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During development, cells interpret complex and often conflicting signals to make optimal decisions. Plant stomata, the cellular interface between a plant and the atmosphere, develop according to positional cues, which include a family of secreted peptides called epidermal patterning factors (EPFs). How these signalling peptides orchestrate pattern formation at a molecular level remains unclear. Here we report in Arabidopsis that Stomagen (also called EPF-LIKE9) peptide, which promotes stomatal development, requires ERECTA (ER)-family receptor kinases and interferes with the inhibition of stomatal development by the EPIDERMAL PATTERNING FACTOR 2 (EPF2)-ER module. Both EPF2 and Stomagen directly bind to ER and its co-receptor TOO MANY MOUTHS. Stomagen peptide competitively replaced EPF2 binding to ER. Furthermore, application of EPF2, but not Stomagen, elicited rapid phosphorylation of downstream signalling components in vivo. Our findings demonstrate how a plant receptor agonist and antagonist define inhibitory and inductive cues to fine-tune tissue patterning on the plant epidermis.

Implant-associated infections can have severe effects on the longevity of implant devices and they also represent a major cause of implant failures. Treating these infections associated with implants by antibiotics is not always an effective strategy due to poor penetration rates of antibiotics into biofilms. Additionally, emerging antibiotic resistance poses serious concerns. There is an urge to develop effective antibacterial surfaces that prevent bacterial adhesion and proliferation. A novel class of bacterial therapeutic agents, known as antimicrobial peptides (AMP’s), are receiving increasing attention as an unconventional option to treat septic infection, partly due to their capacity to stimulate innate immune responses and for the difficulty of microorganisms to develop resistance towards them. While host- and bacterial- cells compete in determining the ultimate fate of the implant, functionalization of implant surfaces with antimicrobial peptides can shift the balance and prevent implant infections. In the present study, we developed a novel chimeric peptide to functionalize the implant material surface. The chimeric peptide simultaneously presents two functionalities, with one domain binding to a titanium alloy implant surface through a titanium-binding domain while the other domain displays an antimicrobial property. This approach gains strength through control over the bio-material interfaces, a property built upon molecular recognition and self-assembly through a titanium alloy binding domain in the chimeric peptide. The efficiency of chimeric peptide both in-solution and absorbed onto titanium alloy surface was evaluated in vitro against three common human host infectious bacteria, S. mutans, S. epidermidis, and E. coli. In biological interactions such as occurs on implants, it is the surface and the interface that dictate the ultimate outcome. Controlling the implant surface by creating an interface composed chimeric peptides may therefore open up new possibilities to cover the implant site and tailor it to a desirable bioactivity.

Understanding the phenotypic development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a prerequisite to advancing regenerative cardiac therapy, disease modeling, and drug screening applications. Lack of consistent hiPSC-CM in vitro data can be largely attributed to the inability of conventional culture methods to mimic the structural, biochemical, and mechanical aspects of the myocardial niche accurately. Here, we present a nanogrid culture array comprised of nanogrooved topographies, with groove widths ranging from 350 to 2000 nm, to study the effect of different nanoscale structures on the structural development of hiPSC-CMs in vitro. Nanotopographies were designed to have a biomimetic interface, based on observations of the oriented myocardial extracellular matrix (ECM) fibers found in vivo. Nanotopographic substrates were integrated with a self-assembling chimeric peptide containing the Arg-Gly-Asp (RGD) cell adhesion motif. Using this platform, cell adhesion to peptide-coated substrates was found to be comparable to that of conventional fibronectin-coated surfaces. Cardiomyocyte organization and structural development were found to be dependent on the nanotopographical feature size in a biphasic manner, with improved development achieved on grooves in the 700–1000 nm range. These findings highlight the capability of surface-functionalized, bioinspired substrates to influence cardiomyocyte development, and the capacity for such platforms to serve as a versatile assay for investigating the role of topographical guidance cues on cell behavior. Such substrates could potentially create more physiologically relevant in vitro cardiac tissues for future drug screening and disease modeling studies.

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo .

Development of versatile and flexible assembly systems for fabrication of functional hybrid nanomaterials with well-defined hierarchical and spatial organization is of a significant importance in practical nanobiotechnology applications. Here we demonstrate a bio-enabled self-assembly technique for fabrication of multi-layered protein and nanometallic assemblies utilizing a modular gold-binding (AuBP1) fusion tag. To accomplish the bottom-up assembly we first genetically fused the AuBP1 peptide sequence to the C′-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs. Using various spectroscopic techniques, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), we verified the exceptional binding and self-assembly characteristics of AuBP1 peptide. The AuBP1 peptide tag can direct the organization of recombinant MBP protein on various gold surfaces through an efficient control of the organic–inorganic interface at the molecular level. Furthermore using a combination of soft-lithography, self-assembly techniques and advanced AuBP1 peptide tag technology, we produced spatially and hierarchically controlled protein multi-layered assemblies on gold nanoparticle arrays with high molecular packing density and pattering efficiency in simple, reproducible steps. This model system offers layer-by-layer assembly capability based on specific AuBP1 peptide tag and constitutes novel biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies and devices with controlled organization, packing density and architecture. Biotechnol. Bioeng. 2012; 109:1120–1130. © 2011 Wiley Periodicals, Inc.

Fluorescence from quantum dots (QDs) sandwiched between colloidal gold nanoparticles and lithographically created metal nanoarrays is studied using engineered peptides as binding agents. For optimized structures, a 15-fold increase is observed in the brightness of the QDs due to plasmon-enhanced fluorescence. This enhanced brightness is achieved by systematically tuning the vertical distance of the QD from the gold nanoparticles using solid-specific peptide linkers and by optimizing the localized surface plasmon resonance by varying the geometric arrangement of the patterned gold nanoarray. The size and pitch of the patterned array affect the observed enhancement, and sandwiching the QDs between the patterned features and colloidal gold nanoparticles yields even larger enhancements due to the increase in local electromagnetic hot spots induced by the increased surface roughness. The use of bifunctional biomolecular linkers to control the formation of hot spots in sandwich structures provides new ways to fabricate hybrid nanomaterials of architecturally induced functionality for biotechnology and photonics.

Despite extensive recent reports on combinatorially selected inorganic-binding peptides and their bionanotechnological utility as synthesizers and molecular linkers, there is still only limited knowledge about the molecular mechanisms of peptide binding to solid surfaces. There is, therefore, much work that needs to be carried out in terms of both the fundamentals of solid-binding kinetics of peptides and the effects of peptide primary and secondary structures on their recognition and binding to solid materials. Here we discuss the effects of constraints imposed on FliTrx-selected gold-binding peptide molecular structures upon their quantitative gold-binding affinity. We first selected two novel gold-binding peptide (AuBP) sequences using a FliTrx random peptide display library. These were, then, synthesized in two different forms: cyclic (c), reproducing the original FliTrx gold-binding sequence as displayed on bacterial cells, and linear (l) dodecapeptide gold-binding sequences. All four gold-binding peptides were then analyzed for their adsorption behavior using surface plasmon resonance spectroscopy. The peptides exhibit a range of binding affinities to and adsorption kinetics on gold surfaces, with the equilibrium constant, Keq, varying from 2.5x10(6) to 13.5x10(6) M(-1). Both circular dichroism and molecular mechanics/energy minimization studies reveal that each of the four peptides has various degrees of random coil and polyproline type II molecular conformations in solution. We found that AuBP1 retained its molecular conformation in both the c- and l-forms, and this is reflected in having similar adsorption behavior. On the other hand, the c- and l-forms of AuBP2 have different molecular structures, leading to differences in their gold-binding affinities.

Production of antibodies has been revolutionized by the development of modern molecular biology methods for the expression of recombinant DNA. Phage display technology represents one of the most powerful tools for production and selection of recombinant antibodies and has been recognized as a valuable alternative way for the preparation of antibodies of a desired specificity. In comparison to poly- and monoclonal antibodies, recombinant antibodies using the phage display technology can be prepared faster, in more automatic process and with reduced consumption of laboratory animals. This review summarizes current trends of phage display technol - ogy with focus on the generation of hapten-specific recombinant antibodies and gives the examples of successful applications of phage display in the environmental analysis of low molecular weight compound.

Biological systems have developed a wide range of ingenious solutions, which serve as valuable sources for inspiration in designing new materials and systems. The evolutionary pathways through which biological systems have been formed build upon the biomolecular machinery bridging multiple length scales to exhibit a multitude of diverse outstanding properties. With a growing understanding of the molecular processes involved, biological principles are regularly revisited for developing new bio-enabled approaches to materials engineering. A number of biomolecules play important roles in biological systems by performing various tasks based on their functional specificity and their precise molecular recognition capability. Proteins are specifically involved in both collecting and transporting raw materials and interacting with ions. Proteins systematically undergo self- and co-assembly to yield short- and long-range ordered nuclei, substrates and other cellular organelles, as well as to catalyze reactions. The precise molecular recognition and the self-assembly exhibited in these interactions are an outcome of evolutionary process, where proteins have undergone cycles of structural fittings that lead to improved specific interactions. In the last decade, peptides have been utilized as critical building blocks to mimic biomolecular capabilities of proteins and to develop unique novel hybrid materials for a variety of practical applications. Here in, we summarize the inspirations that allow engineers to mimic biomolecular processes and the utility of combinatorial biology-based library systems to screen peptides for materials. Finally, we provide examples of addressable assembly on a variety of surfaces leading to self-organized hybrid systems that employ peptides fused to different functional proteins as building blocks for materials specificity.

Genetically engineered solid binding peptides, because of their unique affinity and specificity for solid materials, represent a promising molecular toolbox for nanoscience and nanotechnology. Despite their potential, the physicochemical determinants of their high affinity for surfaces remain, in most cases, poorly understood. Here we present experimental data and classical atomistic molecular dynamics simulations for two gold binding dodecapeptides (AuBP1 and AuBP2, Hnilova, M. et al. Langmuir 2008, 24, 12440) and a control peptide that does not bind to gold, to unravel the key microscopic differences among them. In particular, by means of extensive sampling via replica exchange simulations, we show here that the conformational ensemble of the three peptides in solution and on the gold surface can be examined, and that the role played by their different conformational flexibility can be analyzed. We found, specifically, that AuBP1 and AuBP2 are much more flexible than the control peptide, which allows all the potential Au-binding amino acids present in these AuBPs to concurrently bind to the gold surface. On the contrary, the potential Au-binding amino acids in the rigid control peptide cannot contact the surface all at the same time, hampering the overall binding. The role of conformational flexibility has been also analyzed in terms of the configurational entropy of the free and adsorbed peptides. Such analysis suggests a possible route to improve upon current flexible gold binding peptides. © 2013 American Chemical Society.

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein-mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure-function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss.

Valves on the plant epidermis called stomata develop according to positional cues, which likely involve putative ligands (EPIDERMAL PATTERNING FACTORS [EPFs]) and putative receptors (ERECTA family receptor kinases and TOO MANY MOUTHS [TMM]) in Arabidopsis. Here we report the direct, robust, and saturable binding of bioactive EPF peptides to the ERECTA family. In contrast, TMM exhibits negligible binding to EPF1 but binding to EPF2. The ERECTA family forms receptor homomers in vivo. On the other hand, TMM associates with the ERECTA family but not with itself. While ERECTA family receptor kinases exhibit complex redundancy, blocking ERECTA and ERECTA-LIKE1 (ERL1) signaling confers specific insensitivity to EPF2 and EPF1, respectively. Our results place the ERECTA family as the primary receptors for EPFs with TMM as a signal modulator and establish EPF2–ERECTA and EPF1–ERL1 as ligand–receptor pairs specifying two steps of stomatal development: initiation and spacing divisions.

This study constitutes a demonstration of the biological route to controlled nano-fabrication via modular multi-functional inorganic-binding peptides. Specifically, we use gold- and silica-binding peptide sequences, fused into a single molecule via a structural peptide spacer, to assemble pre-synthesized gold nanoparticles on silica surface, as well as to synthesize nanometallic particles in situ on the peptide-patterned regions. The resulting film-like gold nanoparticle arrays with controlled spatial organization are characterized by various microscopy and spectroscopy techniques. The described bio-enabled, single-step synthetic process offers many advantages over conventional approaches for surface modifications, self-assembly and device fabrication due to the peptides’ modularity, inherent biocompatibility, material specificity and catalytic activity in aqueous environments. Our results showcase the potential of artificially-derived peptides to play a key role in simplifying the assembly and synthesis of multi-material nano-systems in environmentally benign processes.

Biological fabrication routes can provide a way to overcome the limitations presented by current chemistry-based nanoparticle arrangement and assembly methods. Many recent assembly strategies utilize DNA as the templating molecule by patterning gold nanoparticles on DNA through chemical conjugation via, for example, a sulfhydryl bond. Reliance upon this chemistry, however, limits applications because it acts indiscriminately on several different metals and is only useful for some noble-metal nanoparticles. Strategies that covalently link nanoparticles to proteins or DNA risk denaturation or distortion of native protein, distortion of DNA, and/or disruption of the plasmonic or photonic properties unique to nanoparticles. We present a strategy for nanoparticle patterning on DNA that utilizes the biologically based self-assembly properties of DNA-binding proteins to facilitate the targeted immobilization of nanoparticles on DNA. Here we show that a derivative of the DNA-binding protein TraI spontaneously organizes colloidal gold nanoparticles on DNA through an engineered gold-binding peptide motif. This system, based solely on specific, noncovalent, biologically determined interactions, represents significant progress on the route to spontaneously ordered assembly of nanoparticles important for downstream applications in nanoelectronics and photonics. TraI (192 kDa, 1756 residues) is the E. coli F-plasmid-encoded relaxase/helicase that harbors sequence-specific single-stranded-DNA-binding activity (relaxase domain) and nonspecific single and double-stranded-DNA-binding activity (helicase domain). We engineered TraI with a gold-binding motif at an internal permissive site after residue Q369 to direct the assembly of gold nanoparticles (AuNPs) on DNA through noncovalent interactions. Permissive sites, regions of proteins that tolerate a wide variety of amino acid additions without disrupting native protein function, were previously identified through transposon/epitope tag mutagenesis in TraI. This study utilizes TraI’s nonspecific DNA-binding activity as the first step toward optimization of this biologically based nanoparticletemplating strategy. Because TraI is also capable of sequencespecific DNA binding, this work paves the path to the final step of the biologically based strategy: addressable, targeted immobilization of nanoparticles on DNA. Inorganic binding peptides identified by several groups have demonstrated potential as molecular tools due to their high material affinity and specificity. Proteins non-specifically interact with gold, but our strategy relies on increased goldbinding specificity by including a gold-binding motif. By incorporating a gold-binding peptide while maintaining native DNA-binding function, we create a potent bifunctional molecular building block. The gold-binding peptide GBP1 (MHGKTQATSGTIQS), selected from a combinatorial peptide-display library, was incorporated into TraI in tandem repeats because previous research found that five (5 ) or seven (7 ) repeats of GBP1 exhibited a higher affinity for gold particles than a single copy. Se quences coding for the gold-binding peptides GBP1-5 , GBP17 , and the silica-binding control peptide QBP (LPDWWPPPQLYH) were inserted through molecular cloning techniques into the traI gene at a permissive site after codon 369 (Experimental Section); this permissive site lies in a region that codes for a large surface-exposed linker between the protein’s functional domains. The engineered TraI derivatives were tested for maintenance of their in vivo DNA-binding activity by comparison to wildtype TraI in a bacterial-mating assay. E. coli F’DtraI donor strains carrying plasmids with the complementing traI+, traI369GBP1-5 , traI369GBP1-7 , and traI369QBP alleles were proficient for F-plasmid transfer to a recipient strain, indicating that the essential DNA-binding activities (relaxase and helicase) of the encoded proteins were intact. The engineered TraI proteins were purified as previously described. Purified proteins exhibited DNA-dependent ATPase activity, indicating that these protein derivatives maintained their DNA-binding activity in vitro (Experimental Section). In our prior study, TraI with a cuprous oxide binding peptide (CN225) incorporated at a different permissive site near its C terminus was shown to precipitate cuprous oxide on DNA. However, the TraI inorganic binding derivatives of this study are the first to utilize internal permissive sites, as opposed to Nor C-terminal sites. Use of an internal permissive site circumvents the risk of degradation of inorganic binding repeats from the end of the protein and the specificity for gold instead of cuprous oxide provides an opportunity for electronic and photonic applications. Localized surface plasmon resonance (LSPR) analysis was used to measure the binding of the protein derivatives to AuNPs. Noble metal nanoparticles exhibit characteristic UV– visible absorption bands that result from their LSPR spectra. The peak extinction wavelength, lmax, of the LSPR spectrum [a] R. Hall Sedlak, E. Gachelet, L. Przybyla, Prof. B. Traxler Department of Microbiology, University of Washington Seattle, WA 98195 (USA) Fax: (+1)206-543-8297 E-mail : btraxler@u.washington.edu [b] M. Hnilova, Prof. M. Sarikaya, Prof. C. Tamerler Department of Materials Science and Engineering University of Washington Seattle, WA 98195 (USA) [c] D. Dranow, Prof. T. Gonen Department of Biochemistry, Howard Hughes Medical Institute University of Washington Seattle, WA 98195 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201000407.

In nature, the viability of biological systems is sustained via specific interactions among the tens of thousands of proteins, the major building blocks of organisms from the simplest single-celled to the most complex multicellular species. Biomolecule-material interaction is accomplished with molecular specificity and efficiency leading to the formation of controlled structures and functions at all scales of dimensional hierarchy. Through evolution, Mother Nature developed molecular recognition by successive cycles of mutation and selection. Molecular specificity of probe-target interactions, e.g., ligand-receptor, antigen-antibody, is always based on specific peptide molecular recognition. Using biology as a guide, we can now understand, engineer, and control peptide-material interactions and exploit them as a new design tool for novel materials and systems. We adapted the protocols of combinatorially designed peptide libraries, via both cell surface or phage display methods; using these we select short peptides with specificity to a variety of practical materials. These genetically engineered peptides for inorganics (GEPI) are then studied experimentally to establish their binding kinetics and surface stability. The bound peptide structure and conformations are interrogated both experimentally and via modeling, and self-assembly characteristics are tested via atomic force microscopy. We further engineer the peptide binding and assembly characteristics using a computational biomimetics approach where bioinformatics based peptide-sequence similarity analysis is developed to design higher generation function-specific peptides. The molecular biomimetic approach opens up new avenues for the design and utilization of multifunctional molecular systems in a wide-range of applications from tissue engineering, disease diagnostics, and therapeutics to various areas of nanotechnology where integration is required among inorganic, organic and biological materials. Here, we describe lessons from biology with examples of protein-mediated functional biological materials, explain how novel peptides can be designed with specific affinity to inorganic solids using evolutionary engineering approaches, give examples of their potential utilizations in technology and medicine, and, finally, provide a summary of challenges and future prospects.

Biocombinatorially selected solid-binding peptides, through their unique material affinity and selectivity, are a promising platform for building up complex hierarchical assemblies of nanoscale materials and molecular probes, targeted to specific practical solid surfaces. Here, we demonstrate the material-specific characteristics of engineered gold-binding and silica-binding peptides through co-assembly onto micro- and nano-patterned gold surfaces on silica substrates. To build hierarchical nanostructures on patterned solid surfaces, we utilize peptides as molecular tools and monitor their behavior by either conjugating biotin to them for specific affinity to streptavidin-coated QDot nanoparticles or labelling them with small fluorescent labels. This biomimetic peptide-based approach could be used as an alternative to conventional chemical coupling and surface functionalization techniques with substantial advantages, allowing simultaneous assembly of two or more inorganic nano-entities and/or molecular probes onto patterned inorganic solid substrates. The results have significant implications in a wide range of potential applications, including controlled assembly of hybrid nanostructures in bionanophotonic and biosensing devices.