Role of protein aggregate structure on the strength and underwater performance of barnacle-inspired adhesives.

Nature employs protein aggregates when strong materials are needed to adhere surfaces in extreme environments, allowing organisms to survive conditions ranging from harsh intertidal coasts to open oceans. Amyloids and amyloid-like materials are prevalent and amongst the most densely bonded aggregate structures, though how they contribute to wet adhesion is not well understood. In this work, waterborne protein solutions of individual whey proteins are cured in place using varied temperature to produce model adhesives enriched in amyloid or non-amyloid aggregates. Dry adhesive strengths range from 0.2-1.5 MPa, while wet adhesive strengths range from 0-0.5 MPa across the tested proteins and processing conditions, highlighting that both proper protein selection and controlled aggregation extent are necessary for successful underwater performance. For bovine serum albumin, the amyloid-enriched adhesive was able to retain ca. 500 kPa bond strength underwater throughout extended immersion and thermal degradation testing, while the non-amyloid adhesive weakened by up to 80%. As freestanding gels, higher temperature processing improved underwater stability for all the protein materials, with amyloid-rich structures remaining mostly water-insoluble after 30 days submerged in water. Protein-based adhesives with a controlled aggregate structure shed light on the ability of amyloid-containing materials to remain adhered underwater, a necessary trait for the survival of many organisms.

Extremophilic behavior of catalytic amyloids sustained by backbone structuring.

Enzyme function relies on the placement of chemistry defined by solvent and self-associative hydrogen bonding displayed by the protein backbone. Amyloids, long-range multi-peptide and -protein materials, can mimic enzyme functions while having a high proportion of stable self-associative backbone hydrogen bonds. Though catalytic amyloid structures have exhibited a degree of temperature and solvent stability, defining their full extremophilic properties and the molecular basis for such extreme activity has yet to be realized. Here we demonstrate that, like thermophilic enzymes, catalytic amyloid activity persists across high temperatures with an optimum activity at 81 °C where they are 30-fold more active than at room temperature. Unlike thermophilic enzymes, catalytic amyloids retain both activity and structure well above 100 °C as well as in the presence of co-solvents. Changes in backbone vibrational states are resolved in situ using non-linear 2D infrared spectroscopy (2DIR) to reveal that activity is sustained by reorganized backbone hydrogen bonds in extreme environments, evidenced by an emergent vibrational mode centered at 1612 cm-1. Restructuring also occurs in organic solvents, and facilitates complete retention of hydrolysis activity in co-solvents of lesser polarity. We support these findings with molecular modeling, where the displacement of water by co-solvents leads to shorter, less competitive, bonding lifetimes that further stabilize self-associative backbone interactions. Our work defines amyloid properties that counter classical proteins, where extreme environments induce mechanisms of restructuring to support enzyme-like functions necessary for synthetic applications.

High-Throughput Screening of Heterologous Functional Amyloids Using Escherichia coli.

Escherichia coli remains one of the most widely used workhorse microorganisms for the expression of heterologous proteins. The large number of cloning vectors and mutant host strains available for E. coli yields an impressively wide array of folded globular proteins in the laboratory. However, applying modern functional screening approaches to interrogate insoluble protein aggregates such as amyloids requires the use of nonstandard expression pathways. In this chapter, we detail the use of the curli export pathway in E. coli to express a library of gene fragments and variants of a functional amyloid protein to screen sequence traits responsible for aggregation and the formation of nanoscale materials.

Transfer of printed electronic structures using graphene oxide and gelatin enables reversible and biocompatible interface with living cells.

We present a low-cost, easy-to-implement platform for printing materials and interfacing them with eukaryotic cells. We show that thermal or chemical reduction of a graphene oxide thin film allows water-assisted delamination of the film from glass or plastic. The chemical and physical properties and permeability of the resulting film are dependent on the method of reduction and deposition of the graphene oxide, with thermal reduction removing more oxidized carbon functionality than chemical reduction. We also developed a method to attach the films onto cell surfaces using a thin layer of gelatin as an adhesive. In general, the films are highly impermeable to nutrients and we observed a significant amount of cell death when gelatin was not used; gelatin enables diffusion of nutrients for sustained cell viability. The combination of nanoscale membranes with a low melting point biopolymer allows us to reversibly interface cells with cargo transferred by graphene oxide while maintaining cell viability. To demonstrate delivery of electronic structures, we modified a commercial off-the-shelf printer to print a silver-based ink directly onto the reduced graphene oxide films which we then transferred to the surface of the cells.

Engineered Escherichia coli Biofilms Produce Adhesive Nanomaterials Shaped by a Patterned 43 kDa Barnacle Cement Protein.

Barnacles integrate multiple protein components into distinct amyloid-like nanofibers arranged as a bulk material network for their permanent underwater attachment. The design principle for how chemistry is displayed using adhesive nanomaterials, and fragments of proteins that are responsible for their formation, remains a challenge to assess and is yet to be established. Here, we use engineered bacterial biofilms to display a library of amyloid materials outside of the cell using full-length and subdomain sequences from a major component of the barnacle adhesive. A staggered charged pattern is found throughout the full-length sequence of a 43 kDa cement protein (AACP43), establishing a conserved sequence design evolved by barnacles to make adhesive nanomaterials. AACP43 domain deletions vary in their propensity to aggregate and form fibers, as exported extracellular materials are characterized through staining, immunoblotting, scanning electron microscopy, and atomic force microscopy. Full-length AACP43 and its domains have a propensity to aggregate into nanofibers independent of all other barnacle glue components, shedding light on its function in the barnacle adhesive. Curliated Escherichia coli biofilms are a compatible system for heterologous expression and the study of foreign functional amyloid adhesive materials, used here to identify the c-terminal portion of AACP43 as critical in material formation. This approach allows us to establish a common sequence pattern between two otherwise dissimilar families of cement proteins, laying the foundation to elucidate adhesive chemistries by one of the most tenacious marine fouling organisms in the ocean.

Direct Observation of Corrosive Wear by In Situ Scanning Probe Microscopy.

Tribocorrosion involves mechanical wear in a corrosive environment, damaging the protective oxide layer of passivating alloys and increasing material loss rates. Here, we develop a nanoscale, in situ technique using scanning probe microscopy in an electrochemical cell to explore the phase-by-phase tribocorrosion behavior of a heat-treated duplex stainless-steel alloy with secondary phases. We found that under anodic potentials well within the passive oxide region, sliding mechanical contact initiated pitting corrosion and increased electrochemical cell current localized to regions undergoing pitting. Secondary phases were most vulnerable to pitting corrosion during sliding, particularly secondary austenite which is chromium-depleted relative to the matrix steel phases. Under certain conditions, even sigma phases of high nobility were damaged from pits that originate from chromium nitrides. Initiation sites coincide with nanoscale surface voids created at chromium nitride inclusions under a threshold contact stress. Below the initiation stress, no pitting or corrosive wear was observed on sensitized phases. Material loss ceased to propagate when sliding stresses were removed but accelerated when sliding contact stresses were increased. Wear rates and current in the cell were both linearly correlated with material loss. Electrochemical current data were used to monitor oxide penetration spatially but could not be used to quantify material loss. In situ tribocorrosion using a scan probe tip is a viable platform to resolve mechanisms of failure that originate at the nanoscale on actively passivated metal surfaces.

Adhesion of acorn barnacles on surface-active borate glasses.

Concerns about the bioaccumulation of toxic antifouling compounds have necessitated the search for alternative strategies to combat marine biofouling. Because many biologically essential minerals have deleterious effects on organisms at high concentration, one approach to preventing the settlement of marine foulers is increasing the local concentration of ions that are naturally present in seawater. Here, we used surface-active borate glasses as a platform to directly deliver ions (Na+, Mg2+ and BO43-) to the adhesive interface under acorn barnacles (Amphibalanus (=Balanus) amphitrite). Additionally, surface-active glasses formed reaction layers at the glass-water interface, presenting another challenge to fouling organisms. Proteomics analysis showed that cement deposited on the gelatinous reaction layers is more soluble than cement deposited on insoluble glasses, indicating the reaction layer and/or released ions disrupted adhesion processes. Laboratory experiments showed that the majority (greater than 79%) of adult barnacles re-attached to silica-free borate glasses for 14 days could be released and, more importantly, barnacle larvae did not settle on the glasses. The formation of microbial biofilms in field tests diminished the performance of the materials. While periodic water jetting (120 psi) did not prevent the formation of biofilms, weekly cleaning did dramatically reduce macrofouling on magnesium aluminoborate glass to levels below a commercial foul-release coating. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Molecular Recognition of Structures Is Key in the Polymerization of Patterned Barnacle Adhesive Sequences.

The permanent adhesive produced by adult barnacles is held together by tightly folded proteins that form amyloid-like materials distinct among marine foulants. In this work, we link stretches of alternating charged and noncharged linear sequences from a family of adhesive proteins to their role in forming fibrillar nanomaterials. Using recombinant proteins and short barnacle cement derived peptides (BCPs), we find a central sequence with charged motifs of the pattern [Gly/Ser/Val/Thr/Ala-X], where X are charged amino acids, to exert specific control over timing, structure, and morphology of fibril formation. While most BCPs remain dormant, the core segment demonstrates rapid polymerization as well as an ability to template other peptides with no propensity for self-assembly. Patterned charge domains assemble dormant peptides through a specific antiparallel ß-sheet structure as measured by FTIR. While charged domains favor an antiparallel structure, BCPs without charged domains switch fibril assembly to favor simpler parallel ß-sheet aggregates. In addition to activation, charged domains direct nanofibers to grow into discrete microns long fibrils similar to the natural adhesive, while segments without such domains only form short branched aggregates. The assembly of adhesive sequences through recognition of structured templates outlines a strategy used by barnacles to control physical mechanisms of underwater adhesive delivery, activation, and curing based on molecular recognition between proteins.

High-performance nanomaterials formed by rigid yet extensible cyclic ß-peptide polymers.

Organisms have evolved biomaterials with an extraordinary convergence of high mechanical strength, toughness, and elasticity. In contrast, synthetic materials excel in stiffness or extensibility, and a combination of the two is necessary to exceed the performance of natural biomaterials. We bridge this materials property gap through the side-chain-to-side-chain polymerization of cyclic ß-peptide rings. Due to their strong dipole moments, the rings self-assemble into rigid nanorods, stabilized by hydrogen bonds. Displayed amines serve as functionalization sites, or, if protonated, force the polymer to adopt an unfolded conformation. This molecular design enhances the processability and extensibility of the biopolymer. Molecular dynamics simulations predict stick-slip deformations dissipate energy at large strains, thereby, yielding toughness values greater than natural silks. Moreover, the synthesis route can be adapted to alter the dimensions and displayed chemistries of nanomaterials with mechanical properties that rival nature.

Electrochemical Control of Peptide Self-Organization on Atomically Flat Solid Surfaces: A Case Study with Graphite.

The nanoscale self-organization of biomolecules, such as proteins and peptides, on solid surfaces under controlled conditions is an important issue in establishing functional bio/solid soft interfaces for bioassays, biosensors, and biofuel cells. Electrostatic interaction between proteins and surfaces is one of the most essential parameters in the adsorption and self-assembly of proteins on solid surfaces. Although the adsorption of proteins has been studied with respect to the electrochemical surface potential, the self-assembly of proteins or peptides forming well-organized nanostructures templated by lattice structure of the solid surfaces has not been studied in the relation to the surface potential. In this work, we utilize graphite-binding peptides (GrBPs) selected by the phage display method to investigate the relationship between the electrochemical potential of the highly ordered pyrolytic graphite (HOPG) and peptide self-organization forming long-range-ordered structures. Under modulated electrical bias, graphite-binding peptides form various ordered structures, such as well-ordered nanowires, dendritic structures, wavy wires, amorphous (disordered) structures, and islands. A systematic investigation of the correlation between peptide sequence and self-organizational characteristics reveals that the presence of the bias-sensitive amino acid modules in the peptide sequence has a significant effect on not only surface coverage but also on the morphological features of self-assembled structures. Our results show a new method to control peptide self-assembly by means of applied electrochemical bias as well as peptide design-rules for the construction of functional soft bio/solid interfaces that could be integrated in a wide range of practical implementations.

Static and Time-Resolved Terahertz Measurements of Photoconductivity in Solution-Deposited Ruthenium Dioxide Nanofilms.

Thin-film ruthenium dioxide (RuO2) is a promising alternative material as a conductive electrode in electronic applications because its rutile crystalline form is metallic and highly conductive. Herein, a solution-deposition multi-layer technique is employed to fabricate ca. 70 ± 20 nm thick films (nanoskins) and terahertz spectroscopy is used to determine their photoconductive properties. Upon calcining at temperatures ranging from 373 K to 773 K, nanoskins undergo a transformation from insulating (localized charge transport) behavior to metallic behavior. Terahertz time-domain spectroscopy (THz-TDS) indicates that nanoskins attain maximum static conductivity when calcined at 673 K (σ = 1030 ± 330 S·cm-1). Picosecond time-resolved Terahertz spectroscopy (TRTS) using 400 nm and 800 nm excitation reveals a transition to metallic behavior when calcined at 523 K. For calcine temperatures less than 523 K, the conductivity increases following photoexcitation (ΔE < 0) while higher calcine temperatures yield films composed of crystalline, rutile RuO2 and the conductivity decreases (ΔE > 0) following photoexcitation.

Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes.

Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive enzyme assays on isolated cement. A novel full-length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Brady's reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43.

Sequence basis of Barnacle Cement Nanostructure is Defined by Proteins with Silk Homology.

Barnacles adhere by producing a mixture of cement proteins (CPs) that organize into a permanently bonded layer displayed as nanoscale fibers. These cement proteins share no homology with any other marine adhesives, and a common sequence-basis that defines how nanostructures function as adhesives remains undiscovered. Here we demonstrate that a significant unidentified portion of acorn barnacle cement is comprised of low complexity proteins; they are organized into repetitive sequence blocks and found to maintain homology to silk motifs. Proteomic analysis of aggregate bands from PAGE gels reveal an abundance of Gly/Ala/Ser/Thr repeats exemplified by a prominent, previously unidentified, 43 kDa protein in the solubilized adhesive. Low complexity regions found throughout the cement proteome, as well as multiple lysyl oxidases and peroxidases, establish homology with silk-associated materials such as fibroin, silk gum sericin, and pyriform spidroins from spider silk. Distinct primary structures defined by homologous domains shed light on how barnacles use low complexity in nanofibers to enable adhesion, and serves as a starting point for unraveling the molecular architecture of a robust and unique class of adhesive nanostructures.

Bioelectronic interfaces by spontaneously organized peptides on 2D atomic single layer materials.

Self-assembly of biological molecules on solid materials is central to the "bottom-up" approach to directly integrate biology with electronics. Inspired by biology, exquisite biomolecular nanoarchitectures have been formed on solid surfaces. We demonstrate that a combinatorially-selected dodecapeptide and its variants self-assemble into peptide nanowires on two-dimensional nanosheets, single-layer graphene and MoS2. The abrupt boundaries of nanowires create electronic junctions via spatial biomolecular doping of graphene and manifest themselves as a self-assembled electronic network. Furthermore, designed peptides form nanowires on single-layer MoS2 modifying both its electric conductivity and photoluminescence. The biomolecular doping of nanosheets defined by peptide nanostructures may represent the crucial first step in integrating biology with nano-electronics towards realizing fully self-assembled bionanoelectronic devices.

Imaging Active Surface Processes in Barnacle Adhesive Interfaces.

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces.

Self-Assembly of Protein Nanofibrils Orchestrates Calcite Step Movement through Selective Nonchiral Interactions.

The recognition of atomically distinct surface features by adsorbed biomolecules is central to the formation of surface-templated peptide or protein nanostructures. On mineral surfaces such as calcite, biomolecular recognition of, and self-assembly on, distinct atomic kinks and steps could additionally orchestrate changes to the overall shape and symmetry of a bulk crystal. In this work, we show through in situ atomic force microscopy (AFM) experiments that an acidic 20 kDa cement protein from the barnacle Megabalanus rosa (MRCP20) binds specifically to step edge atoms on {101̅4} calcite surfaces, remains bound and further assembles over time to form one-dimensional nanofibrils. Protein nanofibrils are continuous and organized at the nanoscale, exhibiting striations with a period of ca. 45 nm. These fibrils, templated by surface steps of a preferred geometry, in turn selectively dissolve underlying calcite features displaying the same atomic arrangement. To demonstrate this, we expose the protein solution to bare and fibril-associated rhombohedral etch pits to reveal that nanofibrils accelerate only the movement of fibril-forming steps when compared to undecorated steps exposed to the same solution conditions. Calcite mineralized in the presence of MRCP20 results in asymmetric crystals defined by frustrated faces with shared mirror symmetry, suggesting a similar step-selective behavior by MRCP20 in crystal growth. As shown here, selective surface interactions with step edge atoms lead to a cooperative regime of calcite modification, where templated long-range protein nanostructures shape crystals.

Controlling the surface chemistry of graphite by engineered self-assembled peptides.

The systematic control over surface chemistry is a long-standing challenge in biomedical and nanotechnological applications for graphitic materials. As a novel approach, we utilize graphite-binding dodecapeptides that self-assemble into dense domains to form monolayer-thick long-range-ordered films on graphite. Specifically, the peptides are rationally designed through their amino acid sequences to predictably display hydrophilic and hydrophobic characteristics while maintaining their self-assembly capabilities on the solid substrate. The peptides are observed to maintain a high tolerance for sequence modification, allowing control over surface chemistry via their amino acid sequence. Furthermore, through a single-step coassembly of two differently designed peptides, we predictably and precisely tune the wettability of the resulting functionalized graphite surfaces from 44° to 83°. The modular molecular structures and predictable behavior of short peptides demonstrated here give rise to a novel platform for functionalizing graphitic materials that offers numerous advantages, including noninvasive modification of the substrate, biocompatible processing in an aqueous environment, and simple fusion with other functional biological molecules.

Electrical detection of biomolecular adsorption on sprayed graphene sheets.

The binding affinities of graphite-binding peptides to a graphite surface were electrically characterized using sprayed graphene field effect transistors (SGFETs) fabricated with solution exfoliated graphene. The binding affinities of these peptides were also characterized using atomic force microscopy (AFM) and mechanically exfoliated graphene field effect transistors (GFETs) to confirm the validity of the SGFET platform. Binding constants obtained via GFET and AFM were comparable with those observed using SGFETs. The sprayed graphene film serves as a scalable platform to study biomolecular adsorption to graphitic surfaces.

Controlling self-assembly of engineered peptides on graphite by rational mutation.

Self-assembly of proteins on surfaces is utilized in many fields to integrate intricate biological structures and diverse functions with engineered materials. Controlling proteins at bio-solid interfaces relies on establishing key correlations between their primary sequences and resulting spatial organizations on substrates. Protein self-assembly, however, remains an engineering challenge. As a novel approach, we demonstrate here that short dodecapeptides selected by phage display are capable of self-assembly on graphite and form long-range-ordered biomolecular nanostructures. Using atomic force microscopy and contact angle studies, we identify three amino acid domains along the primary sequence that steer peptide ordering and lead to nanostructures with uniformly displayed residues. The peptides are further engineered via simple mutations to control fundamental interfacial processes, including initial binding, surface aggregation and growth kinetics, and intermolecular interactions. Tailoring short peptides via their primary sequence offers versatile control over molecular self-assembly, resulting in well-defined surface properties essential in building engineered, chemically rich, bio-solid interfaces.

Chemically self-assembled antibody nanorings (CSANs): design and characterization of an anti-CD3 IgM biomimetic.

A number of clever recombinant methodologies have been developed that recapitulate the valencies of IgG's (bivalent) and IgA's (tetravalent). Although higher synthetic valencies have been achieved by conjugation of either monoclonal antibodies or single-chain antibodies to nanoparticles and liposomes, a method for the preparation of recombinant antibodies with valencies similar to IgM's (decavalent) but considerably less than what is generally found after antibody particle conjugation has yet to be devised. Recently, we have developed a methodology for the design of bivalent Chemically Self-Assembled Antibody Nanorings (CSANs). We now report the crystal structure of the nanoring subunit composed of the E. coli DHFR dimer and a methotrexate dimerizer (MTX2-C9) containing a visible nine methylene linker and a protocol for the preparation of CSANs from this subunit with valencies similar to IgM's, ranging from 8-10 single chain antibodies (scFvs). The multivalent CSANs were reversibly assembled from a fusion protein dihydrofolate reductase (DHFR)-DHFR-antiCD3 scFv containing a single glycine linker between the two DHFR scaffolding proteins. We also demonstrate that, similar to the parental bivalent anti-CD3 monoclonal antibody (mAB), anti-CD3 CSANs selectively bind to CD3+ leukemia cells and undergo rapid internalization through a caveolin-independent pathway that requires cholesterol, actin polymerization, and protein tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell activation and nearly complete loss (i.e., 90%) of the surface displayed T-cell receptor (TCR), only 25-30% of the TCR down regulate and no significant T-cell proliferation is observed after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs. Consistent with the proliferation findings, 15-25% less CD25 (IL-2 receptor) was found on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs, respectively, than on PBMCs treated with the parental mAB. Comparative experiments with F(ab')2 derived from the mAB confirm that the activation of the T-cells by the mAB is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory cells, such as macrophages. Taken together, our results demonstrate that anti-CD3 CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug, or potentially oligonucleotide delivery to T-cells without, as has been observed for other antibody conjugated nanoparticles, the deleterious effects of activation observed for mAB. Further the CSAN construct may be adapted for the preparation of other multivalent scFvs.