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1.  Materiomics: biological protein materials, from nano to macro 
Materiomics is an emerging field of science that provides a basis for multiscale material system characterization, inspired in part by natural, for example, protein-based materials. Here we outline the scope and explain the motivation of the field of materiomics, as well as demonstrate the benefits of a materiomic approach in the understanding of biological and natural materials as well as in the design of de novo materials. We discuss recent studies that exemplify the impact of materiomics – discovering Nature’s complexity through a materials science approach that merges concepts of material and structure throughout all scales and incorporates feedback loops that facilitate sensing and resulting structural changes at multiple scales. The development and application of materiomics is illustrated for the specific case of protein-based materials, which constitute the building blocks of a variety of biological systems such as tendon, bone, skin, spider silk, cells, and tissue, as well as natural composite material systems (a combination of protein-based and inorganic constituents) such as nacre and mollusk shells, and other natural multiscale systems such as cellulose-based plant and wood materials. An important trait of these materials is that they display distinctive hierarchical structures across multiple scales, where molecular details are exhibited in macroscale mechanical responses. Protein materials are intriguing examples of materials that balance multiple tasks, representing some of the most sustainable material solutions that integrate structure and function despite severe limitations in the quality and quantity of material building blocks. However, up until now, our attempts to analyze and replicate Nature’s materials have been hindered by our lack of fundamental understanding of these materials’ intricate hierarchical structures, scale-bridging mechanisms, and complex material components that bestow protein-based materials their unique properties. Recent advances in analytical tools and experimental methods allow a holistic view of such a hierarchical biological material system. The integration of these approaches and amalgamation of material properties at all scale levels to develop a complete description of a material system falls within the emerging field of materiomics. Materiomics is the result of the convergence of engineering and materials science with experimental and computational biology in the context of natural and synthetic materials. Through materiomics, fundamental advances in our understanding of structure–property–process relations of biological systems contribute to the mechanistic understanding of certain diseases and facilitate the development of novel biological, biologically inspired, and completely synthetic materials for applications in medicine (biomaterials), nanotechnology, and engineering.
PMCID: PMC3781696  PMID: 24198478
biological materials; hierarchies; multiscale; materiomics; deformation; failure; functional material properties; protein; peptide; universality; diversity
2.  Hypothesis: Bones Toughness Arises from the Suppression of Elastic Waves 
Scientific Reports  2014;4:7538.
Bone and other natural material exhibit a combination of strength and toughness that far exceeds that of synthetic structural materials. Bone's toughness is a result of numerous extrinsic and intrinsic toughening mechanisms that operate synergistically at multiple length scales to produce a tough material. At the system level however no theory or organizational principle exists to explain how so many individual toughening mechanisms can work together. In this paper, we utilize the concept of phonon localization to explain, at the system level, the role of hierarchy, material heterogeneity, and the nanoscale dimensions of biological materials in producing tough composites. We show that phonon localization and attenuation, using a simple energy balance, dynamically arrests crack growth, prevents the cooperative growth of cracks, and allows for multiple toughening mechanisms to work simultaneously in heterogeneous materials. In turn, the heterogeneous, hierarchal, and multiscale structure of bone (which is generic to biological materials such as bone and nacre) can be rationalized because of the unique ability of such a structure to localize phonons of all wavelengths.
PMCID: PMC4269876  PMID: 25518898
3.  Category Theoretic Analysis of Hierarchical Protein Materials and Social Networks 
PLoS ONE  2011;6(9):e23911.
Materials in biology span all the scales from Angstroms to meters and typically consist of complex hierarchical assemblies of simple building blocks. Here we describe an application of category theory to describe structural and resulting functional properties of biological protein materials by developing so-called ologs. An olog is like a “concept web” or “semantic network” except that it follows a rigorous mathematical formulation based on category theory. This key difference ensures that an olog is unambiguous, highly adaptable to evolution and change, and suitable for sharing concepts with other olog. We consider simple cases of beta-helical and amyloid-like protein filaments subjected to axial extension and develop an olog representation of their structural and resulting mechanical properties. We also construct a representation of a social network in which people send text-messages to their nearest neighbors and act as a team to perform a task. We show that the olog for the protein and the olog for the social network feature identical category-theoretic representations, and we proceed to precisely explicate the analogy or isomorphism between them. The examples presented here demonstrate that the intrinsic nature of a complex system, which in particular includes a precise relationship between structure and function at different hierarchical levels, can be effectively represented by an olog. This, in turn, allows for comparative studies between disparate materials or fields of application, and results in novel approaches to derive functionality in the design of de novo hierarchical systems. We discuss opportunities and challenges associated with the description of complex biological materials by using ologs as a powerful tool for analysis and design in the context of materiomics, and we present the potential impact of this approach for engineering, life sciences, and medicine.
PMCID: PMC3169555  PMID: 21931622
4.  Nanostructure and molecular mechanics of spider dragline silk protein assemblies 
Spider silk is a self-assembling biopolymer that outperforms most known materials in terms of its mechanical performance, despite its underlying weak chemical bonding based on H-bonds. While experimental studies have shown that the molecular structure of silk proteins has a direct influence on the stiffness, toughness and failure strength of silk, no molecular-level analysis of the nanostructure and associated mechanical properties of silk assemblies have been reported. Here, we report atomic-level structures of MaSp1 and MaSp2 proteins from the Nephila clavipes spider dragline silk sequence, obtained using replica exchange molecular dynamics, and subject these structures to mechanical loading for a detailed nanomechanical analysis. The structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly beta-sheet crystal domains, while disorderly regions are formed by glycine-rich repeats that consist of 31-helix type structures and beta-turns. Our structural predictions are validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots, alpha-carbon atomic distances, as well as secondary structure content. Mechanical shearing simulations on selected structures illustrate that the nanoscale behaviour of silk protein assemblies is controlled by the distinctly different secondary structure content and hydrogen bonding in the crystalline and semi-amorphous regions. Both structural and mechanical characterization results show excellent agreement with available experimental evidence. Our findings set the stage for extensive atomistic investigations of silk, which may contribute towards an improved understanding of the source of the strength and toughness of this biological superfibre.
PMCID: PMC2988266  PMID: 20519206
biological material; molecular modelling; materiomics; spider silk; deformation; failure
5.  High-performance spider webs: integrating biomechanics, ecology and behaviour 
Spider silks exhibit remarkable properties, surpassing most natural and synthetic materials in both strength and toughness. Orb-web spider dragline silk is the focus of intense research by material scientists attempting to mimic these naturally produced fibres. However, biomechanical research on spider silks is often removed from the context of web ecology and spider foraging behaviour. Similarly, evolutionary and ecological research on spiders rarely considers the significance of silk properties. Here, we highlight the critical need to integrate biomechanical and ecological perspectives on spider silks to generate a better understanding of (i) how silk biomechanics and web architectures interacted to influence spider web evolution along different structural pathways, and (ii) how silks function in an ecological context, which may identify novel silk applications. An integrative, mechanistic approach to understanding silk and web function, as well as the selective pressures driving their evolution, will help uncover the potential impacts of environmental change and species invasions (of both spiders and prey) on spider success. Integrating these fields will also allow us to take advantage of the remarkable properties of spider silks, expanding the range of possible silk applications from single threads to two- and three-dimensional thread networks.
PMCID: PMC3061126  PMID: 21036911
biomaterials; biomimetics; dragline silk; ecology; evolution; orb-web spider
6.  Protein-protein nanoimprinting of silk fibroin films 
Control of the interface between biological tissue and high technology materials is paramount for the development of future applications in biomedicine, especially in the case of implantable integrated devices for signal transduction.[1]-[3] Such work requires careful materials design to develop devices that can efficiently perform technological functions while retaining biocompatibility and biological integration. Silk fibroin protein from the Bombyx mori silkworm has come of considerable interest in this context, owing to its attractive mechanical,[4]-[7] biological, [8][9] and optical properties.[10][11] Recent work has shown adaptation of common micro- and nano-fabrication tools to silk films,[12]-[18] as well as silk protein secondary structure patterning techniques,[19] leading to biocompatible and degradable electronic and photonic devices which can simultaneously act as a carrier and stabilizer for protein pharmaceuticals and other bioactive reagents.[20]-[23] In particular, silk based nanoscale photonic devices face the challenge of sub-wavelength resolution fabrication on a soft polymeric substrate.[15][24] Previous work introduced the possibility of direct, rapid nanoimprinting in silk for the fabrication of photonic structures by leveraging the material properties of this protein.[25]
PMCID: PMC3752341  PMID: 23483712
silk; biopolymer; protein; nanoimprinting; fabrication
7.  Increasing silk fibre strength through heterogeneity of bundled fibrils 
Can naturally arising disorder in biological materials be beneficial? Materials scientists are continuously attempting to replicate the exemplary performance of materials such as spider silk, with detailed techniques and assembly procedures. At the same time, a spider does not precisely machine silk—imaging indicates that its fibrils are heterogeneous and irregular in cross section. While past investigations either focused on the building material (e.g. the molecular scale protein sequence and behaviour) or on the ultimate structural component (e.g. silk threads and spider webs), the bundled structure of fibrils that compose spider threads has been frequently overlooked. Herein, I exploit a molecular dynamics-based coarse-grain model to construct a fully three-dimensional fibril bundle, with a length on the order of micrometres. I probe the mechanical behaviour of bundled silk fibrils with variable density of heterogenic protrusions or globules, ranging from ideally homogeneous to a saturated distribution. Subject to stretching, the model indicates that cooperativity is enhanced by contact through low-force deformation and shear ‘locking’ between globules, increasing shear stress transfer by up to 200 per cent. In effect, introduction of a random and disordered structure can serve to improve mechanical performance. Moreover, addition of globules allows a tuning of free volume, and thus the wettability of silk (with implications for supercontraction). These findings support the ability of silk to maintain near-molecular-level strength at the scale of silk threads, and the mechanism could be easily adopted as a strategy for synthetic fibres.
PMCID: PMC3627094  PMID: 23486175
computational mechanics; spider silk; fibre bundles; strength; shear transfer; free volume
8.  Hierarchical flexural strength of enamel: transition from brittle to damage-tolerant behaviour 
Hard, biological materials are generally hierarchically structured from the nano- to the macro-scale in a somewhat self-similar manner consisting of mineral units surrounded by a soft protein shell. Considerable efforts are underway to mimic such materials because of their structurally optimized mechanical functionality of being hard and stiff as well as damage-tolerant. However, it is unclear how different hierarchical levels interact to achieve this performance. In this study, we consider dental enamel as a representative, biological hierarchical structure and determine its flexural strength and elastic modulus at three levels of hierarchy using focused ion beam (FIB) prepared cantilevers of micrometre size. The results are compared and analysed using a theoretical model proposed by Jäger and Fratzl and developed by Gao and co-workers. Both properties decrease with increasing hierarchical dimension along with a switch in mechanical behaviour from linear-elastic to elastic-inelastic. We found Gao's model matched the results very well.
PMCID: PMC3350718  PMID: 22031729
hierarchical structures; mechanical properties; biological materials; dental enamel
9.  Flexibility regeneration of silk fibroin in vitro 
Biomacromolecules  2012;13(7):2148-2153.
Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were firstly prepared by slow drying process. Then the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared to the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nano-assembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.
PMCID: PMC3393754  PMID: 22632113
silk; mechanical regeneration; flexibility; bound water
10.  Silk Self-Assembly Mechanisms and Control-From Thermodynamics to Kinetics 
Biomacromolecules  2012;13(3):826-832.
Silkworms and spiders generate fibres that exhibit high strength and extensibility. The underlying mechanisms involved in processing silk proteins into fiber form remain incompletely understood, resulting in the failure to fully recapitulate the remarkable properties of native fibers in vitro from regenerated silk solutions. In the present study, the extensibility and high strength of regenerated silks were achieved by mimicking the natural spinning process. Conformational transitions inside micelles, followed by aggregation of micelles and their stabilization as they relate to the metastable structure of silk are described. Subsequently, the mechanisms to control the formation of nanofibrous structures were elucidated. The results clarify that the self-assembly of silk in aqueous solution is a thermodynamically driven process where kinetics also play a key role. Four key factors, molecular mobility, charge, hydrophilic interactions and concentration underlie the process. Adjusting these factors can balance nanostructure and conformational composition, and be used to achieve silk-based materials with properties comparable to native fibers. These mechanisms suggest new directions to design silk-based multifunctional materials.
PMCID: PMC3302850  PMID: 22320432
Silk Self-Assembly; Biomaterials; Kinetics; Nanostructure
11.  Multi-tissue transcriptomics of the black widow spider reveals expansions, co-options, and functional processes of the silk gland gene toolkit 
BMC Genomics  2014;15(1):365.
Spiders (Order Araneae) are essential predators in every terrestrial ecosystem largely because they have evolved potent arsenals of silk and venom. Spider silks are high performance materials made almost entirely of proteins, and thus represent an ideal system for investigating genome level evolution of novel protein functions. However, genomic level resources remain limited for spiders.
We de novo assembled a transcriptome for the Western black widow (Latrodectus hesperus) from deeply sequenced cDNAs of three tissue types. Our multi-tissue assembly contained ~100,000 unique transcripts, of which > 27,000 were annotated by homology. Comparing transcript abundance among the different tissues, we identified 647 silk gland-specific transcripts, including the few known silk fiber components (e.g. six spider fibroins, spidroins). Silk gland specific transcripts are enriched compared to the entire transcriptome in several functions, including protein degradation, inhibition of protein degradation, and oxidation-reduction. Phylogenetic analyses of 37 gene families containing silk gland specific transcripts demonstrated novel gene expansions within silk glands, and multiple co-options of silk specific expression from paralogs expressed in other tissues.
We propose a transcriptional program for the silk glands that involves regulating gland specific synthesis of silk fiber and glue components followed by protecting and processing these components into functional fibers and glues. Our black widow silk gland gene repertoire provides extensive expansion of resources for biomimetic applications of silk in industry and medicine. Furthermore, our multi-tissue transcriptome facilitates evolutionary analysis of arachnid genomes and adaptive protein systems.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-365) contains supplementary material, which is available to authorized users.
PMCID: PMC4200122  PMID: 24916340
de novo assembly; Spidroin; Gene family; Molecular evolution; Latrodectus hesperus
12.  Bioprospecting Finds the Toughest Biological Material: Extraordinary Silk from a Giant Riverine Orb Spider 
PLoS ONE  2010;5(9):e11234.
Combining high strength and elasticity, spider silks are exceptionally tough, i.e., able to absorb massive kinetic energy before breaking. Spider silk is therefore a model polymer for development of high performance biomimetic fibers. There are over 41.000 described species of spiders, most spinning multiple types of silk. Thus we have available some 200.000+ unique silks that may cover an amazing breadth of material properties. To date, however, silks from only a few tens of species have been characterized, most chosen haphazardly as model organisms (Nephila) or simply from researchers' backyards. Are we limited to ‘blindly fishing’ in efforts to discover extraordinary silks? Or, could scientists use ecology to predict which species are likely to spin silks exhibiting exceptional performance properties?
We examined the biomechanical properties of silk produced by the remarkable Malagasy ‘Darwin's bark spider’ (Caerostris darwini), which we predicted would produce exceptional silk based upon its amazing web. The spider constructs its giant orb web (up to 2.8 m2) suspended above streams, rivers, and lakes. It attaches the web to substrates on each riverbank by anchor threads as long as 25 meters. Dragline silk from both Caerostris webs and forcibly pulled silk, exhibits an extraordinary combination of high tensile strength and elasticity previously unknown for spider silk. The toughness of forcibly silked fibers averages 350 MJ/m3, with some samples reaching 520 MJ/m3. Thus, C. darwini silk is more than twice tougher than any previously described silk, and over 10 times better than Kevlar®. Caerostris capture spiral silk is similarly exceptionally tough.
Caerostris darwini produces the toughest known biomaterial. We hypothesize that this extraordinary toughness coevolved with the unusual ecology and web architecture of these spiders, decreasing the likelihood of bridgelines breaking and collapsing the web into the river. This hypothesis predicts that rapid change in material properties of silk co-occurred with ecological shifts within the genus, and can thus be tested by combining material science, behavioral observations, and phylogenetics. Our findings highlight the potential benefits of natural history–informed bioprospecting to discover silks, as well as other materials, with novel and exceptional properties to serve as models in biomimicry.
PMCID: PMC2939878  PMID: 20856804
13.  A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning 
Nature protocols  2009;4(3):341-355.
The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.
PMCID: PMC2720753  PMID: 19229199
14.  Chromosome Mapping of Dragline Silk Genes in the Genomes of Widow Spiders (Araneae, Theridiidae) 
PLoS ONE  2010;5(9):e12804.
With its incredible strength and toughness, spider dragline silk is widely lauded for its impressive material properties. Dragline silk is composed of two structural proteins, MaSp1 and MaSp2, which are encoded by members of the spidroin gene family. While previous studies have characterized the genes that encode the constituent proteins of spider silks, nothing is known about the physical location of these genes. We determined karyotypes and sex chromosome organization for the widow spiders, Latrodectus hesperus and L. geometricus (Araneae, Theridiidae). We then used fluorescence in situ hybridization to map the genomic locations of the genes for the silk proteins that compose the remarkable spider dragline. These genes included three loci for the MaSp1 protein and the single locus for the MaSp2 protein. In addition, we mapped a MaSp1 pseudogene. All the MaSp1 gene copies and pseudogene localized to a single chromosomal region while MaSp2 was located on a different chromosome of L. hesperus. Using probes derived from L. hesperus, we comparatively mapped all three MaSp1 loci to a single region of a L. geometricus chromosome. As with L. hesperus, MaSp2 was found on a separate L. geometricus chromosome, thus again unlinked to the MaSp1 loci. These results indicate orthology of the corresponding chromosomal regions in the two widow genomes. Moreover, the occurrence of multiple MaSp1 loci in a conserved gene cluster across species suggests that MaSp1 proliferated by tandem duplication in a common ancestor of L. geometricus and L. hesperus. Unequal crossover events during recombination could have given rise to the gene copies and could also maintain sequence similarity among gene copies over time. Further comparative mapping with taxa of increasing divergence from Latrodectus will pinpoint when the MaSp1 duplication events occurred and the phylogenetic distribution of silk gene linkage patterns.
PMCID: PMC2943472  PMID: 20877726
15.  The quest for stiff, strong and tough hybrid materials: an exhaustive exploration 
How to arrange soft materials with strong but brittle reinforcements to achieve attractive combinations of stiffness, strength and toughness is an ongoing and fascinating question in engineering and biological materials science. Recent advances in topology optimization and bioinspiration have brought interesting answers to this question, but they provide only small windows into the vast design space associated with this problem. Here, we take a more global approach in which we assess the mechanical performance of thousands of possible microstructures. This exhaustive exploration gives a global picture of structure–property relationships and guarantees that global optima can be found. Landscapes of optimum solutions for different combinations of desired properties can also be created, revealing the robustness of each of the solutions. Interestingly, while some of the major hybrid designs used in engineering are absent from the set of solutions, the microstructures emerging from this process are reminiscent of materials, such as bone, nacre or spider silk.
PMCID: PMC3808548  PMID: 24068176
biological materials; bioinspired composites; optimization; hybrid materials; bone; nacre
16.  The elaborate structure of spider silk 
Prion  2008;2(4):154-161.
Biomaterials, having evolved over millions of years, often exceed man-made materials in their properties. Spider silk is one outstanding fibrous biomaterial which consists almost entirely of large proteins. Silk fibers have tensile strengths comparable to steel and some silks are nearly as elastic as rubber on a weight to weight basis. In combining these two properties, silks reveal a toughness that is two to three times that of synthetic fibers like Nylon or Kevlar. Spider silk is also antimicrobial, hypoallergenic and completely biodegradable.
This article focuses on the structure-function relationship of the characterized highly repetitive spider silk spidroins and their conformational conversion from solution into fibers. Such knowedge is of crucial importance to understanding the intrinsic properties of spider silk and to get insight into the sophisticated assembly processes of silk proteins. This review further outlines recent progress in recombinant production of spider silk proteins and their assembly into distinct polymer materials as a basis for novel products.
PMCID: PMC2658765  PMID: 19221522
biomimetics; biotechnology; protein folding; protein assembly; spinning
17.  Module-Based Analysis of Robustness Tradeoffs in the Heat Shock Response System 
PLoS Computational Biology  2006;2(7):e59.
Biological systems have evolved complex regulatory mechanisms, even in situations where much simpler designs seem to be sufficient for generating nominal functionality. Using module-based analysis coupled with rigorous mathematical comparisons, we propose that in analogy to control engineering architectures, the complexity of cellular systems and the presence of hierarchical modular structures can be attributed to the necessity of achieving robustness. We employ the Escherichia coli heat shock response system, a strongly conserved cellular mechanism, as an example to explore the design principles of such modular architectures. In the heat shock response system, the sigma-factor σ32 is a central regulator that integrates multiple feedforward and feedback modules. Each of these modules provides a different type of robustness with its inherent tradeoffs in terms of transient response and efficiency. We demonstrate how the overall architecture of the system balances such tradeoffs. An extensive mathematical exploration nevertheless points to the existence of an array of alternative strategies for the existing heat shock response that could exhibit similar behavior. We therefore deduce that the evolutionary constraints facing the system might have steered its architecture toward one of many robustly functional solutions.
Biological systems maintain phenotypic stability in the face of various perturbations arising from environmental changes, stochastic fluctuations, and genetic variations. This robustness, which seems to be an inherent property of such systems, is still poorly understood at the molecular level. At the same time, systems approaches that were used with great success in the study and design of complex engineered systems provide a unique opportunity for investigating the basic tenants of robustness in cellular mechanisms. This is motivated by the fact that at the system level, biology and engineering seem to have a large number of common features despite their extremely different physical implementations. The heat shock response is one such robust cellular system, which interestingly achieves its seemingly simple objective of refolding or eliminating heat-denatured proteins through a complicated set of interactions. In analogy to engineering control architectures, the complex regulation strategies seem to be a specifically designed solution to generate robustness against different types of perturbations.
PMCID: PMC1523291  PMID: 16863396
18.  Interactions between Spider Silk and Cells – NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames 
PLoS ONE  2010;5(8):e12032.
Several materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature.
Methodology/Principal Findings
Steel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen- and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01).
Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres.
PMCID: PMC2918503  PMID: 20711495
19.  Multiscale structural gradients enhance the biomechanical functionality of the spider fang 
Nature Communications  2014;5:3894.
The spider fang is a natural injection needle, hierarchically built from a complex composite material comprising multiscale architectural gradients. Considering its biomechanical function, the spider fang has to sustain significant mechanical loads. Here we apply experiment-based structural modelling of the fang, followed by analytical mechanical description and Finite-Element simulations, the results of which indicate that the naturally evolved fang architecture results in highly adapted effective structural stiffness and damage resilience. The analysis methods and physical insights of this work are potentially important for investigating and understanding the architecture and structural motifs of sharp-edge biological elements such as stingers, teeth, claws and more.
Fangs are segments of the spider mouthparts, which are used to inject venom into prey and are required to sustain large mechanical loads. Here, the authors perform experiment-driven simulations, so to understand the correlation between the multiscale structural gradients and the biomechanical function of the fang.
PMCID: PMC4050259  PMID: 24866935
20.  Recombinant Minimalist Spider Wrapping Silk Proteins Capable of Native-Like Fiber Formation 
PLoS ONE  2012;7(11):e50227.
Spider silks are desirable biomaterials characterized by high tensile strength, elasticity, and biocompatibility. Spiders produce different types of silks for different uses, although dragline silks have been the predominant focus of previous studies. Spider wrapping silk, made of the aciniform protein (AcSp1), has high toughness because of its combination of high elasticity and tensile strength. AcSp1 in Argiope trifasciata contains a 200-aa sequence motif that is repeated at least 14 times. Here, we produced in E. coli recombinant proteins consisting of only one to four of the 200-aa AcSp1 repeats, designated W1 to W4. We observed that purified W2, W3 and W4 proteins could be induced to form silk-like fibers by shear forces in a physiological buffer. The fibers formed by W4 were ∼3.4 µm in diameter and up to 10 cm long. They showed an average tensile strength of 115 MPa, elasticity of 37%, and toughness of 34 J cm−3. The smaller W2 protein formed fewer fibers and required a higher protein concentration to form fibers, whereas the smallest W1 protein did not form silk-like fibers, indicating that a minimum of two of the 200-aa repeats was required for fiber formation. Microscopic examinations revealed structural features indicating an assembly of the proteins into spherical structures, fibrils, and silk-like fibers. CD and Raman spectral analysis of protein secondary structures suggested a transition from predominantly α-helical in solution to increasingly β-sheet in fibers.
PMCID: PMC3509139  PMID: 23209681
21.  A modular gradient-sensing network for chemotaxis in Escherichia coli revealed by responses to time-varying stimuli 
Combining in vivo FRET with time-varying stimuli, such as steps, ramps, and sinusoids allowed deduction of the molecular mechanisms underlying cellular signal processing.The bacterial chemotaxis pathway can be described as a two-module feedback circuit, the transfer functions of which we have characterized quantitatively by experiment. Model-driven experimental design allowed the use of a single FRET pair for measurements of both transfer functions of the pathway.The adaptation module's transfer function revealed that feedback near steady state is weak, consistent with high sensitivity to shallow gradients, but also strong steady-state fluctuations in pathway output.The measured response to oscillatory stimuli defines the frequency band over which the chemotaxis system can compute time derivatives.
In searching for better environments, bacteria sample their surroundings by random motility, and make temporal comparisons of experienced sensory cues to bias their movement toward favorable directions (Berg and Brown, 1972). Thus, the problem of sensing spatial gradients is reduced to time-derivative computations, carried out by a signaling pathway that is well characterized at the molecular level in Escherichia coli. Here, we study the physiology of this signal processing system in vivo by fluorescence resonance energy transfer (FRET) experiments in which live cells are stimulated by time-varying chemoeffector signals. By measuring FRET between the active response regulator of the pathway CheY-P and its phosphatase CheZ, each labeled with GFP variants, we obtain a readout that is directly proportional to pathway activity (Sourjik et al, 2007). We analyze the measured response functions in terms of mechanistic models of signaling, and discuss functional consequences of the observed quantitative characteristics.
Experiments are guided by a coarse-grained modular model (Tu et al, 2008) of the sensory network (Figure 1), in which we identify two important ‘transfer functions': one corresponding to the receptor–kinase complex, which responds to changes in input ligand concentration on a fast time scale, and another corresponding to the adaptation system, which provides negative feedback, opposing the effect of ligand on a slower time scale. For the receptor module, we calibrate an allosteric MWC-type model of the receptor–kinase complex by FRET measurements of the ‘open-loop' transfer function G([L],m) using step stimuli. This calibration provides a basis for using the same FRET readout (between CheY-P and CheZ) to further study properties of the adaptation module.
It is well known that adaptation in E. coli's chemotaxis system uses integral feedback, which guarantees exact restoration of the baseline activity after transient responses to step stimuli (Barkai and Leibler, 1997; Yi et al, 2000). However, the output of time-derivative computations during smoothly varying stimuli depends not only on the presence of integral feedback, but also on what is being integrated. As this integrand can in general be any function of the output, we represent it by a black-box function F(a) in our model, and set out to determine its shape by experiments with time-varying stimuli.
We first apply exponential ramp stimuli—waveforms in which the logarithm of the stimulus level varies linearly with time, at a fixed rate r. It was shown many years ago that during such a stimulus, the kinase output of the pathway changes to a new constant value, ac that is dependent on the applied ramp rate, r (Block et al, 1983). A plot of ac versus r (Figure 5A) can thus be considered as an output of time-derivative computations by the network, and could also be used to study the ‘gradient sensitivity' of bacteria traveling at constant speeds.
To obtain the feedback transfer function, F(a), we apply a simple coordinate transformation, identified using our model, to the same ramp-response data (Figure 5B). This function reveals how the temporal rate of change of the feedback signal m depends on the current output signal a. The shape of this function is analyzed using a biochemical reaction scheme, from which in vivo kinetic parameters of the feedback enzymes, CheR and CheB, are extracted. The fitted Michaelis constants for these enzymatic reactions are small compared with the steady-state abundance of their substrates, thus indicating that these enzymes operate close to saturation in vivo. The slope of the function near steady state can be used to assess the strength of feedback, and to compute the relaxation time of the system, τm. Relaxation is found to be slow (i.e. large τm), consistent with large fluctuations about the steady-state activity caused by the near-saturation kinetics of the feedback enzymes (Emonet and Cluzel, 2008).
Finally, exponential sine-wave stimuli are used to map out the system's frequency response (Figure 5C). The measured data points for both the amplitude and phase of the response are found to be in excellent agreement with model predictions based on parameters from the independently measured step and ramp responses. No curve fitting was required to obtain this agreement. Although the amplitude response as a function of frequency resembles a first-order high-pass filter with a well-defined cutoff frequency, νm, we point out that the chemotaxis pathway is actually a low-pass filter if the time derivative of the input is viewed as the input signal. In this latter perspective, νm defines an upper bound for the frequency band over which time-derivative computations can be carried out.
The two types of measurements yield complementary information regarding time-derivative computations by E. coli. The ramp-responses characterize the asymptotically constant output when a temporal gradient is held fixed over extended periods. Interestingly, the ramp responses do not depend on receptor cooperativity, but only on properties of the adaptation system, and thus can be used to reveal the in vivo adaptation kinetics, even outside the linear regime of the kinase response. The frequency response is highly relevant in considering spatial searches in the real world, in which experienced gradients are not held fixed in time. The characteristic cutoff frequency νm is found by working within the linear regime of the kinase response, and depends on parameters from both modules (it increases with both cooperativity in the receptor module, and the strength of feedback in the adaptation module).
Both ramp responses and sine-wave responses were measured at two different temperatures (22 and 32°C), and found to differ significantly. Both the slope of F(a) near steady state, from ramp experiments, and the characteristic cutoff frequency, from sine-wave experiments, were higher by a factor of ∼3 at 32°C. Fits of the enzymatic model to F(a) suggest that temperature affects the maximal velocity (Vmax) more strongly than the Michaelis constants (Km) for CheR and CheB.
Successful application of inter-molecular FRET in live cells using GFP variants always requires some degree of serendipity. Genetic fusions to these bulky fluorophores can impair the function of the original proteins, and even when fusions are functional, efficient FRET still requires the fused fluorophores to come within the small (<10 nm) Förster radius on interactions between the labeled proteins. Thus, when a successful FRET pair is identified, it is desirable to make the most of it. We have shown here that combined with careful temporal control of input stimuli, and appropriately calibrated models, a single FRET pair can be used to study the structure of multiple transfer functions within a signaling network.
The Escherichia coli chemotaxis-signaling pathway computes time derivatives of chemoeffector concentrations. This network features modules for signal reception/amplification and robust adaptation, with sensing of chemoeffector gradients determined by the way in which these modules are coupled in vivo. We characterized these modules and their coupling by using fluorescence resonance energy transfer to measure intracellular responses to time-varying stimuli. Receptor sensitivity was characterized by step stimuli, the gradient sensitivity by exponential ramp stimuli, and the frequency response by exponential sine-wave stimuli. Analysis of these data revealed the structure of the feedback transfer function linking the amplification and adaptation modules. Feedback near steady state was found to be weak, consistent with strong fluctuations and slow recovery from small perturbations. Gradient sensitivity and frequency response both depended strongly on temperature. We found that time derivatives can be computed by the chemotaxis system for input frequencies below 0.006 Hz at 22°C and below 0.018 Hz at 32°C. Our results show how dynamic input–output measurements, time honored in physiology, can serve as powerful tools in deciphering cell-signaling mechanisms.
PMCID: PMC2913400  PMID: 20571531
adaptation; feedback; fluorescence resonance energy transfer (FRET); frequency response; Monod–Wyman–Changeux (MWC) model
22.  Elucidating Metabolic Pathways for Amino Acid Incorporation Into Dragline Spider Silk using 13C Enrichment and Solid State NMR 
Spider silk has been evolutionarily optimized for contextual mechanical performance over the last 400 million years. Despite precisely balanced mechanical properties, which have yet to be reproduced, the underlying molecular architecture of major ampullate spider silk can be simplified being viewed as a versatile block copolymer. Four primary amino acid motifs: polyalanine, (GA)n, GPGXX, and GGX (X = G,A,S,Q,L,Y) will be considered in this study. Although synthetic mimetics of many of these amino acid motifs have been produced in several biological systems, the source of spider silk’s mechanical integrity remains elusive. Mechanical robustness may be a product not only of the amino acid structure but also of the tertiary structure of the silk. Historically, solid state Nuclear Magnetic Resonance (ssNMR) has been used to reveal the crystalline structure of the polyalanine motif; however, limitations in amino acid labeling techniques have obscured the structures of the GGX and GPGXX motifs thought to be responsible for the structural mobility of spider silk. We describe the use of metabolic pathways to label tyrosine for the first time as well as to improve the labeling efficiency of proline. These improved labeling techniques will allow the previously unknown tertiary structures of major ampullate silk to be probed.
PMCID: PMC3624022  PMID: 21334448
23.  The New Toxicology of Sophisticated Materials: Nanotoxicology and Beyond 
Toxicological Sciences  2010;120(Suppl 1):S109-S129.
It has long been recognized that the physical form of materials can mediate their toxicity—the health impacts of asbestiform materials, industrial aerosols, and ambient particulate matter are prime examples. Yet over the past 20 years, toxicology research has suggested complex and previously unrecognized associations between material physicochemistry at the nanoscale and biological interactions. With the rapid rise of the field of nanotechnology and the design and production of increasingly complex nanoscale materials, it has become ever more important to understand how the physical form and chemical composition of these materials interact synergistically to determine toxicity. As a result, a new field of research has emerged—nanotoxicology. Research within this field is highlighting the importance of material physicochemical properties in how dose is understood, how materials are characterized in a manner that enables quantitative data interpretation and comparison, and how materials move within, interact with, and are transformed by biological systems. Yet many of the substances that are the focus of current nanotoxicology studies are relatively simple materials that are at the vanguard of a new era of complex materials. Over the next 50 years, there will be a need to understand the toxicology of increasingly sophisticated materials that exhibit novel, dynamic and multifaceted functionality. If the toxicology community is to meet the challenge of ensuring the safe use of this new generation of substances, it will need to move beyond “nano” toxicology and toward a new toxicology of sophisticated materials. Here, we present a brief overview of the current state of the science on the toxicology of nanoscale materials and focus on three emerging toxicology-based challenges presented by sophisticated materials that will become increasingly important over the next 50 years: identifying relevant materials for study, physicochemical characterization, and biointeractions.
PMCID: PMC3145386  PMID: 21177774
nanotechnology; nanotoxicology; engineered nanomaterials; biokinetics; biointeractions; dose; physicochemical characterization
24.  Biological liquid crystal elastomers. 
Liquid crystal elastomers (LCEs) have recently been described as a new class of matter. Here we review the evidence for the novel conclusion that the fibrillar collagens and the dragline silks of orb web spiders belong to this remarkable class of materials. Unlike conventional rubbers, LCEs are ordered, rather than disordered, at rest. The identification of these biopolymers as LCEs may have a predictive value. It may explain how collagens and spider dragline silks are assembled. It may provide a detailed explanation for their mechanical properties, accounting for the variation between different members of the collagen family and between the draglines in different spider species. It may provide a basis for the design of biomimetic collagen and dragline silk analogues by genetic engineering, peptide- or classical polymer synthesis. Biological LCEs may exhibit a range of exotic properties already identified in other members of this remarkable class of materials. In this paper, the possibility that other transversely banded fibrillar proteins are also LCEs is discussed.
PMCID: PMC1692931  PMID: 11911772
25.  Nanomechanics of collagen microfibrils 
Collagen constitutes one third of the human proteome, providing mechanical stability, elasticity and strength to organisms and is thus the prime construction material in biology. Collagen is also the dominating material in the extracellular matrix where its stiffness controls cell differentiation, growth and pathology. We use atomistic-based hierarchical multiscale modeling to describe this complex biological material from the bottom up. This includes the use and development of large-scale computational modeling tools to investigate several aspects related to collagen-based tissues, including source of visco-elasticity and deformation mechanisms at the nanoscale level. The key innovation of this research is that until now, collagen materials have primarily been described at macroscopic scales, without explicitly understanding the mechanical contributions at the molecular and fibrillar levels. The major impact of this research will be the development of fundamental models of collagenous tissues, important to the design of new scaffolding biomaterials for regenerative medicine as well as for the understanding of collagen-related diseases.
PMCID: PMC3676161  PMID: 23885342
atomistic simulations; biomechanics; collagen; fibril; nanomechanics

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