Marketa Hnilova

Direct interaction of ligand–receptor pairs specifying stomatal patterning

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.

Effect of Molecular Conformations on the Adsorption Behavior of Gold-Binding Peptides

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.

A novel knowledge-based approach to design inorganic-binding peptides

MOTIVATION The discovery of solid-binding peptide sequences is accelerating along with their practical applications in biotechnology and materials sciences. A better understanding of the relationships between the peptide sequences and their binding affinities or specificities will enable further design of novel peptides with selected properties of interest both in engineering and medicine. RESULTS A bioinformatics approach was developed to classify peptides selected by in vivo techniques according to their inorganic solid-binding properties. Our approach performs all-against-all comparisons of experimentally selected peptides with short amino acid sequences that were categorized for their binding affinity and scores the alignments using sequence similarity scoring matrices. We generated novel scoring matrices that optimize the similarities within the strong-binding peptide sequences and the differences between the strong- and weak-binding peptide sequences. Using the scoring matrices thus generated, a given peptide is classified based on the sequence similarity to a set of experimentally selected peptides. We demonstrate the new approach by classifying experimentally characterized quartz-binding peptides and computationally designing new sequences with specific affinities. Experimental verifications of binding of these computationally designed peptides confirm our predictions with high accuracy. We further show that our approach is a general one and can be used to design new sequences that bind to a given inorganic solid with predictable and enhanced affinity.

Competitive binding of antagonistic peptides fine-tunes stomatal patterning

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.

Molecular biomimetics: GEPI‐based biological routes to technology

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.

Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells

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.

Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

ABSTRACT 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.

Peptide-directed co-assembly of nanoprobes on multimaterial patterned solid surfaces

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.

Multifunctional Protein-Enabled Patterning on Arrayed Ferroelectric Materials

This study demonstrates a biological route to programming well-defined protein-inorganic interfaces with an arrayed geometry via modular peptide tag technology. To illustrate this concept, we designed a model multifunctional fusion protein, which simultaneously displays a maltose-binding protein (MBP), a green fluorescence protein (GFPuv) and an inorganic-binding peptide (AgBP2C). The fused combinatorially selected AgBP2C tag controls and site-directs the multifunctional fusion protein to immobilize on silver nanoparticle arrays that are fabricated on specific domain surfaces of ferroelectric LiNbO(3) via photochemical deposition and in situ synthesis. Our combined peptide-assisted biological and ferroelectric lithography approach offers modular design and versatility in tailoring surface reactivity for fabrication of nanoscale devices in environmentally benign conditions.

Fabrication of hierarchical hybrid structures using bio‐enabled layer‐by‐layer self‐assembly

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.