha-helix.

A molecular adaptor for interfacing environmentally sensitive, soluble polymers and antibody molecules has been developed. The gene coding for the minimally sized, 55 amino acid IgG binding domain from protein G has been constructed by total gene synthesis. This domain is thermally stable, exhibits a highly reversible folding and unfolding equilibrium, and recognizes IgG and Fab molecules with high affinity. These properties make the protein G domain a potentially useful adaptor for non-covalent immobilization of antibodies to soluble polymers and hydrogels. Engineered single-chain Fv antibody fragments have also been constructed and a method for expanding the usefulness of the protein G adaptor to these molecules is proposed. The engineered antibodies also provide a model system for developing general immobilization strategies aimed at maximizing binding affinities and therapeutic responses. The overall goal is to develop optimized engineering designs for functionally optimized antibody-material hybrids.

A new approach to materials design is presented, utilizing specific recognition and assembly at the molecular level. The approach described exploits the control over polymer chain microstructure afforded by biosynthesis to produce proteinbased materials with precisely defined physical properties. Incorporated into these materials are recognition elements that stringently control the placement and organization of each chain within higher order superstructures. The proteins, designated Recognin A2 through Recognin E2, are recombinant polypeptides designed de novo from both natural consensus sequences and an appreciation of the physical principles governing biological recognition. These materials are designed to examine the forces involved in specific recognition and complexation. through control of charge identity and placement, a pattern for specific interaction can be introduced. A subset of these materials are programmed to spontaneously assemble into complex, multicomponent structures and represent the first step in a rational approach to nanometer-scale structural design.

A gene encoding one complete [C+SR] core repeat from spIa, a 1000-kDa silk protein from Chironomus tentans, was synthesized and its recombinant protein expressed to high levels in bacterial cells. We observed that reducing agents significantly alter the electrophoretic mobility of this protein. A variety of data indicate that the purified recombinant protein is folded and its structure stabilized by two intramolecular disulfide bonds.

Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were cloned and expressed in Escherichia coli. The bioadhesive precursor (BP, Mx−25,000) was expressed from one 600-bp gene comprised of a 30-bp unit repeat. The repetitious gene appeared stable in a T7-based host/vector system.

Using the T7 expression system for induction, BP was produced at levels approaching 60% of total cell protein. BP was found both in intracellular inclusions and in the soluble fraction. Interestingly, methionine was processed from the N-terminus of the purified protein to give an authentic consensus precursor protein.

Two different biomimetic strategies were utilized in the formation of magnetite fibers. The first strategy utilized natural (Sphaerotilus natans sheaths) or synthetic (hollow fibers) matrices for magnetite formation. The second strategy made use of an iron-hydroxide intermediate that was subsequently chemically converted to magnetite within the biomimetic matrix. The formation of magnetite was determined by both visual and x-ray diffraction analysis. This process has advantages over conventional routes because of the expense and handling problems associated with the production of ceramic whiskers and fibers. The magnetite formed by this process may prove to have unique properties due to its unusual fiber structure.

Angular-resolved ESCA was used to study single cadmium arachidate monolayers transferred to Si (100) wafers by the Langmuir-Blodgett technique. We find the monolayers to be of high integrity with respect to those defects which enhance the escape probability of substrate photoelectrons through the overlayer. The inelastic mean-free pathlengths of Si (2p) and C (1s) electrons were calculated to be 49±6 Å and 45±6 Å for the kinetic energies of 1388 eV and 1202 eV, respectively. The overall ordering of the hydrocarbon chains is less than for alkane thiols assembled on noble metals. We find that the precision of the Tyler algorithm to deconvolute angular-resolved ESCA data into depth profiles is accurate within 10% for predicting the thickness of the hydrocarbon overlayer but less precise for intermediate layers.

DNA is a tractable medium for controlling the structure of matter on the nanometer scale. We have explored ligating together stable branched DNA molecules to form geometrical objects. By this means, we have assembled a 3-connected molecule whose helix axes have the connectivity of a cube. The construct is a hexacatenane, each of whose cyclic strands corresponds to a face of the object. Each of its twelve edges contains a unique recognition site for cleavage by a restriction enzyme; these sites are used to demonstrate the assembly of the object. The plectonemic structure of DNA also permits the directed synthesis of molecular knots. Recently, we have constructed trefoil knots from B-DNA and an amphichiral figure-8 knot whose helical domains contain both B-DNA and Z-DNA.

We have developed a solid-support methodology for the synthesis of geometrical objects. This approach provides greater control over products and topological purity, and lends itself better to automation. Branched molecules containing 3–6 double helical arms can be formed from equimolar mixtures of their component strands, thereby enabling the construction of 3–6 connected networks. The goals of this work include the construction of periodic multiply-connected networks of DNA. The aims of these DNA constructions include using them as scaffolding to build periodic macromolecular arrays for diffraction purposes, as well as directing the assembly of molecular electronic devices. There are wellcharacterized molecular transformations of DNA that make nano-scale machines feasible to build in this molecular context. These materials are likely to be useful for understanding crystallization processes and structure-function relationships.

The polycation conducting polymer, oxidized polypyrrole (PPy), possesses the ability to form complexes with DNA. Our previously proposed diffusion limited binding model for double helical DNA was also found to be applicable to single stranded DNA in this study. Single stranded DNA was found to bind PPy at a nearly identical level to that of double helical DNA. An investigation of electropolymerized PPy film morphology using SEM revealed two distinctly differing surface morphologies for the Platinum (Pt) electrode face (smooth) and polymeric growth face (rough). The DNA uptake levels were found to be consistently different on either surface, being higher on the rough surface. DNA penetrated into the disk interior with increasing time periods of exposure while a similar phenomenon but to a lesser extent was observed for single stranded DNA.

A novel hierarchical biomaterial capable of incorporating any biotinylated biomolecule has been created. Our strategy is to biotinylate one-dimensional electroactive polymers and use a bridging streptavidin protein on Langmuir-Blodgett (LB) organized films. The following copolymeric system which enables functionalization of other molecules and formation of good monolayers was employed. Biotinylated poly(3-methanolthiophene-co-3-undecylthiophene) (B-PMUT) demonstrated a significantly better isotherm implying superior molecular packing compared to poly(3-methanolthiophene-co-3-undecylthiophene) (PMUT) on the LB airwater surface. The isotherm showed significant area expansion when streptavidin was injected below the B-PMUT monolayer in 0.1mM NaH2PO4/0.1 M NaCl buffer (pH 6.8) subphase. We then incorporated biotinylated phycoerythrin (B-PE) into this novel biomaterial by binding the unoccupied biotin binding sites on the bound streptavidin (4 sites total). The pressure-area isotherm of the protein injected monolayer showed area expansion. A characteristic fluorescent emission peak at 576nm was detected from the monolayer transferred onto a solid substrate. These observations demonstrated the function of B-PMUT in hierarchical monolayer assembly of molecules incorporating the biotin / streptavidin interaction.

Enzymatic polymerization of a number of monomers in an ordered lattice on a Langmuir trough (L-T) was investigated. The assembly and polymerization of two mixed monomeric systems consisting of 4-tetradecyloxyphenol (C14PP) with phenol, and 4-hexadecylaniline (C16PA) with aniline, in various ratios were carried out. Polymerization was obtained with C14PP and phenol (in the ratio 1:10), and C16PA and aniline (in the ratio 1:2) on buffered MilliQ water (pH 7.5) using the enzyme horseradish peroxidase (HRP) at 20°C. Polymerized monolayers were then transferred to appropriate substrates for UV-Vis, third order non linear optical (NLO) properties, and thickness measurements. Thermogravimetric analysis (TGA) was performed on the final polymers. Results suggest that the lattice controlled polymerization results in highly ordered conjugated polymers with improved functional, electronic, NLO and processability properties, which form a basis for intelligent materials design and applications.

Supramolecular organizates of liposomes and Langmuir-Blodgett (LB) films are described which are capable of specifically binding to pathogens such as influenza virus. The specific interaction is between the hemagglutinin protein of the virus and an α-C-glycoside of sialic acid expressed on the surface of the liposome or LB film. Sialic acidcontaining liposomes were found to inhibit influenza virus infectivity in cell culture, and may provide a basis for new materials which act as therapeutic agents. Sialic acid-containing LB films also specifically bind to the influenza virus. In this case, the conjugated polymer backbone acts as a built-in reporter of the binding event, measurable by a chromatic change in the visible absorption spectrum. These films may provide a basis for new materials which act as direct detectors of binding

Poly(γ-benzyl-L-glutamate) (PBLG) may be derivatized on its periphery by covalently attaching χ-active NLO chromophores at the termini of its sidechains and thereby give a new class of SHG materials. The inherent liquid crystalline properties of concentrated solutions of α-helical PBLG may be exploited to establish unique supramolecular structures prior to E-field poling. When PBLG is derivatized at its N-terminus with lipoic acid, it will self-assemble on gold to give a thin film. Angle-dependent XPS, ellipsometry, contact angle measurements, and grazing angle IR reflection-absorption spectroscopy give quantitative information about the orientation of the polypeptide α-helices relative to the substrate surface. Consequently, polypeptides, in particular, Merrifield-synthesized or recombinant DNA-expressed synthetic polypeptides, present a novel fabrication route to thin films wherein molecular-engineered functionalities (chemical, electrical, or optically active species) may be encoded into the macromolecule's primary structure and subsequently expressed spatially via the spontaneous self-organization of these rod-like polymers.

Biopolymer self assembly in heterogeneous lipid monolayers at the air-water interface was investigated. Fluorescence microscopy allowed the visualization of protein recognition and binding to these microstructured membranes. Mimicking proteininduced membrane microstructuring and protein docking was achieved in ternary mixed lipid monolayer systems.