Membrane-mediated protein-protein and protein-lipid interactions, membrane protein localization, and related dynamics, modulate membrane protein function [1]. So far membrane structure and dynamics could not be studied altogether lacking the technique that analyzes unlabelled proteins at submolecular lateral and high temporal resolution. Here we used high-speed atomic force microscopy (HS-AFM, [2]) to characterize the movements and interactions of unlabelled porin OmpF [3] and aquaporins [4] in native membranes. We are able to describe essential novel aspects that govern membrane protein assembly and membrane superstructure. Protein motion scales roughly with membrane crowding. However molecules display individuality of diffusion behavior ranging from fast moving to immobile molecules trapped by favorable protein-protein associations. We derive the molecular interaction probability landscapes and assembly rationales that we compare with coarse-grained molecular dynamics and Monte Carlo simulations. HS-AFM may open a novel research avenue that bridges structure of individual membrane proteins and supramolecular membrane architecture. References: [1] D.M. Engelman, Nature 438, 578 (2005) [2] T. Ando, et al., Proceedings of the National Academy of Sciences 98, 12468 (2001) [3] I. Casuso, et al., Nature Nanotechnology, in press [4] A. Colom, et al., in revision

Several investigations reported that cellular-substrate interactions are associated with the surface topography, chemical composition, surface energy, and surface charge of biomaterials. Zeolites are perspective inorganic nanomaterials for potential biomedical applications due to their unique properties, such as their high surface areas, tunable surface properties, chemical composition, and surface charge with controllable hydrophilic/hydrophobic nature. In this work, an array of line varying in size composed of zeolites was patterned on silicon substrate to study the effect of the zeolite patterns on cellular behavior. Zeolite A and silicalite were patterned on 1 cm x 1 cm wafer pieces by photolithography with patterned areas of 0.125, 0.08825, and 0.04167 cm2. Viability of MG 63 osteoblasts and NIH 3T3 fibroblasts was assessed through the MTT assays after 24, 48, and 72 hours of incubation with an initial cell concentration of 2 x 104 cells/sample of zeolite A- and silicalite-patterned surfaces. In addition, the calcined and as-synthesized zeolite A and silicalite nanoparticles were patterned in the same manner in order not only to investigate the effect of zeolite type, but also the effect of zeolite amount and calcination of zeolites on cell proliferation. Results showed that the cell proliferation was higher on zeolites with respect to the control group. Silicalite- patterned samples showed however the highest proliferation rate among all samples. This could be associated with the present of positive charge on the surface and interestingly, calcination increased the total number of both osteoblasts and fibroblasts. As known, calcination increases the internal surface area by cleaning the pores from organic species. Therefore, our result suggests that the pores play a significant role in the cell proliferation. As such, changing different zeolitic surfaces would lead to different cell behavior, and cell proliferation can readily be modulated by changing the amount and type of zeolites.

One-dimensional nanostructures are ideal building blocks for functional nanoscale assembly. Peptide based nanofibers have great potential in building smart hierarchical structures due to their tunable structures at the single residue level and their ability to reconfigure themselves in response to environmental stimuli. We observed that pre-adsorbed silk-elastin based protein polymers (SELP) self-assemble into nanofibers through conformational changes on a mica substrate. Furthermore, we demonstrated that the rate of self-assembly was significantly enhanced by applying a nanomechanical stimulus using a tapping mode atomic force microscopy (AFM). The orientation of the newly grown nanofibers was mostly perpendicular to the scanning direction, implying that the new nanofiber assembly was locally activated with directional control. Here we investigate the effects of the frequency and density of nanomechanical stimulus on nucleation of SELP nanofibers by controlling scan speeds and the number of scan line. It is noted that nucleation areas where nanofibers are grown are formed immediately during AFM scanning and their shape is elongated along the AFM scanning direction. Controlling tapping frequency and density of tapping showed that repetitive tapping is crucial to create nucleation areas, hence nanofiber growth. We also observe that self-assembly of SELP nanofiber is induced in a lateral force mode of AFM. Lateral force profiles show a significant correlation between frictional force and nucleation formation, indicating that strong interaction between SELP and AFM tip is a prerequisite for nucleation formation. Underlying molecular mechanisms are proposed based upon the characteristics of the force profiles, nucleation areas and their growth behaviors observed in time lapse AFM. Finally mechanically guided nanofiber patterns were successfully created on a mica substrate using this technique.

Collagen constitutes one third of the human proteome, providing mechanical stability, elasticity and strength to organisms. Normal type I collagen is a heterotrimer and consists of two alpha-1 chains and one alpha-2 chain. A variation of the natural type I collagen molecule is the type I homotrimer, which consists of three alpha-1 chains, and has been found in fetal tissues, fibrotic tissues, carcinomas, and fetal and cancer cells in human. A mouse model of the genetic brittle bone disease, osteogenesis imperfect (oim), is characterized by a replacement of the alpha-2 chain by a alpha-1 chain, resulting in a homotrimer collagen molecule. Experimental studies of oim mice tendon and bone have shown reduced mechanical strength compared to normal mice. Atomistic studies have revealed that the homotrimers form local kinks at molecular level. How the local kinks affect the packing of collagen molecules at the microfibril level and the relationship between the molecular content and the decrease in strength is, however, still not clear. Here we present a comprehensive study of the molecular and mechanical differences between the heterotrimer and homotrimer collagen microfibril models through a molecular simulation approach, coupled with experimental analysis. The collagen microfibril models are generated based on the in situ structure of full length collagen type I molecule with the actual amino acid sequence of real mouse collagen sequence (Collagen, type I, alpha 1 and alpha 2 chain precursor [Mus musculus]). In silico mechanical tests and structural analyses of collagen microfibril models are performed to understand the effects of the replacement of alpha-2 chain of type I collagen at the microfibril level and to address the origin of brittle bone disease. The results reveal nonlinear stress-strain relations for both normal and oim microfibril models, which are consistent with previous experiments that have shown the toe, heel and linear regions in the stress-strain curve of a single type I collagen fibril. We find that the homotrimer microfibril has an increased lateral spacing between collagen molecules in both dehydrated and hydrated conditions, which is coincided with experiments and suggests that the increased lateral spacing is not solely due to the higher hydrophobicity of the alpha-2 chain. Furthermore, mechanical tests show a higher modulus of the normal (heterotrimer) compared to the oim (homotrimer) microfibril, which suggests a molecular mechanism that the increase of lateral distance in oim fibril, due to local kink formations, causes a decrease of the modulus. Our studies provide fundamental insight of the effect of the loss of alpha-2 chain at the molecular level and help understanding the molecular origin of the bone brittle disease at much larger length-scales.

Ordered solid phases of proteins in native conformations: crystals, sickle cell hemoglobin polymers, and others, underlie physiological processes and pathological conditions, and laboratory procedures and technological designs. It has been shown that crucial precursors for the nucleation of these phases are metastable mesoscopic clusters. The clusters are composed of protein-rich liquid and exist both in the homogenous region of the solution phase diagram and in the region supersaturated with respect to an ordered solid phase. These clusters are of interest because their existence challenges our understanding of phases and phase equilibria. It has been demonstrated that the clusters largely consist of transient protein complexes that form at high protein concentrations. Towards understanding the nature of the transient complexes, we explore two potential mechanisms of their formation. Experiments reveal that the clusters exist both as high and at negligible ionic strength with similar properties. We computationally evaluate the potential of mean force (PMF) between two folded protein molecules. We account for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. The obtained PMF between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. The mechanism of cluster formation due to the conformation flexibility of the protein molecules, leading to the exposure of hydrophobic surfaces and enhanced intermolecular binding, was tested in experiments. We show that additives known to destabilize the native protein structure lead to enhanced cluster formation. NMR characterization reveals that in solutions, in which clusters are present at concentrations allowing each protein molecule pass through a cluster within an hour, the protein conformational variability is significantly enhanced in comparison to solutions without clusters. These results indicate that protein conformational flexibility might be the mechanism behind the metastable mesoscopic complexes and, hence, behind the clusters and new-phase nucleation

Tropoelastin is a molecular precursor that assembles and cross-links to form elastin, a protein key to providing elasticity and recoil to diverse tissues. Here we report a joint experimental-computational analysis of tropoelastin, achieved by combining molecular mechanics and x-ray scattering, to identify structure-property links in this critical biomaterial. The tropoelastin molecule contains a highly-coiled, extensible N-terminal region, separated from the cell-interactive C-terminal by a bridge region. A model has been proposed implicating the bridge region in assembly function due to a highly conserved domain 26, which is surrounded by hydrophobic domains involved in association and contains a protease-susceptible arginine residue. Three-dimensional geometries of wild type tropoelastin and a mutated form with a single alanine-for-arginine substitution were identified with small-angle x-ray scattering. These configurations served as templates for an elastic network bead-spring model to identify native conformational changes resulting primarily from molecular geometry. Fluctuations about the native molecular state were discretized through normal mode analysis to identify dominant dynamic response. Qualitative visualization in addition to quantitative stress analysis identified the central role of the bridge region in assembly driving motion.

Silk is a natural marvel: it is biodegradable, recyclable, and renowned for its outstanding toughness and extensibility. Attempts to produce artificial silk that mimics the qualities of natural silk have been met with only limited success, for the fabrication of natural silk not only requires silk protein, fibroin; but also involves finely tuned processing within the ducts of the silk-producing animal. As shearing forces are widely accepted to be crucial in the processing of silk, we imaged B. mori silk protein samples prepared from dopes at different concentrations and shear conditions using non-contact mode atomic force microscopy (NC-AFM). We observed the self-assembly of silk proteins into several distinct morphologies, some of which had not been reported before. When we sheared high concentrations of native silk fibroin harvested directly from B. mori specimens, we found straight nanofibrils 20-25 nm in diameter and multiple micrometers in length that were oriented parallel to the direction of shear. They are similar to the nanofibrillar structures known to occur on the surface of silk fibers, which are possibly due to shear at the walls of the B. mori spigot. We also tested reconstituted silk fibroin (RSF), a popular substitute for natural silk fibroin that is more easily obtained than native dope via dissolution of B. mori cocoons. Under similar concentrations and shearing conditions, RSF produced fibrils that were also 20-25 nm in diameter. But in notable contrast to the native silk nanofibrils, RSF fibrils were highly branched, less than a micrometer in length, and lacking in organization relative to the axis of shear. These striking differences in self-assembly between native silk dope and RSF indicate a fragile balance of interactions in these systems. We envision our approach to become a powerful tool to further investigate self-assembly of silk and other proteins under different conditions.

The structural integrity of tissues relies on the mechanical properties of the extracellular matrix (ECM) network. Mechanical forces regulate ECM assembly over multiple spatial scales; however, the impact of secondary protein structure on the mechanical properties of a network is still poorly understood. We asked how secondary protein structure influences the bulk mechanical properties of a manufactured Fibronectin (FN) textile. FN textiles are built in the form of nanometer thick fabrics by releasing micropatterned FN from a thermosensitive substrate. When these FN fabrics are uniaxially loaded, they deformed elastically when stretched less than two times their original length. Beyond this threshold, they exhibited plastic deformation. Fabric mechanics are modeled using an eight-chain network and two-state model to account for unfolding, revealing that the elastic properties of FN primarily depend upon the conformational changes of the protein and that the plastic deformation depends on domain unfolding. Thus, our results reveal how the mechanical properties of centimeter fabrics can be regulated by their molecular architecture.

Cephalopods are masters at manipulating light for camouflage, intra-species communication and threat display. Squid reflectin was previously identified as a protein with properties that made it well suited for light manipulation. Reflectin films demonstrate qualitative properties such as iridescence and light scattering, with the protein demonstrating the ability to self organize into photonic structures, such as diffraction gratings. Here we show that films made of reflectin1b can be made to scatter light in a wavelength-dependent manner when exposed to humidity. To address what portions of reflectin are important for this phenomenon, we have analyzed truncated variants of the protein to identify the minimal region important for light scattering. From this study, we have created concatemeric constructs with multiple repeats of the identified minimal reflectin region and analyzed the film morphology when the film is induced to scatter light with pulsed vapor. AFM analysis indicates that the films self organize into nanoscale, reticulated structures reminiscent of structures formed in the barbs of bird feathers that display structural coloration. The significance of these structures in the scattering of light at specific wavelengths is discussed.

This talk focuses on the material properties of beta-sheet rich protein nanofibrils. These structures possess numerous functional roles in nature but fibrils of this type are also connected to a range of protein misfolding disorders. We will discuss the nanoscale mechanics of protein fibrils, and use these measurements to shed light on the material selection criteria that bias molecular evolution to chose certain materials rather than others for functional purposes. Finally we discuss approaches to exploit the characteristics of these structures in artificial functional materials.

The ongoing pursuit to construct an artificial functional nanorobot was already achieved by biology long ago; nature perfected nanotechnology before researchers turned within us for bioinspiration. Many proteins act at the nano-scale as rotation or translation biological motors, being responsible for fundamental processes such as mitosis, cellular transport and muscle contraction. Among these proteins, the building blocks of self-assembled, highly-efficient natural motors, kinesin is sought as a promising tool in the development of synthetic nano-machines. Kinesin protein is a well known naturally occurring molecular machine capable of cargo transport upon interaction with cytoplasmic systems of fibers, known as microtubules. Conversion of chemical energy into mechanical work, harnessed by the hydrolysis of adenosine-5'-triphosphate (ATP), propels kinesin movement along microtubules. Even though recent efforts were made to engineer tailor-made artificial nanotransport systems using kinesin, no studies systematic investigated how those systems are built from the bottom up. Relying on the Surface Plasmon Resonance (SPR) technique, we will show for the first time that it is possible to quantitatively evaluate how each component of such nanoscopic machines is assembled by monitoring the individual association of its components, namely, the kinesin association to microtubule as well as the cargo-kinesin association.

Clathrin, a protein that plays a key role in the dynamic remodeling of the cell membrane during endocytosis, provides a framework that offers access to a variety of self-assembled cage-like architectures outside of a cell, such as spheres, barrels, tetrahedra, and cubes. This structural diversity using only a single protein building block makes clathrin an attractive model system to study the kinetic and thermodynamic principles of self-assembly. We combine experimental and theoretical approaches to study clathrin self-assembly in solution. Dynamic light scattering measurements and cryo electron microscopy are used to assess the dependence of self-assembly on environmental pH and ionic strength which reveals two distinct kinetic routes of assembly. A theoretical model has been developed that captures clathrin assembly behavior in Brownian dynamics simulations and has been used to study the effects of intermolecular binding energies and intramolecular elasticity on clathrin self-assembly and remodeling. Taken together, the results suggest several design rules for self-assembly which are expected to be generalizable to other self-assembling systems.

Self-assembly of proteins into extended ordered structures is a widespread paradigm in biological production of functional materials. During biomineral formation, protein matrices often serve to impose order on a nucleating mineral phase. In bone, collagen monomers form into triple helices, which then self-assemble into well-organized fibrils. Within these fibrils co-oriented hydroxyapatite (Hap) crystals nucleate and grow with a specific crystal face in contact with the collagen. The structural complexity and mechanical properties made possible through matrix-mediated mineralization are unparalleled in current synthetic processes. To understand the underlying physical controls governing both matrix assembly and mineralization, we are using in situ AFM, dynamic force spectroscopy (DFS) and molecular dynamics (MD) to investigate these processes. AFM investigations into the assembly of both collagen and microbial S-layer membrane proteins reveal the key role of conformational transformations in controlling the assembly pathways and kinetics. The large barriers associated with these transformation renders them rate-limiting. Consequently, before the ordered state can emerge, these systems must be driven to condense into metastable, liquid-like clusters in which protein-protein contact times are large. The emergence of order within these clusters catalyzes the further transformation and attachment of the monomeric proteins. DFS measurements show that subtle changes in the binding free energy between the proteins and with the substrates result in radically different architectures. Moreover, the pathway to the final ordered state can pass through transient, less-ordered conformational states. Thus the concept of a folding funnel with kinetic traps used to describe folding of individual proteins is also applicable to protein matrix self-assembly. In situ AFM and optical measurements of mineral nucleation dynamics on protein matrices show that these surfaces promote nucleation through a reduction in the interfacial energy. However, in the calcium phosphate-on-collagen system, nucleation of the amorphous phase is observed at supersaturations too low to be explained by classical nucleation theory (CNT). This leads to a formation pathway starting with amorphous calcium phosphate and passing through octacalcium phosphate before ending at the final Hap phase. Pre-nucleation clusters are shown to provide a low-barrier pathway to crystallization that circumvents the large barriers predicted by CNT. DFS measurements demonstrate that that collagen binding to HAP is highly anisotropic and the preferred directions are in good agreement with MD simulations providing a rationale for the control of the organized protein matrix over mineral orientation to form a hierarchical structure. Taken together, these results provide new insights into the physical mechanisms controlling self-assembly of protein matrices and matrix-directed mineralization.

One of the major challenges in bionanotechnology is the development of new methods for creating extended ordered self-assembled nanostructures. We have demonstrated that monomolecular protein lattices (S-layers) fulfil most key requirements for the development of new supramolecular materials enabling the bottom up design of a broad range of nanoscale devices [1]. Crystalline S(urface)-layers are the most commonly observed cell surface structures in prokaryotic organisms (bacteria and archaea) [1]. S-layers are highly porous protein meshworks with unit cell sizes in the range of 3 to 30 nm, and thicknesses of sim;10 nm. One of the key features of S-layer proteins is their intrinsic capability to form self-assembled monolayers in solution and at interfaces. Basic research on S layer proteins enabled us to make use of the unique self-assembly properties of native and, in particular, genetically functionalized S-layer protein lattices as matrices for the binding of molecules and the templated synthesis of nanomaterials. In addition, most recently S-layer proteins were used as scaffolds for making hybrid organic-inorganic nanostructures. Furtheron, advances in understanding the atomistic structure of S-layer proteins and simulating the self-assembly process will lead to the design of new materials for a diverse range of applications. In summary, the overall goal of our research is dedicated towards the development of an S-layer-based biomolecular construction kit for basic and applied research in the life and non-life sciences. References: [1] Sleytr, U.B., Schuster, B., Egelseer, E.M., Pum, D., Horejs, C.M., Tscheliessnig, R., Ilk, N. 2011. Nanotechnology with S-Layer Proteins as Building Blocks pp.277-352. In: Horworka, S. (Ed.), Prog. in Molecular Biology and Translational Science 103, Academic Press, Burlington, MA (USA). Acknowledgements: Part of this work was funded by the Air Force Office of Scientific Research (AFOSR) Agreement Awards FA9550-09-0342 and FA9550-10-0223, and the Erwin Schödinger Society for Nanobiosciences, Vienna, Austria.

Self-assembled protein architectures exhibit a wide range of structural motifs with functions that include selective transport, structural scaffolding, mineral templating and propagation of pathogenesis. Although the primary sequences of the individual proteins define their governing interactions, their functions depend on the quaternary architecture that emerges from self-assembly. Proteins that naturally self-assemble into extended structures of oligomers with long-range order often adopt conformations that are distinct from those of the individual monomeric protein. When individual proteins fold from an unstructured state to the final stable state, the concept of a folding funnel with kinetic traps, in which the protein exhibits non-equilibrium structures for extended period of time, is used to describe the pathway. Unfortunately, these transient states are difficult to observe at the single molecular level largely due to the limitations of spatial resolution of conventional optical techniques. Consequently, detailed information about the dynamics and energetics of protein collapsing down the folding funnel is limited. Despite the fact that folding transformations are part and parcel of protein self-assembly, this concept of the folding funnel has not been considered in that context. Here, we investigate the connection between these two phenomena and explore the dynamics and energetics of folding transformations by utilizing the inherent single molecule resolution of in situ AFM to follow 2D crystallization of S-layers on atomically flat mica surfaces at molecular-scale. We demonstrate the emergence of binary states of S-layer organization in 2D. We find this system possesses a kinetic trap associated with a conformational difference between a long-lived transient state and the final stable state. Both ordered tetrameric states emerge from clusters of an amorphous precursor phase, however, they then track along two different pathways. Over time, we show that the trapped state transforms into the stable state. By analyzing the time and temperature dependencies of formation and transformation, we find that the energy barriers to formation of either state are quite similar, differing by a mere ~1.6 kJ/mol. However, once the higher energy state forms, the barrier to transformation to the lower energy state is much larger at ~61 kJ/mol, leading to the slow transformation process. In conclusion, our findings demonstrate the importance of kinetic traps in determining the pathway of S-layer crystallization and suggest that the concept of the folding funnel for individual proteins can be equally applied to assembly of extended protein superstructures.

Collagen is the major structural protein of bone, dentine and the extracellular matrix and can template growth of numerous mineral phases. Both its molecular-scale conformation and mesoscale architecture are critical for its activity. Because both are influenced by interactions with substrates, understanding those interactions and the mechanisms of assembly on surfaces may enable us to direct assembly and hence engineer bioactive surfaces. We studied the dynamics and structure of collagen type I self-assembly on mica by AFM. At acidic conditions, K+ ions critically affected the collagen-mica interaction leading to assembled structures that evolved from 2D films of randomly oriented fibers to co-aligned fibers and finally to ordered 3D bundles as the K+ concentration increased from 100 to 300mM. High-resolution AFM showed the bundles consisted of intertwined single collagen triple-helices. XPS and NEXAFS showed the concentration of surfacial K+ within the collagen layer increases and the intensity of absorption peak due to π*(C=O) resonance decreases as the K+ concentration increased. This indicates K+ interacts with collagen via complexing with carbonyl groups and its effect on collagen-collagen bridging is the likely source of bundle formation. The magnitude of collagen-mica (C-M) and collagen-collagen (C-C) interactions at 200 and 300 mM K+ were measured by dynamic force spectroscopy. The binding free energy for C-M and C-C at 200 mM K+ were 13.7kT and 1.4kT respectively, while Gb at 300 mM K+ were 5.7kT and 12.3kT, respectively. The observed reversal in the sequence of C-C and C-M binding energies explains why the architecture switches from a 2D film to 3D bundles. Transformations between different assemblies were studied by in-situ AFM. Pre-organized films of ordered fibers transformed into ordered 3D bundles upon incubation in 300mM K+ solution. Because Gb of C-M binding at 300mM K+ was only 5.7kT, collagen fibers were still partially mobile with a diffusion coefficient D1~4×10-17cm2s-1. The stronger C-C interaction (12.3kT) drove bundle nucleation, which occurred through lateral movement and twisting of individual fibers. This result confirms that the film-substrate interactions are too weak to enforce a direct registry with the substrate lattice, while intrafilm interactions drive reorganization, giving surface-templated quasiepitaxial growth. For 2D co-aligned fibers, the time dependence of surface coverage followed a simple Langmuir adsorption, consistent with the observation that there is no cooperativity between fibers. However, for the bundles, assembly followed a highly non-linear time dependence in which acceleration in assembly rate correlated with bundle size. Thus bundle assembly on mica proceeds in three steps: (1) adsorption of a fibers that serve as the “nutrient”; (2) surface diffusion of the adsorbed fibers; and (3) nucleation of the ordered bundle through reorganization of aggregated fibers.

Visualizing dynamic processes occurring with biological molecules at high spatiotemporal resolution is one of the holy grails. To materialize this dream, we have developed high-speed atomic force microscopy (HS-AFM) instruments and techniques, including small cantilevers, an optical beam deflection detector for the small cantilevers, fast scanners, active vibration damping methods for the scanners, a new feedback controller, and so on. The feedback bandwidth of our current HS-AFM system exceeds 100 kHz, which provides a new opportunity of imaging dynamic molecular interaction and self-assembly processes at a rate of several tenths of ms per frame without disturbing their structure and dynamics (Ando et al. Prog. Surf. Sci. 2008). For example, the following dynamic processes were successfully captured on video using this system: planar lipid bilayer formation (Giocondi et al. BBA-Biomembranes 2009), growth and assembly of amyloid-like fibrils (Milhiet et al. PLoS One 2009), diffusion of point defects in two-dimensional crystal of a protein (Yamamoto et al. Nanotechnology 2008), and dynamic equilibrium occurring at the liquid-crystal interface in the purple membrane (Yamashita et al. J. Struct. Biol. 2009). Various dynamic events of molecules appear in an AFM movie without planned selection. Moreover, the dynamic events that appear on video can often be interpreted straightforwardly without sophisticated analyses, helping obtain convincing conclusions. These excellent general features of HS-AFM imaging markedly facilitate and accelerate our understanding of the mechanism of self-assembly of biological molecules.

Cellulose occurs in nature as long thin microfibrils. The self assembly (liquid crystalline phase formation) of cellulose microfibrils from various sources has been widely studied. For this, in order to get rid of entanglement and bundling/branching present in native cellulose microfibrils, various treatments have been used, most common being surface oxidation and acid hydrolysis. Surface oxidation eliminates the branching issue but does not get rid of entanglement. On the other hand, acid hydrolysis more efficiently gets rid of entanglement but drastically shortens the length of microfibrils to yield nanowhiskers. However, most of the effort towards self assembly till date has been on post biogenesis strategies. Therefore we have focussed on in-situ modification strategies. Amongst the various sources of cellulose, bacterial cellulose is advantageous due to its high purity, high crystallinity and thin long endless microfibrils. Bacterial cellulose yields larger aspect ratio nanowhiskers after acid hydrolysis in comparison to plants based sources. Moreover, bacterial cellulose has long been used to understand the biogenesis of cellulose in general as it provides opportunity of observations and modifications during synthesis. Here, we report in-situ modification strategies in bacterial cellulose in order to optimise microstructure for obtaining nanowhiskers with increased aspect ratio and thus improved self assembly. The effect of modification by additives like CMC, PEG etc on microstructure and on the aspect ratio of nanowhiskers obtained after acid hydrolysis is reported and analysed. Additives effective in altering length of microfibrils between branching points which in turn seems to affect the length of nanowhiskers have been identified. Thus, an effort has been made to control various aspects of cellulose microfibrils like width of microfibrils and length between branching points by in-situ modification.

The rational design of self-assembled materials requires an understanding of how intermolecular interactions are directed by the three-dimensional patterns in which functional groups are displayed from a molecular backbone. For some molecular systems, such as those interacting through nucleotide base pairing, remarkably complex structures (e.g., DNA origami) can be designed simply through specification of the subunit sequence. For many other molecular systems, including those that associate through “hydrophobic” interactions (as measured, for example, using the surface force apparatus and the atomic force microscope), an understanding of how patterns of nonpolar, uncharged polar and ionic functional groups combine to direct intermolecular associations in aqueous solution remains to be fully developed. Helical oligomers of β-peptides represent a particularly promising type of organic nanostructure for investigations of intermolecular forces that direct assembly processes because (i) the helical secondary structure can be designed to be very stable and because (ii) control of the β-amino acid sequence can lead to precise patterning of chemical functional groups over the surfaces of the helices. This presentation will describe the use of force spectroscopy to quantify the interactions of single β-peptide oligomers, each of which display stable and well-defined three-dimensional chemical nanopatterns, with hydrophobic surfaces. Whereas many prior reports of single molecule force measurements of oligo-α-peptides and macromolecules exist - the secondary and/or tertiary structures of these species are not preserved during their interactions at interfaces, and thus the three-dimensional chemical patterns that underlie previously reported force measurements are generally not known. By using β-peptide oligomers that display the same chemical functional groups in stable and distinct spatial nano-patterns, we have demonstrated that it is possible to relate changes in measured forces to changes in three-dimensional chemical nano-patterns. Overall, the results to be described in this presentation will show how β-peptide oligomers can be used to study intermolecular interactions that arise from precisely defined chemical nano-patterns. A particular focus of the talk will be directed to understanding hydrophobic interactions, thus providing insights into the mechanisms through which changes in chemical patterns presented by organic nanoscopic objects can dramatically affect their self-assembly behavior in aqueous environments.

We have previously identified peptides from a combinatorial library that are capable of binding to graphene. These identified peptides have shown their distinctive binding affinity to graphene/graphene nanostrip edges and planes. Here, we use a chemical vapor deposition (CVD) technique to produce an apposite graphene coating for studying the binding kinetics of the peptides using tools such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). The assembling characteristics of the graphene edge and plane binding peptide assembly is contrasted to their influence on the electronic properties of the CVD graphene using field effect transistor and resonance Raman spectroscopy measurements.

The self-assembly of peptides into different nanostructures has been the focus of intense research. In particular, the self-assembly mechanisms of amyloid β peptides have been extensively investigated due to their association with Alzheimer's disease. The resemblance of the obtained nanostructures to structures required for modern electronic devices motivates us to explore the possibility of integrating them in such applications. In this talk, the role of peptide sequence and the obtained morphology in determining electronic conduction along self-assembled peptide nanofilament networks will be demonstrated using an extension of a core sequence from the amyloid β peptide (AAKLVFF). I will present methodological studies that demonstrate changes induced to the morphology of amyloid β peptide self-assembled filaments by the incorporation of thienylalanine, a non-natural amino acid, into the peptide sequence and by changing the solvent used in the assembly process. I will further show that these sequence and structural changes affect the electrical conductance of networks of these materials. Finally, I will show that the conductance depends exponentially on the relative humidity in a structure dependent manner. Our results demonstrate that, in similar to the behavior of natural systems, the assembly and folding of peptides could be of great importance for optimizing their function as components of future electronic devices. 1) Moran Amit, Ge Cheng, Ian W. Hamley, and Nurit Ashkenasy, Soft Matter, accepted.

Designed self-assembling peptide hydrogels are a new class of materials that have gained considerable research interest for tissue engineering applications over the last decade. P11-4 (Ac-QQRFEWEFEQQ-Am) and P11-8 (Ac-QQRFOWOFEQQ-Am) are two such peptides. Each peptide is capable of self-assembling into beta sheet fibrils, forming hydrogels in physiological conditions by adding the dry peptide directly to cell medium, slightly adjusting the pH if necessary, and mixing. One feature common to both P11-4 and P11-8 is that, at neutral pH, each has a net charge of 2. To gain a better understanding of why these two peptides are particularly well suited to physiological conditions, we investigated the effect of charge on self-assembly. Using a proton NMR method, the concentration dependence of self-assembly was studied for P11-4, P11-8 and several other peptides derived from the same core sequence of P11-2 (Ac-QQRFQWQFEQQ-Am). This gave a range of net peptide charges from -6 to +4. Peptide secondary structures were additionally analysed with FTIR spectroscopy to verify the NMR study. NMR experiments showed that the critical aggregation concentration (c*) of the peptides increases with increasing net peptide charge. A lower c* may be particularly desirable so that gel breakdown is slower in vivo. (Non-aggregated peptide present at equilibrium will diffuse away from the implantation site). However, to ensure aggregates remain soluble, a small, non-zero net charge is required. These findings help to explain the observation that the ±2 charge peptides, P11-4 and P11-8, are ideal for use in physiological conditions. From the NMR data, the fraction of aggregated peptide can be determined as a function of concentration. This data, together with c* values and TEM-measured fibril widths, has been fitted to the statistical mechanical model for hierarchical peptide self-assembly previously developed by Aggeli et al.[1] From these fits, we are now able to derive thermodynamic parameters of self-assembly for this group of peptides and better rationalise the experimental findings. Design criteria determined in studies like these are particularly useful. They can be harnessed when designing new peptides, and in identifying those peptides that may be commercially important in the future. [1] PNAS (2001), 98, pp11857-11862

The rational design and synthesis of protein-mimetic materials based on the folding of non-natural polymers into defined nanostructures is a fundamental challenge in materials science. Two levels of "synthesis" are required: (1) the covalent structure - the conventional multi-step organic synthesis of the precise monomer sequence in the chain, and (2) the non-covalent structure - the folding/assembly of the chain into a higher order structure. Both of these synthetic processes have been studied in detail for the production of peptoid nanosheets in high yield. Two-dimensional nanomaterials play a critical role in biology (e.g., lipid bilayers) and electronics (e.g., graphene) but are difficult to directly synthesize with a high level of precision. Peptoid nanosheet bilayers are a versatile synthetic platform for constructing multifunctional, precisely ordered two-dimensional nanostructures. Here we show that nano sheet formation occurs through an unusual monolayer intermediate at the airwater interface. Lateral compression of a self-assembled peptoid monolayer beyond a critical collapse pressure results in the irreversible production of nanosheets. An unusual thermodynamic cycle is employed on a preparative scale, where mechanical energy is used to buckle an intermediate monolayer into a more stable nanosheet. Detailed physical studies of the monolayer-compression mechanism revealed a simple preparative technique to produce nanosheets in 95% overall yield by cyclical monolayer compressions in a rotating closed vial. Compression of monolayers into stable, free-floating products may be a general and preparative approach to access 2D nanomaterials.

Directed assembly is a commonplace phenomenon in nature and is frequently responsible for development of one, two and three-dimensional functional structures. For example, protein self-assembly plays a significant role in the growth and organization of the mineral in hard tissues (e.g. tooth and bone). Peptoids, or poly-N-substituted glycines, are a novel class of non-natural polymers developed to mimic both structures and functionalities of polypeptides, and bridge the gap between biopolymers and bulk polymers. As with peptides, sequence-specific peptoids can be efficiently synthesized by using automated solid-phase synthesis. Moreover, peptoids exhibit much higher protease and thermal stabilities than polypeptides. Recently, we successfully demonstrated that peptoids were able to mimic mineralization polypeptides for control over both CaCO3 crystal morphology and growth kinetics. Therefore, directed assembly of functional peptoids and an understanding of their assembly pathways and mechanisms can be significant for developing biomimetic materials for applications. Here, we report the recent investigation of oriented assembly of acidic peptoids both on mica surfaces and at the water-air interface. On freshly cleaved mica, peptoid-calcium(II) complexes self-assemble into well-aligned nano-rods or -fibers with hexagonally symmetric patterns. Peptoid-calcium(II) complexes first appear as amorphous structures, and then form uniform nanoparticles before eventually assembling into the well-oriented nano-fibers. Interestingly, we found that these patterns of fibers are capable of directing both formation and orientation of superfibers up to ~1.4 mu;m in width and ~14 mu;m in length. High-resolution AFM images showed that these superfibers follow the same orientation on mica as the nano-fiber precursors. At the water-air interface, peptoids self-assemble into two-dimensional sheet-like superstructures comprising peptoid fibers. Assembled peptoid sheets reached sizes in excel of 20 × 20 mu;m2 and their thicknesses varied from ~3.0 nm to more than 100 nm. In order to provide further insights into the assembly mechanisms of peptoid-calcium(II) complexes, we performed dynamic force spectroscopic (DFS) studies of interactions between peptoid-calcium(II) complexes and both mica and the assembled peptoid fibers. At a single pulling speed, the rupture forces of peptoid-mica and peptoid-peptoid fibers in the presence of 11 mM CaCl2 solution are 771.5 pN and 120.0 pN respectively. The binding free energy extracted from DFS studies indicates that peptoid-calcium(II) complexes bind much more strongly to mica surfaces than do the peptoid fibers. While the presence of 120 pN rupture force between peptoid-calcium(II) complexs and peptoid fibers indicates peptoid can also bind to each other in the presence of CaCl2 for further assembly. Therefore, our DFS results provide an explanation for the observation of two different assembly phenomena.

In spite of the growing interest in nanobiotechnology, fundamental understanding of the interactions of biological elements (e.g., peptides, proteins, nucleic acids, cells) with nanomaterials to control structure, properties and functionality at the nano-bio interfaces remains challenging. Particularly, specific interactions between peptide and gold concern potential applications of their hybrids in the new realm of nanobiotechnology, namely biofunctionalization of nanomaterials for biological and chemical detection, and nanomedicine. Here, we demonstrate and propose a mechanism for dynamic coil formation of cysteine-free gold-binding peptide (A3) that has been screened by phage-display to bind to gold, upon adsorption on to Au (111) surface as revealed by atomic force microscopy (AFM). Concentration-dependent adsorption morphologies of A3 oligomers as well as dramatic changes in their persistance length with adsorption time will be presented. Our results suggest that A3 fibrillation continued with coil formation on Au (111) via surface-diffusion was supported by intermolecular and intramolecular interactions. Our observations provide new insight into adsorption and surface behavior of material-binding peptides and lay path forward for understanding their interaction with nanomaterials.

The chiral biasing of proteins with only L-amino acid configurations is a fundamental paradigm in biology. To date, there has been some evidence to suggest that differential hydration, preferential crystallization, chiral selection by minerals, and the presence of right circularly polarized light on earth may have had an impact in the evolutionary selection of L- over D-amino acids. Alternatively, we believe that inorganic matrices may have played a substantial role in producing L-amino acid containing protein structures in nature beyond simple chiral selection by acting both as a catalytically active and chiral surface for D-amino acid containing peptides and proteins. In this study, we show that a right-handed peptide composed of all D-amino acids undergoes an optical and chemical transition to the left-handed L-form in the presence of gold nanoparticles. This chiral conversion activity of nanoparticles will likely expand the tunability and scope of metamaterials as well as provide a platform for chiral sensing.

The interface between minerals (hard) and organic molecules, arrays and scaffolds (soft) can exercise control in two directions. First, the binding of large molecules on surfaces can induce conformational folding and consequent effects on molecular function. Understanding these effects is essential for understanding the attachment of cells and bacteria (and consequently biofilms) to surfaces. On the other hand, soft matter in the form of molecules,arrays or scaffolds can control the nucleation and growth of crystals. The resulting materials have complex structures, often with distinctive features at different length-scales. We present a number of examples from our recent work showing how a combination of theory and experiment can shed light on the fundamental mechanisms involved from the atomic to the macroscopic level. These examples include the effect of binding to quartz and aragonite on the conformation of biomolecules, the effect of biomolecules on crystal nucleation, inhibition of crystal growth by molecules such as polysaccharides, the control of crystal orientation and growth by self-assembled monolayers, the effect of incorporation of "inclusion compounds" on crystal properties, and how the sequence of amino acids in biopolymers controls the mineralisation of biominerals such as coral.

Hydroxyapatite (HAP) based polymer composites have been effectively used to prepare scaffolds for bone tissue engineering. In our recent research studies, organically modified nanoclay particles are introduced to improve the mechanical properties and bone tissue engineering applications of scaffolds. A novel biomimetic materials design route is used to mineralize hydroxyapatite in the intercalated galleries of nanoclays. This mineralization route mimics biomineralization in human bone. In this study we report simulations of this nanoclays-hydroxyapatite-polymer system and molecular interactions therein. Representative models are constructed systematically to represent organically modified clay (OMMT) with HAP and polycapralactone (PCL) polymer composite. The representative models are validated by combining conventional computational criteria like minimum energy and density of composite system with experimental techniques such as photoacoustic spectroscopy and XRD. The molecular dynamics simulation studies reveal quantitatively interactions between the clay, modifier, HAP and PCL system. The interaction energy maps are obtained that reveals mechanisms and interactions between different constituents in the system. These models provide useful information about the functions of backbone chain and functional groups, orientation and conformation changes, hydrogen bonding in modifier, chelation bonds, orientation of HAP, mechanism of HAP mineralisation in OMMT-HAP and the role of different constituents and their quantitative contributions in OMMT-HAP-PCL system. We have investigated the mechanical behaviour by stress deformation responses of the composite system using molecular dynamics simulations. Mechanical properties of composites are also studied by nanoindentation and other mechanical experiments. These simulation and experiments provide insight into a simulation based design of biomaterials that mimic biology.

Molecular self-assembly has become a widely used method for fabricating biological and biocompatible structures at the nano- and micrometer range. In particular, amphiphilic peptides have been shown to self-assemble into a variety of nano-mesoscale structures, going from fibrils to ribbons, with diameter of a few nanometer and length in the micrometer scale. In the present paper, the self-organizing behavior on surfaces of two classes of amphiphilic peptides, both composed of alanine (A), Aspartic acid (D) and Lysine (D), with different structure has been studied by using Atomic Force Microscopy. In particular, a 5-terms peptide AcA4DOH, featuring a single tail, and a 10-terms peptide (AcA4)2KDOH, structured with a double tail, have been studied. The peptides showed different organization patterns ranging from unstructured round shape aggregates to 1D-wires. In particular, single tail peptides were not able to self-organize, while double tail molecules showed a dramatic self-organization behavior. The role of surfaces in promoting self-organization by tuning the orientation of the single molecules will be focused, going from the primary alignment step to the aggregation process at the mesoscopic scale. Thus, the effect of pH, surface free energy and charge state of molecules and surfaces has been investigated for two positively charged polymeric polyelectrolyte substrates, polyethylenimine and poly(diallyl dimethylammonium) chloride, and a negative one, poly(sodium styrene sulfonate). A molecular model, based on the role of the primary structure of the two peptide classes and the charge state evolution of the molecules and surfaces, will be proposed highlighting the predominance of the electrostatic interactions.

Aptamers are oligonucleic acids that can be selected to specifically interact with in principle any kind of target molecule. An important asset of aptamer conjugates is the fact that upon specific binding, their spatial conformation may change drastically, depending on a fine equilibrium between mainly electrostatic and hydrophobic interactions. Recently, if has been shown that this specific conformational change can be exploited for controlled release applications in particles and membranes, where aptamers serve as a “nanovalve” which selectively triggers the opening or closing of a nanopore depending on the presence of a target molecule. Such systems add an important degree of freedom in the design of stimuli-responsive systems which conventionally respond to bulk stimuli such as pH, temperature, light or electrical and magnetic fields. In this work particular attention is given to the potential of using aptamers to selectively interact with small molecules, such as adenosine-triphosphate (ATP), which has hardly been explored until today for nanodevices. Recently it has been demonstrated that target-specific aptamers can be a promising alternative to existing methods of creating selective, stimuli-responsive interfaces, particularly with respect to their versatility and their potential to be designed for virtually any target molecule [1]. For elucidating interactions and conformational changes that lead to the function of aptamer-based nanovalves, some analytical techniques will be critically discussed, namely surface plasmon resonance (SPR), dual polarization interferometry (DPI) and the quartz crystal microbalance with dissipation monitoring (QCM-D). It will be shown how these techniques can help us to verify the function of aptamer films, but also how much one needs to be aware of underlying measurement principles in order to avoid possible pitfalls. 1.Özalp, V.C., and Schäfer , T. Chem. Eur. J., 17(36), 9893-9896, 2011

A major challenge in materials science is the synthesis of polymers with determinate properties for the production of high-performance materials. Recent advances in synthetic organic chemistry have allowed for refinement of molecular design. However, the complexity and functionality of synthetic materials is limited by an inability to precisely control chain length and location of functional groups. In contrast, biologically derived polymers have an unmatched ability to assemble into functionally precise three-dimensional structures via sequence-specific chain folding, but these “biomaterials” are evolutionary constrained to their natural functions. Therefore, our approach aims to broaden the capabilities of existing biopolymers to supplant “model” synthetic polymers for the production of high precision materials. In this work, we report a highly tunable system capable of rapid and cost-effective production of monodisperse and stereochemically precise nucleotidomimetic polymers. Based on a top-down approach, we developed a facile synthesis platform to precisely incorporate a wide-variety of functional group modifications in a simple two-step process. First, we utilize the natural ability of DNA polymerase to enzymatically incorporate chemically-modified monomers in a template-directed fashion. In this way, we are able to explicitly position non-natural (deoxy)ribonucleotide triphosphates (dNTPs) along the ssDNA backbone. Second, we employ copper-catalyzed azide-alkyne cycloaddition (CuAAC) to integrate the desired chemical functionality. The mild reaction conditions, unique bioorthogonality, high yields and broad functional group acceptance of CuAAC make it an ideal reaction to modify ssDNA. This overall synthetic strategy allows for the systematic variation of oligomer length, stoichiometry, concentration and environmental conditions to rapidly explore nucleotidomimetic polymer phase behavior for materials discovery.

Photosystem II contains an oxygen-evolving center (OEC) which catalyzes the oxidation reaction of water to molecular oxygen. This protein, having 700kDa molecular weights, consists of a tetra-nuclear Mn cluster and has a double-bridged di-nuclear Mn structure (Mn(mu;-O)2Mn). In 2011, a remarkably high-resolution X-ray analysis of its crystal structure has been established by J.-R. Shen et al. This result provides a good opportunity to clarify the mechanism of the water splitting reactions. The X-ray structure shows the detailed information of S1 state from which the OEC must forward four more steps (JK cycle) to generate molecular oxygen. It is very difficult to obtain structural information for S2, S3, S4 and S0. Considering the difficulty of this natural complexity, new spectroscopic approaches and theoretical studies are in order. In an attempt to understand the mechanism in OEC on a molecular level, we have carried out studies of following four approaches: (1) Analysis of artificial Mn2 complexes which catalyze also water oxidation reaction, (2) QM/MM quantum chemical study of Mn4O5Ca cluster, (3) Classical molecular dynamics simulation of total protein structure PSII, and (4) Obtain CD spectroscopic data during the JK cycle. Amongst some artificial Mn-complexes by which water oxidation reactions are reported, we have chosen two well defined experimental results. The first one is reported by A. K. Poulsen et al., a synthetic di-nuclear manganese complex of the ligand N-methyl-N0-carboxymethyl- N,N0-bis(2-pyridylmethyl)ethane-1,2-diamine. The second one is by J. Limburg et al, a synthesized Mn complex with similar double-bridged (Mn(mu;-O)2Mn) structures of [H2O(terpy)Mn(mu;-O)2 Mn(terpy)OH2]3+ (terpy denoting 2,2':6',2''-terpyridine). Notice that OEC oxidizes water by using light energy, on the other hand these artificial Mn2 complexes need oxidant such as HOCl or tBuOOH. We have shown the mechanism how molecular oxygen evolves by obtaining the transition state structures and energies. Based on the knowledge of these complexes, we have analyzed the mechanism of the Mn cluster by using quantum chemical QM/MM methods. We have compared the experimental FTIR data with calculated frequency analysis data through which we infer the mechanism of JK cycle. Remarkable results are the field fluctuation effect, which is obtained by MD on total PSII. The results suggest that Mn cluster functions on the assistance of the field soft modes of the protein.

Laser induced molecular self-assembled sophorolipid structure is realized which shows remarkably strong fluorescence in the visible which is totally absent in the original sophorolipid molecule. The sophorolipid was mixed in water and sonicated for few hours for complete dispersion and was then subjected to pulsed excimer laser (248 nm) irradiation at an energy density of ~166 mJ/cm2 and pulse repetition rate of 10 Hz for 1h. It was observed that the sophorolipid solution turns slightly yellowish after completion of reaction and shows nanoparticle formation. Their size can be tuned by adjusting the parameters like concentration, stirring and duration of laser irradiation. Study of the kinetics of nanoparticle formation process reveals the mechanism of lipid conversion into vesicles. These samples were characterized by various techniques such as polarized microscope, scanning electron microscopy (SEM), high resolution transmission electron microscopy (HR-TEM), and photoluminescence spectroscopy. For the study of photoluminescence of the unirradiated and irradiated sophorolipid, these samples were excited at the same wavelength of 330 nm. In the case of the unirradiated sophorolipid sample a very weak excitonic emission is observed in UV at 370 nm, while in the case of the irradiated sample a strong emission is seen to occur in the visible at 510 nm. Importantly the laser synthesized nanostructures can be easily redispersed in aqueous medium after being dried. We could also render these sophorolipid nanostructures magnetic by embedding chemically synthesized iron oxide nanoparticles for hyperthermia treatment. The optical properties, biocompatibility, magnetic property and cost effectiveness make these nanostructures excellent candidates for bioimaging and other therapeutic applications.

The development of multilayer thin films containing biomolecules has attracted much attention in biomaterial science and technology as one of the biomedical applications of layer-by-layer assembly. Recently, the chance that the medical equipments such as endoscopes and catheters are used in the inside of the body has increased. However, when coming in contact with blood, the medical equipment recognized the foreign body for the human body causes the generation of the thrombus. As a result, the performance of the medical equipment decreased and sometime it cause a secondary disease such as the blocking of blood vessel. Therefore, it is strongly required to control the generation of the thrombus to medical equipment. In this study, we prepared polyelectrolyte multilayer films for inhibition of protein absorption because protein absorption is the first factor for formation of thrombus. By dipping hydrophilic substrate into cationic polyelectrolyte solution and anionic solution alternately, thin polymer layer was fabricated. We investigated the relationship between inhibition of protein adsorption and surface wettability. As anionic solution, heparin, which is actually used in clinical practice as anticoagulant agent was selected. Albumin, which is a component of blood plasma was chosen for cationic solution. After fabricating polymer film, water resistance and mechanical durability were enhanced by chemical crosslinking. Hydrophilic agent was added into polymer solution, and surface wettability was changed. We showed the possibility that an effect of inhibition of protein absorption has strong relationship with surface wettability.

Collagen is a triple helical molecule and the most abundant protein in vertebrates, provides mechanical stability, elasticity and strength to organisms. Collagen cleavage, facilitated by collagenases of the matrix metalloproteinase (MMP) family, is crucial for many physiological and pathological processes such as wound healing, tissue remodeling, cancer invasion and organ morphogenesis. Normal physiological remodelling processes involve precisely regulated collagen degradation, where excessive or deficient degradation has been associated with numerous diseases. Earlier work has shown that mechanical force alters the cleavage rate of collagen. However, experimental results yielded conflicting data on whether applying force accelerates or slows down the degradation rate. Normal type I collagen is a heterotrimer and consists of two alpha-1 chains and one alpha-2 chain. A variation of the natural type I collagen molecule is the type I homotrimer, which consists of three alpha-1 chains, and has been found in fetal tissues, fibrotic tissues, carcinomas, and fetal and cancer cells in human. It is also found in a mouse model of the genetic brittle bone disease, osteogenesis imperfecta (OI), the oim mutation of type I collagen. Previous studies have shown that the type I heterotrimer and homotrimer have distinct degradation behaviors. Type I homotrimers are found to be resistant to all mammalian collagenases, with a cleavage rate much slower for homotrimers than for heterotrimers. The MMP resistance of homotrimers may play an important role in homotrimer-related diseases or in early development, during which necessary collagen degradation may be hindered with detrimental results. Here we explain these discrepancies and propose a molecular mechanism by which mechanical force might change the rate of collagen cleavage. We find that a type I collagen heterotrimer is unfolded in its equilibrium state and loses its triple helical structure at the cleavage site without applied force, possibly enhancing enzymatic breakdown as each chain is exposed and can directly undergo hydrolysis. Under application of force, the naturally unfolded region refolds into a triple helical structure, potentially protecting the molecule against enzymatic breakdown. In contrast, a type I collagen homotrimer retains a triple helical structure even without applied force, making it more resistant to enzyme cleavage. In the case of the homotrimer, the application of force may directly lead to molecular unwinding, resulting in a destabilization of the molecule under increased mechanical loading. Our study explains how force may regulate the formation and breakdown of collagenous tissue.

It is well known that liposome is composed of phospholipid bilayer, which is the basic structure of the cell-wall, and is applied to the micro-capsule in the drug delivery system (DDS) and in cosmetics. Since the function of biological soft matter usually depends on its three-dimensional conformation, nanoscale observation of biological soft matter such as protein and DNA, etc. has been studied intensively. Especially, transmission electron microscopy (TEM) offers high resolution images up to sub nanometer, it is one of excellent methods for observation. There is, however, inevitable problem, i.e., the specimen should be settled in the vacuum. This means that the observation of raw biological soft matter is difficult. Therefore, the direct nanoscale observation of biological soft matter is important technique which should be developed and, in this study, we try to use the liposome as a microcapsule for TEM observation of wet biomaterial. First of all, conventional liposome was synthesized by using dipalmitoylphosphatidylcholine (DPPC). However these liposomes were little resistance to electron radiation. Accordingly, the liposome reinforced with cholesterol was prepared as follows: DPPC, pluronic F-18, and cholesterol were firstly mixed in the physiological saline. Subsequently it was evaporated, incubated, sonicated and finally incubated again. For the TEM observation, these liposome were dropped on an amorphous carbon mesh and negatively stained with platinum blue (TI blue) for 60 min. The liposomes reinforced with cholesterol were spherical in shape with 100-500 nm in diameter and still stable in the vacuum. Some of them included the physiological saline, the rest of them included TI blue, which is sometimes crystallized in the liposome. As the result, it can be mentioned that the capsule of the liposome reinforced with cholesterol shows enough strength to hold wet biological material in vacuum and could be applied for the TEM observation. It is also noted that the capsule also supplies a field of the crystal growth.

Collagen-apatite (Col-Ap) composite resembling the composition of natural bone has been studied extensively and considered as a promising bone tissue engineering material . In the current study, a procedure combining biomimetic precipitation and controlled freeze casting has been developed successfully to produce porous Col-Ap composite scaffolds with tunable compositions and microstructures. A simple biomimetic co-precipitation method has been established where a bone-like hierarchical structure was achieved through controllable mineralization of collagen fibrils. Rheological study found that the kinetic of collagen assembly and apatite precipitation was controlled by pH value and temperature. A temperature ramp process at certain pH value was developed to make the collagen self-assembly synchronously with the nucleation and growth of apatite crystals onto the internal surfaces of collagen fibrils and external surfaces of collagen bundle. In addition, the mechanism of freeze casting was investigated in-depth, thereby establishing the freezing regimes for equiaxed and lamellar solidification. Col-Ap scaffolds with an isotropic equiaxed structure and a unidirectional lamellar structure were prepared by controllable freeze casting, and the pore size of which could be easily adjusted. Thus prepared Col-Ap scaffolds well supported bone repair at a mouse calvarial site.

An artificial cell membrane was developed using biomembrane components. The cell membrane with a lipid bilayer was observed using surface plasmon resonance (SPR) for identification of its hydrolysis by phospholipase (PL). The observation utilizing the simultaneous atomic force microscope (AFM) images shows that the PLs enables the nanometer-scale hydrolysis of the artificial lipid biomembranes through enzymatic hydrolysis. In addition, it was confirmed that PL with melittin obtained the controlling factor for enzyme hydrolysis on degradation of lipid bilayer. As for the expected melittin&’s hydrolysis activating effect, any difference from non-treated lipid membrane could not be seen in AFM images. It is assumed that the partitioning of melittin into the membrane might prevent the hydrolysis or binding of Phospholipase A2 (PLA2). This study would provide a basic knowledge to a new approach of patterning the bio-mimicking lipid membranes on the nano-scale.

The inorganic nanoparticles possess a range of tunable optical fluorescence or absorption properties depending on their chemical composition and their shape (semi-conductor (QD) and metallic gold) whereas the surface ligand can be optimized to tailor interactions with the surroundings. Their properties can be used collectively within nanostructured materials. Nanoparticles (QD, Au) are also ideal building blocks for the construction of ordered 3D structures. We present here different strategies to solubilise, chemically-functionalize nanocrystals into water and to organize them in a controlled manner at a macroscale. The first one is based on the self-assembling properties of synthetic gallate amphiphiles (Boulmedais et al, Langmuir 2006 ; Roullier, V. et al Chem. Mat. 2008; Amela-Cortes et al. Chem. Comm. 2011) and the use of controlled water drying to self organize the nanoparticles. In the second approach, biological molecules and molecular self-assemblies are used as templates to organize well-defined inorganic nanostructures. The interaction between anionic peptidic quantum dots and cationic vesicles results in the formation of either hybrid QD vesicles or a well-defined lamellar hybrid condensed phase in which the QDs are densely packed in the plan of lamellas (Dif A. et al J. Am. Chem. Soc. 2008). The addition of the well-known anionic actin protein to this system induced the formation of fluorescent 3D crystalline fibers. We demonstrate the ability of a self-assembled 3D crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots (Henry E; Dif A. et al Nanoletters 2011). Functionnalized tricystein peptidic Quantums Dots, QDs, are incorporated during the dynamical self-assembly of this actin /lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5x18.9x35.5 nm3 are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a larger range of distance parameters.

While many small viruses assemble regular cages according the quasi-equivalence principles formulated by Caspar and Klug 50 years ago, other form spherical shells of a different nature. This seems to be the case for the immature Hiv1 particle, which is formed from thousands of copies of the Gag polyprotein. In this talk, we address the question of the nature of the ground state of the non- icosahedral spherical shell formed by this viral protein as revealed by templated-assembly studies.

Enzyme is one of the most efficient catalysts in nature. The large and complex structure of enzyme enables high specificity to transition states of product to efficiently catalyze the target reaction. If small peptides possessing the same catalytic activity as enzyme are discovered, it can be lead to broad ranges of applications from catalysis to new drug development due to the ease of molecular design with the smaller and simpler structure. However, since peptide does not possess a rigid structure with well-defined catalytic pocket as enzyme, the copied peptide sequence at the catalytic center of enzyme does not always catalyze the target reactions. But by turning weakness to strength, flexible peptides may be able to fit into the conformation matching the catalytic center of enzyme if the proper peptide sequence is folded into the appropriate geometry around the substrate. To discover the enzyme-mimicking peptides, it is desirable to develop technology to screen catalytic peptides from the peptide library. Here we developed a novel methodology to discover catalytic peptides by combining combinatory phage display library and hydrogelation. In this technology, the target catalytic peptides displayed on phage particles create specific covalent bond to crosslink hydrogel precursor molecules, and then it triggers supramoleular gelation around the peptides. The target phage displaying catalytic peptides can be separated from the phage library by centrifugation based of the increase of mass by accumulation of hydrogels. After repeating this panning, the selected phages were subjected to DNA sequencing for the identification of catalytic peptides. In the current work, this methodology was demonstrated to discover the protease/esterase-mimicking catalytic peptides. The assembly of the selected peptide could successfully produce oxide semiconductor nanocrystals through ester-elimination pathway at room temeprature. As compared to esterase enzyme, this selected peptide assembly can grow ZnO with higher crystallinity in methanol. This result could be due to the fact that enzymes tend to denature in organic solvent, and it also indicates that the peptide catalysts are very advantageous to catalyze reactions in non-aqueous environments. This methodology is more generally applicable for other chemical conversions, opening the door to a new method of de novo catalyst design.

Surface functionalization inspired by mussel adhesion has been rapidly emerging because the method is able to modify a variety of surfaces1 such as oxides, noble metals, synthetic organics, ceramics, superhydrophobic surfaces2, and carbon nanomaterials. Catecholamines such as polydopamine1, polynorepinephrine3, and poly(ethylenimine)-catechol4 have demonstrated the powerful abilities in surface functionalization. In this abstract, the catecholamines were used to prepare sticky viruses by surface coating onto adeno-associate viruses (AAV)5. The catecholamine-coated sticky AAVs exhibited excellent surface immobilization capability, which resulted in efficient gene delivery onto overlaying cells. Furthermore, drawing the sticky AAVs were easily patterned on substrates by drawing motions. This enables one to pattern multiple genes in a controlled manner to study a variety of cellular functions and cell-cell communications. Our study herein indicates that other polymeric gene delivery vectors and viruses can be functionalized to be sticky by molecular associations of catecholamines. References: 1. H. Lee et al. Science 2007, 318, 426-430 2. S. M. Kang et al. Angew. Chemie. Int. Ed. 2010, 49, 9401-9404 3. S. M. Kang et al. J. Am. Chem. Soc. 2009, 131, 13224-25 4. S. Ryu et al. Adv. Mater. 2011, 23, 1971-75 5. E. Kim et al. Angew. Chemie. Int. Ed. 2012, 23. 5598-5601

Viral nanoparticles (VNPs) from plants self-assemble into highly organized protein architectures that offer programmability using chemical and genetic engineering methods; these properties render VNPs attractive platforms for targeted drug delivery, imaging and vaccine applications. VNPs are monodisperse and come in a variety of shapes and sizes. This study investigates the in vivo properties including biodistribution and tumor homing and retention of Potato virus X (PVX), a filamentous VNP (measuring 515 by 13 nm) as a potential platform for cancer drug delivery and imaging applications. Nanomaterials with elongated architectures have been shown to possess enhanced vascular interaction in laminar flows; flexible nanorods have been shown to have superior tumor homing capacity. Using in vivo studies involving human tumor xenograft models we illustrate that this is mirrored for VNPs. To investigate the impact of shape on biodistribution and tumor homing, a side-by-side evaluation of PVX and 30 nm-sized Cowpea mosaic virus (CPMV), a model system for spherical nanoparticles, is reported. Differently shaped VNPs, PVX and CPMV, show distinct biodistribution profiles and differ in their tumor homing efficiency. PVX shows enhanced tumor homing and tissue penetration. Maestro imaging of excised tumors and fluorescent intensity measurements of tissue homogenates indicate higher uptake of PEGylated and fluorescently labeled filamentous PVX in tumors compared to CPMV. Immunohistochemical analysis of the tumor sections indicates greater penetration and accumulation of PVX within the tumor tissues. The enhanced tumor homing and retention properties of PVX along with its higher payload carrying capacity makes it a potentially superior platform for applications in cancer drug delivery and imaging applications. Acknowledgements: This work was supported by NIH/NIBIB grants R00 EB009105 and P30 EB011317, and a Case Comprehensive Cancer Center grant P30 CA043703 (NIH/NCI).

In recent years, the availability of low cost, renewable energy sources capable of meeting a significant portion of global energy demands has become a concern. In this report, we investigate the biomineralization of Cu₂S using a viral template. Unlike many second generation solar cell materials which contain scarce and possibly toxic elements (i.e. In, Ga, Te, and Se), Cu₂S is an abundant, non-toxic semiconductor material. Furthermore, its 1.2 eV bulk bandgap is near the theoretical optimum for maximum efficiency of a single junction solar cell. Historically, Cu₂S-based cells have been problematic due to rapid Cu diffusion caused by elevated fabrication temperatures; however our biomineralization approach occurs at room temperature thereby minimizing diffusion. In these biomineralization studies, an 8-mer M13 pVIII combinatorial phage display library was used to select a Cu₂S-binding clone. An abundance of Cu ions in solution was found to reduce phage stability; therefore, the biopanning procedure was modified to minimize the release of Cu ions from the target Cu₂S material. After five rounds of biopanning, six unique peptide sequences were identified. Compared to the library these sequences were rich in basic, hydrophobic, and polar residues, however all the sequences were net negatively charged under biopanning conditions. The peptide fusion DTRAPEIV was found in 16 out of 24 sequenced clones. Notably, this sequence lacks histidine, an amino acid known to bind to both Cu and Cu₂S. A clone with the DTRAPEIV peptide fusion on its major coat protein was selected for mineralization studies. CuCl₂ and Na₂S were used as chemical precursors to mineralize Cu_xS along the length of the M13 viral template. We believe that the Cu ions complex with the selected peptide forming Cu_xS upon addition of Na₂S. The effects of precursor pH and biomineralization time were investigated. Transmission electron microscopy was used to characterize the size, size distribution, coverage, and morphology of the biomineralized nanocrystalline Cu_xS. Electron diffraction indicated that the mineralized material was a mixture of slightly Cu-deficient Cu_xS phases and energy dispersive spectroscopy confirmed a Cu:S ratio near 2:1. The coverage of the biomineralized Cu_xS along the viral template was controlled by precursor pH. Discrete, non-uniformly spaced nanoparticles were formed at low pH and continuous, nanocrystalline material was synthesized at high pH. Furthermore, at low pH, the size and spacing of the template nanoparticles increased with mineralization time, developing larger more widely spaced nanoparticles at longer times. The optical absorption was measured and the effective band edge determined using the Tauc equation for an indirect semiconductor.

Creating precisely defined two- or three-dimensional structures in nano- or micrometer scale is critically important in all fields of science and engineering. Conventional lithography techniques (i.e., photolithography, e-beam lithography, dip-pen nanolithography, nanoimprint lithography and etc.) have been utilized to fabricate various functional devices for electronics, mechanics, and biomedical engineering. Despite their remarkable attributes and capabilities, those fabrication processes often require complicate procedures as well as considerable labors and expenses. However, in nature many hierarchically organized nanostructures (i.e., diatoms, abalone shell, butterfly wing, and moth eyes) possess exquisite structures and functions, which surpassing the capability achievable by current top-down and bottom-up fabrication methods. Moreover, many of these structures are made of a simple basic building block, helical nanofiber (i.e., collagen for animals and cellulose for plants) through self-templated assembly processes. Inspired by nature&’s self-templated assembly processes, here, we developed a novel micropatterning technique to create well-defined two- and three-dimensional hierarchical structures by controlling coalescence of helical nanofiber particles with meniscus forces at the air/liquid/solid interfaces. We utilized M13 bacteriophage (phage) as a model helical nanofiber building block, due to its&’ monodispersity, liquid crystalline property, and genetic flexibility to display functional peptides. By controlling meniscus forces, we could induce formation of the smectic nanofilament phases of the phage and tune the adhesion properties between the nanofilament-to-nanofilament and nanofilament-to-solid substrates. The resulting structures possess hierarchically organized two- and three-dimensional periodic structures with exquisite optical properties. The resulting microstructures could enhance the power of phage-based piezoelectric energy generations. Our facile bio-inspired self-assembly strategy may provide the way to fabricate large-scale advanced micro electronic or optical devices and biomedical applications in the future.

Bacteriophage lambda;, a virus that infects the bacterium Escherichia coli, provides one of the best characterized genetic systems. The standard method for its growth in the laboratory is the plaque assay. In the plaque assay growth of the bacteriophage is contained within a volume of several microliters. These plaques can be considered small vessels allowing any protein encoded by the bacteriophage to be locally produced. We had previously used this property to identify proteins that dissociate aggregated materials and even proteins that dissociate aggregated materials catalytically. Recently we have used this to identify proteins that assemble materials changing their properties.

The use of nanoparticles to deliver magnetic resonance (MR) contrast agents to enhance MR sensitivity has shown great potential in identifying and diagnosing diseases such as cancer and cardiovascular disease. A common challenge is the fabrication of nanoparticles with well-defined properties, such as morphology, size, charge, and surface functionalities. Tobacco mosaic virus (TMV) presents a hollow rod-shaped platform measuring 300 by 18 nm, capable of undergoing chemical conjugation to its interior and exterior surfaces. Recently, it was found that TMV undergoes thermal transition to form RNA-free spherical nanoparticles (SNPs) upon heating for a short time period. Here, we show that TMV can be loaded with thousands of paramagnetic gadolinium (Gd) ions to enhance MR sensitivity. Upon conjugation of chelated Gd compounds, the ionic relaxivity is significantly enhanced. Through thermal transition 160 nm-sized SNPs containing 25,000 Gd ions were formed; such SNPs reach T1 relaxivities of over 400,000 mM-1s-1/SNP. SNPs can undergo further chemical modification to modulate the surface properties for tissue-specific imaging applications.

I will describe and quantify the salient features of charge distributions on viral capsids. By combining the experimentally determined capsid geometry with simple models for ionization of amino acids, I will provide a detailed description of spatial distribution for positive and negative charge across the capsid wall. From the obtained data mean radii of distributions, surface charge densities and dipole moment densities can be extracted. The results will be evaluated and examined in light of previously proposed models of capsid charge distributions, which are shown to have to some extent a very limited value when applied to and compared with real viruses.

Our objective is to design nanostructured biomaterials using the self-assembly of functionalized nanotubes and lipid molecules. In this presentation, we summarize the multiple control parameters which direct the equilibrium morphology of a specific class of nanostructured biomaterials. Individual lipid molecules are composed of a hydrophilic head group and two hydrophobic tails. A bare nanotube encompasses an ABA architecture, with a hydrophobic shaft (B) and two hydrophilic ends (A). We introduce hydrophilic hairs at one end of the tube to enable selective transport through the channel. The dimensions of the nanotube are set to minimize its hydrophobic mismatch with the lipid bilayer. We use a Molecular Dynamics-based mesoscopic simulation technique called Dissipative Particle Dynamics which simultaneously resolves the structure and dynamics of the nanoscopic building blocks and the hybrid aggregate. The amphiphilic lipids and functionalized nanotubes self-assemble into a stable hybrid vesicle or a bicelle in the presence of a hydrophilic solvent. We demonstrate that the morphology of the hybrid structures is directed by factors such as the temperature, the rigidity of the lipid molecules, and the concentration of the nanotubes. We characterize the equilibrium morphology of the hybrid aggregate in terms of the properties of its components.

Silk fibers exhibit exceptional and versatile mechanical properties. Yet, its synthetic imitation has not been fully achieved due to an incomplete knowledge about the interplay between structure, processing and properties. Here, we present a model for studying self-assembly of silk-like polymers into fibers during a microfluidic spinning process that mimics the spider's duct, integrating experimental and computational work. We use a dissipative particle dynamics (DPD) model to simulate hundreds of protein chains, described as a chain of particles with hydrophobic and hydrophilic characteristics in explicit water. Hydrogen bonding between hydrophobic peptides is taken into account by attractive interactions, used to model the strong cross-linking provided by beta-sheet crystals. Reflecting experimentally tested sequences, we demonstrate that under shear flow the cluster size of the hydrophobic domains increases, and that connections between clusters form to a polymer network. Based on the computational results we design new silk sequences that are experimentally synthesized and tested. Our study provides a strategy to better control mechanical properties of the fiber in experiments, as well as an insight on the natural spider silk spinning mechanism.

Amyloid fibrils are a broad class of misfolded protein structures that are the subject of much interest due to both their role in various diseases such as Alzheimer&’s and Parkinson&’s, as well as their potential for functional roles in novel biomaterials. One major challenge in this field lies in understanding how the sheer diversity of amyloid structures affects the mechanical behavior of the fibrils. The characteristic structural motifs that make up the amyloid fibrils range from simple stacked β sheets to more complex geometries involving β-helical domains. We use full-atomistic molecular dynamics simulations to investigate the force-displacement response of amyloid structures with these different structural motifs. We find that the different amyloid structures have very different signatures in their force-displacement behavior. Structures that are held together by only hydrogen bonds exhibit brittle behavior and fail at points with low hydrogen bond density. In contrast, by adding strong intermolecular interactions such as disulfide bonds into the fibrils, the force-displacement curve shows many drops corresponding to the unfolding of the structure followed by an increase once the stronger bonds are stretched. Beyond providing greater insight into the nanomechanics of amyloid fibrils, the results of this work provide a potential method for facile identification of the structure of protein fibrils from simple mechanical testing. We also review coarse-grain modeling of amyloid fibrils assembled into plaques, outlining a computational method to upscale molecular properties to micrometer length-scales. Case studies in different geometries, including a series of analyses of amyloid nanowires, are presented.

The subject of magnetic nanoparticles and their potential uses has drawn much interest over recent years, pulling the attention of research across multi-disciplines. Magnetotactic bacteria produce chains of nano-sized magnetite, Fe₃O₄, particles that operate as internal magnets. This biosynthesis of magnetite is the earliest known example of biomineralisation, having first occurred some two billion years ago. A major step forward in the understanding of magnetite biomineralisation occurred due to the discovery of Magnetotactic bacteria by Blakemore in 1975[1] and subsequent pioneering work. Despite this, much of the detailed atomistic mechanism by which the process occurs is still unknown. Hence, we have begun to develop an atomistic model for the system, in an attempt to understand the processes involved. M.magneticum AMB-1was the Magnetotactic bacteria strain of focus. The control of the morphology and size of magnetite crystals was found to be closely linked to the Mms6 protein which exists within the bacterium. The acidic C-terminal region of this sequence is of particular interest to us and our collaborative experimentalist group. Due to the need to accurately reproduce the interactions between all components involved in the system, the development of an atomistic model for such a system is very complex. Many aspects must be considered when producing force fields including; how the magnetite crystal reacts with itself, the interface between the magnetite and the amino acids and their interaction with water. In order to correctly reproduce the interactions between the amino acids and the magnetite surfaces a force field was developed. For magnetite this forcefield was based on ClayFF [2] and for the amino acids ff99SB was used [3]. The individual amino acids involved in the acidic C-terminal group were solvated and attached to the magnetite {1 0 0} and {1 1 1} surfaces. Constrained dynamics were subsequently performed using an amended version of DL_POLY [4] to describe their interactions with the mineral surface and how they compare. Future work will validate the interactions between the amino acids and the magnetite {1 0 0} and {1 1 1} surface, enabling us to succesfully fit the required organic/ inorganic cross parameters to the system. The acidic C-terminal group shall be examined on both surfaces and in a solution of iron to see if there are preferential areas of iron attachment along the sequence. Various peptide chains, 10 amino acids long, extracted from the full Mms6 sequence shall be looked into in order to give an insight into the surface/peptide interactions along the sequence, with the overall aim of progression to the more complex Mms6 protein. References: [1] R. Blakemore, Science, 1975, 190(4212), 377 [2] S. Kerisit, Geo. Cosmo. Acta, 2011, 75(8), 2043 [3] V. Hornak, Proteins: Structure, Function, and Bioinformatics, 2006, 65(3), 712 [4] S. Kerisit et al, J. Am. Chem. Soc., 2004, 126 (32), 10152

The mechanics of biological materials on the molecular level, specifically proteins such as silk and titin, have been under intense investigation over the past few years due to their novel and functional mechanical properties. The mechanical function of these molecules is governed largely by the different levels of structure above the primary sequence of the protein, with features such as beta sheets and alpha helices dictating the force-extension behavior of individual protein domains. The current state of the field relies on phenomenological models based on observations of molecular simulations, providing a platform that can be scaled up to a macroscopic picture. While this approach has elucidated much of the mechanical behavior of specific proteins, we alternatively consider a coarse-grained simulation model that reproduces many of the characteristic features of such molecules. The molecular structures that we envision can be synthetized in straightforward manners and our approach is generic. The main idea behind our approach is based off standard bead-spring polymer models that incorporate time-dependent “associations” that represent the non-ergodic constraints often found in most biological molecules (disulfide bonds, catch bonds, etc.). This model is simple, and we can demonstrate with statistical mechanical theories the ability to reproduce the interesting force-extension features of such molecules. Our theories lead to quantitative predictions that demonstrate the underlying physical mechanisms governing the molecular mechanics of biological polymers in a fashion that is more general and fundamental than previous phenomenological approaches. Subsequently, we consider the ramifications of such a model; we hypothesize that its non-specific coarse-grained nature lends itself to facile incorporation of the principles into synthetic and/or designed macromolecules. By adjusting along only two coordinates, association kinetics and polymer topology, a vast array of interesting single-molecule mechanics can be realized. We will present a number of examples of such behaviors that are designed through the development of a statistical mechanically-informed model that is analogous to traditional spring-dashpot theories.

Blood clotting is the ubiquitous process in our bodies where clotting materials (platelets and polymeric proteins) form self-healing aggregates at the sites of lesion. Such a process becomes harder in the presence of strong flow. However, nature has designed stimuli-responsive biopolymers that enhance the formation of such aggregates in flowing conditions assuring the correct healing of these small vessels that are more likely to fail. Using multi-scale computer simulations, we explore blood clotting-inspired polymer-colloid composites assembly in flow, as well as their properties. We find that shear-flow-induced composite assembly is a universal process that is only related to the shear rates and the interaction potentials between the polymers and colloids. Furthermore, it is observed that reversible and distinct composite structures are easily tailored by only changing the imposed shear rates. To have a fundamental understanding of the composites, we analyze in details the polymer conformations and dynamics within the composites. It is observed that, within the aggregates, the polymer radius of gyration is fixed, contradicting the free polymers that the radius of gyration usually increases with increasing shear rates. Also, it is observed that the polymer relaxation time is much longer within the aggregates, suggesting that the polymers are “locked” within the aggregates. Furthermore, the mechanical properties (internal colloid-colloid bonding moduli and the overall Young&’s modulus of the aggregates) are also tested. These composites are the scaffolds on which new tissue grows to heal the vessels, and thus understanding their properties is of much interest. Our result suggest new paradigms regarding non-equilibrium composite assembly and tailored functional aggregation, which have potential importance in biomedical, pharmaceutical, or rheological applications.

Materials in biology are synthesized, assembled, controlled and used for a variety of purposes—structural support, force generation, catalysis, or energy conversion—despite severe limitations in available energy, quality and quantity of building blocks. We demonstrate that the chemical composition of biology's materials plays a minor role in achieving functional properties. Rather, the way components are connected at different length-scales defines what material properties can be achieved, how they can be altered to meet functional requirements, and how they fail in disease states. We have achieved this by using the world&’s fastest supercomputers to predict properties of complex materials from first principles, in a multiscale approach that spans orders of magnitude in scale. This method, combined with experimental studies, allows us to build virtual “in silico” material models that provide unseen insight into the workings of natural and synthetic materials from the bottom up. We exemplify this materiomics approach in a case study of spider silk, one of the strongest yet most flexible materials in Nature, despite being made out of some the simplest, most abundant and intrinsically weak proteins, including weak hydrogen bonding. We discovered that the great strength and flexibility of spider silk—exceeding that of steel and other engineered materials—can be explained by the material&’s unique structural makeup that involves multiple hierarchical levels. These hierarchical levels span from the genetic information that defines the protein sequence to the structural scale of an entire spider web. Thereby, each level contributes to the overall properties, but the remarkable properties emerge because of the synergistic interaction across the scales where the sum is more than its parts. By translating this insight gained from the study of natural materials such as spider silk to engineered materials such as carbon nanotube fibers, graphene composites or metal-polymer films, our work has resulted in an engineering paradigm that facilitates the design of sustainable materials starting from the molecular level, leading to the formation of hierarchical structures that span all scales from nano to macro. By utilizing a mathematical tool from category theory we illustrate the hierarchical materials design concept by drawing an analogy to a seemingly far and distant field—music. Reminiscent of protein materials, the integrated use of structures at multiple scales is the key to provide superior functional properties despite limitations in available building blocks, a set of musical instruments such as piano, violin or cello. In music, tones are played at different pitch, accentuation or duration and then assembled into melodies. The collective interaction of melodies, played by different instruments and arranged in a particular way, eventually results in the powerful expression of a symphony.

Nanostructure formation by self-assembly of small peptidic molecules is a notoriously tricky concept to model in silico. This is due to the large number of individual molecules required to form a self-assembled structure that is large enough for obtaining statistically relevant information. With the advance of computational power however, simulations of sufficient size are becoming available, although appropriate methods are still in development. Here, we will discuss two different approaches to understand and predict the properties of self-assembled nanostructures of peptides: 1) atomistic simulations using the CHARMM force field for understanding the supramolecular organisation of Fmoc-dipeptides (Fmoc = 9-Fluorenylmethyloxycarbonyl); and 2) coarse grain simulations for the screening of new biomaterials consisting of small (di)peptides using the MARTINI force field. We will show that an insight on the molecular level can explain surprising nanostructure architectures (e.g. two-dimensional sheets versus 1D fibers). The atomistic simulations performed are consistent with experimental data showing π-stacked β-sheets for Fmoc-dipeptides and can correctly predict self-assembly for monomers in various ionization states, even in mixed-molecule structures. Although computationally still quite expensive, this method is able to reproduce experimental features arising from small changes in the self-assembling monomer, such as changing between a serine and threonine amino acid residue. On the other hand, we have developed a method for screening large amounts of peptidic molecules for self-assembling properties. Our coarse-grain screening protocol and scoring method correctly predict whether aggregation (an indicator of the ability to self-assemble) will occur for almost all known examples of dipeptides in literature. Extended simulations can even give insight into the supramolecular structure of a particular peptide (e.g. tubes for the Phe-Phe dipeptide, but fibers for Ile-Phe). Overall, the procedure shows good promise for the discovery of new bio-inspired materials. References: Frederix et al., J. Phys. Chem. Lett., 2011, 2, 2380 Hughes et al. Soft Matter, 2012, 8, 5595

Viral capsids self assembly consists of hundreds to thousands of protein subunits spontaneously forming an icosahedral shell around the viral nucleic acid genome. We model this assembly using Coarse-Grained Brownian Dynamics simulations, specifically focusing on cationic amino acid tails, or Arginine Rich Motifs (ARMs); a common structural element through which viral capsids interact with their nucleic acid genome or a surrogate polyanion. In our dynamic simulations, we find that the presence of a polymer greatly enhances the rate of assembly through a disordered mechanism, wherein many capsomers adhere to the polymer before successful capsid assembly occurs. A complementary set of equilibrium calculations reveals that the thermodynamically optimal polymer length is larger than that which promotes assembly, illustrating an interesting contrast between kinetics and thermodynamics. Through these simulations we are able to investigate the role of several variables, including polymer length, stiffness, and secondary structure, as well as the ARM location and length. Our reductionist computational strategy allows us to isolate the effect of individual physical features, providing insight which complements that gained by experimental study. Supported by Award Number R01AI080791 from the National Institute Of Allergy And Infectious Diseases.

Theories of protein crystallization based on spheres that form close-packed crystals predict optimal assembly within a `slot' of second virial coefficients and enhanced assembly near the metastable liquid-vapor critical point. However, most protein crystals are open structures stabilized by anisotropic interactions. Here, we use theory and simulation to show that assembly of one such structure is not predicted by the second virial coefficient or enhanced by the critical point. Instead, good assembly requires that the thermodynamic driving force be on the order of the thermal energy and that interactions be made as nonspecific as possible without promoting liquid-vapor phase separation.

Patchy particle models have been used to describe the interactions between biomolecules such as proteins to help guide their self-assembly. Although many studies have investigated the effect of the range of the interactions and the size and number of the patches on the phase diagram and on the nucleation dynamics, a common assumption among the models is that all the patches are identical. Yet protein-protein interactions are characterized by a wide spectrum of strength and specificity. How does this diversity affect their crystallization and, more in general, their self-assembly? Because a diversity of interactions can also be artificially constructed in colloids, exploring these questions is of direct interest for material science research as well. We employ advanced Monte Carlo simulations to characterize the phase diagram of a series of Kern-Frenkel patchy particle models where the specifically-interacting patches differ in strength, range and width. We investigate different geometrical distributions of the patches on the surface so that the crystal phase satisfies a P212121 symmetry, i.e. the most common symmetry group among proteins. The results indicate that diversity in strength, and to a lesser extent in range and width, drastically affects the phase diagram, while their geometrical distribution only weakly perturbs the coexistence lines. The free-energy barriers of nucleation for the different models show that the crystallization dynamics is also profoundly affected. Successful strategies to obtain high-quality protein crystals and to design self-assembly materials are discussed.

Recent experiments demonstrate polyelectrolyte adsorption to a conducting surface to become continuous, i.e. to scale linearly with time over hours, upon application of a modest electric potential. Continuous polyelectrolyte adsorption offers the possibility of polymer films of tailored nanoscale thickness realized in a single step, but raises fundamental mechanistic questions. A key feature of one hypothesized mechanism involves a net attraction among like-charged polymers at the electrified interface, caused by a highly correlated counter-ion distribution and/or the presence of image charges. Here, we present a Monte Carlo simulation study of polyelectrolyte-polyelectrolyte interactions, with a goal of developing mechanistic insight into like-charge attraction at the electrified interface, and hence also into the continuous adsorption process. Our system consists of two parallel polymer chains, composed of charged tangent spheres, above a conducting surface, in the presence of spherical counter-ions and salt ions. We observe conditions where counter-ions preferentially locate between the polymers, and act to promote a net polymer-polymer attraction. We discuss the sensitivity of the attractive regime to a Coulombic coupling parameter, to counter-ion size, and to the nature of the underlying substrate. We also compare simulation results to those of a strong Coulomb coupling statistical mechanical model, and to our ongoing experiments.

Keratin, a type of intermediate filament protein, is the key component of hair, nail and skin in vertebrates, including mammals. Hard alpha-keratin intermediate filaments are rich in disulfide bonds and feature a hierarchical structure, ranging from alpha-helical protein, a dimer composed of alpha-helical coiled coils and two globular C- and N-terminal domains, to full-length intermediate filaments embedded in a sulfur-rich protein matrix. At the molecular level, bundles of the keratin filaments in the sulfur-rich protein matrix are stabilized by disulfide bonds that play a defining role in their mechanical and physical stability. Here we report a bottom-up atomistic model of the keratin dimer, using the complete human keratin type k35 and k85 amino acid sequence. A detailed analysis of geometric and mechanical properties through full-atomistic simulation with validation against experimental results is presented. We introduce disulfide cross-links in a keratin tetramer and compare the mechanical behavior of the disulfide bonded systems with a system without disulfide bonds. Disulfide bond results in a higher strength (20% increase) and toughness (49% increase), but the system loses alpha-helical structures under loading, suggesting that disulfide bonds play a significant role in achieving the characteristic mechanical properties of hard alpha-keratin. Our study provides general insight into the effect of disulfide cross-link on mechanical properties. Moreover, the availability of an atomistic model of this protein opens the possibility to study the mechanical properties of hair fibrils and other fibers from a bottom-up perspective. We discuss opportunities of synthesizing peptide materials with higher durability and resistibility against the external loading.

The hierarchical structure of bone enables it to be a light-weight material that can carry relatively large loads. At the nanoscale, interactions between the collagen molecule and hydroxyapatite (HAP) and the amount of mineral plays a significant role in providing strength to the bone. Collagen-HAP composites are not only the basic building blocks of human bone, but they are amongst the most abundant class of bio-mineralized materials in the animal kingdom. This composite exhibits properties of both hard and soft matter, combining the toughness of inorganic material and the flexibility of biological tissues. Although the structure of bone and its mechanical properties are well-studied, the knowledge about how collagen fibrils and hydroxyapatite crystals interact and deform as an integrated system under external stress are not well understood. Indeed, in spite of the fact that collagen is a soft material and hydroxyapatite is stiff but brittle, combined they form a composite of high-mechanical strength and fracture toughness. A deep understanding of the properties of bone building block requires a thorough investigation of the interplay of the organic molecules with the mineral crystals from an atomistic perspective. Here we present an atomistic model of collagen microfibril with varying densities of mineral content. A newly developed method that simulates the incorporation of mineral into collagen and its distribution will be discussed. We perform tensile tests on non-mineralized and mineralized collagen micro fibril models to study the stress-strain behavior, and also determine the modulus of the bone microfibril model as the mineral content increases from 0 to 40%. We specifically investigate the load transfer mechanism between collagen and HAP as the applied stress increases, and study the distribution of stresses in the mineralized collagen fibril. The predictions from this study will be useful to provide design criteria for the development of biomineral based composites. Our model may also help us to better understand the mechanistic origin of various bone diseases, including those associated with varying mineral density and mutations in collagen.

Single strands of DNA can self-assemble to form versatile structures, including 2D and 3D crystals. In these crystals the strands are connected by sticky end links, which are formed by pairing of complimentary unpaired bases at the ends of two DNA molecules. The dissociation of the DNA sticky end links is investigated in this work by molecular dynamics simulations. The stretch of strands connected by sticky end links with different base sequences and lengths is studied and a model is suggested for the structural evolution of these constructs up to failure. The results have implications for the design of DNA self-assembled structures.

Recent work on functionalized nanoparticles has shown that grafting nanoparticles with polymer ligands is an attractive method to tailor the assembly of nanoparticles. We use an integrated theory-simulation approach [1] to demonstrate how the monomer sequence (e.g. alternating or diblock) in the grafted copolymers can be a valuable tuning parameter to control assembly and the structure within the assembled nanoclusters. Our studies [1-3] show a complex interplay of monomer sequence, molecular weight, inter-monomer interactions, polymer grafting density and nanoparticle size and their effect on the conformations of the grafted copolymer chains, and the structural characteristics of the cluster. For example, we observe that alternating AB copolymer grafts produce relatively large isotropic nanoclusters when either A-A or B-B monomers are attractive in the presence of negligible A-B repulsions, but smaller clusters or particle dispersions in the presence of strong A-B repulsions. Diblock AB copolymer grafts produce nanoclusters that are smaller and compact when the block closer to the surface (A-A) is attractive, and larger loosely held together clusters when outer block (B-B) is attractive in the presence of both strong and negligible (A-B) repulsions. Additionally, diblock copolymer grafted particles tend to assemble into anisotropic shapes despite the isotropic grafting of the copolymer chains on the particle surface. We will present in this talk these complex trends in assembly of copolymer grafted nanoparticle, emphasizing the effect of graft monomer sequence and monomer chemistry on the assembled cluster characteristics. References: 1. N. Nair and A. Jayaraman*,'Self-Consistent PRISM Theory-Monte Carlo Simulation Studies of Copolymer Grafted Nanoparticles in a Homopolymer Matrix'Macromolecules 43 (19), pp 8251.8263 (2010) 2. A. Seifpour, P. Spicer, N. Nair, A. Jayaraman. ‘Effect of monomer sequences on conformations of copolymers grafted on spherical nanoparticles: A Monte Carlo simulation study.&’ J. Chem. Phys. 132 164901 (2010) 3. T. Martin, A. Seifpour, and A. Jayaraman. ‘Assembly of Copolymer Functionalized Nanoparticles: A Monte Carlo simulation study.&’ Soft Matter 7,5952-5964 (2011)

Scaffolded DNA origami offers a powerful approach to synthesizing complex nonlinear structures at the nanometer-scale with Angstrom-level resolution for diverse applications in materials science and engineering. Transferring this technology to mainstream research applications requires predictive computational tools that allow non-specialists the capability to design nanostructures in silico to avoid the costly and time-consuming design-synthesis-validate cycle. Here, we present an advanced computational engineering tool for the design of 3D DNA origami structures. We model the canonical twisting, bending, and stretching stiffness of DNA in addition to backbone electrostatic interactions to predict equilibrium 3D solution shape and flexibility. Excellent agreement with Transmission Electron Microscopy images is obtained for a diverse set of nonlinear DNA origami structures.

Lyotropic phases of amphiphilic soft materials are prototypical examples of self-assembly. Their structure is generally determined by amphiphile molecular shape and phase transitions are governed primarily by composition. In this presentation we will show recent synchrotron small-angle X-ray scattering (SAXS) studies that demonstrate a new paradigm for membrane shape control where the electrostatic coupling of charged membranes to sDNA, with tunable temperature-dependent end-to-end interactions, enables switching between the inverse gyroid cubic structure and the inverted hexagonal phase. The SAXS structural investigation of sDNA confined within 1D lipid tubes is an excellent platform to access with molecular detail DNA self-organization via end-to-end coupling; either by base paring or hydrophobic interactions which are nonspecific (sequence-independent) and play an important role in DNA hybridization and ligation as well as large nanostructure manufacturing. _{We acknowledge support by DOE-BES grant number DOE-DE-FG02-06ER46314 (interplay between membrane shape and DNA stacking), NSF DMR-1101900 (phase behavior), and NIH GM-59288. This work made use of the Central Facilities of the Materials Research Laboratory at UCSB which are supported by the MRSEC Program of the NSF under award no. DMR-1121053; a member of the NSF-funded Materials Research Facilities Network (www.mrfn.org). Cecília Leal was funded by the Swedish Research Council (VR) and in part by DOE-BES. The X-ray diffraction work was carried out at the Stanford Synchrotron Radiation Lightsource (SSRL) beam line 4.2. CRS acknowledges useful discussions with KAIST Faculty where he has a WCU (World Class University) Visiting Professor of Physics appointment supported by the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology grant No. R33-2008-000-10163-0.}

A long standing problem in the fabrication of carbon nanotube electronic devices is the formation of monolayer parallel CNT films with nanometer scale CNT density. Such films can increase the performance of CNT field effect transistors and enable fabrication of ultra-dense crossbar circuits and other novel devices, but no previous methods can produce CNT arrays with consistent inter-nanotube spacing down to a few nanometers. Here, we demonstrate the self-assembly of DNA dispersed single walled carbon nanotubes (SWNT) into small, parallel arrays via linker induced surface assembly. In this process, DNA linkers that keep SWNTs dispersed in solution drive the same nanotubes to form parallel arrays during deposition and surface diffussion. Duplex domains on our DNA linkers act as spacers to precisely control the inter-nanotube separation down to < 3 nm. These spacers can also serve as scaffolds to position molecular components such as proteins between adjacent parallel nanotubes. The resulting arrays can then be stamped onto other substrates. Our results demonstrate a simple and scalable route towards preparation of high performance CNT devices via surface assembly driven by biomimetic interactions.

The controlled self-assembly of nanostrucutres on surfaces with nanometer resolution is of great interest in the fields of nanoscience and nanotechnology. We have developed strategies towards the controlled and ordered arrangement of 1D nanoobjects on lithographically patterned, chemically (or biochemically) functionalized surfaces. 1D DNA nanostructures offer biofunctionality and are presently being explored for use as scaffolds and transport agents for both biological and inorganic nanospecies Using electron-beam and nanoimprint lithography we fabricated sub-10nm metal dots arranged in multiple configurations. DNA nanostrucutres have been assembled with high precision via hybridization in situ on properly funnctioanlized nanodots. The DNA-origami employed functioned as a model-system for the bivalent self-assembly of nanorods on metal-nanodot pairs: we characterized the self-assembly via fluorescence microscopy and Atomic Force Microscopy.By varying the length of the ssDNA linker, we were able to study the binding efficiency as a function of interaction strength Using the same basic approach, we have begun studying the selective binding of carbon nanotubes to the nanodot anchors. Fixed-length SWNT segments, wrapped with ssDNA and with an average length of 200 nm, were attached to amine-functionalized nanodot anchors arranged in a square lattice with a 200 nm pitch. Binding occurs via a covalent linkage between the amines on the dots and carboxyl groups formed at the ends of the SWNT segments, which were cut by sonication in water. The binding is preferentially at the ends of the tubes and the yield is high. The combination of high resolution patterning with end-functional chemistry enables the assembly of 1D functional nanostructures in an orderly fashion. The basic requirements for this approach are: molecular-scale lithographic patterning, control over nanostructure length, and facile end-functional chemistry. We believe this may be a viable avenue toward the integration of these materials in complex nanoarchitectures

Inorganic semiconductors combined with bio-organic systems offer the potential for the development of a wide range of novel hybrid devices. In this context, silicon carbide (SiC) is a particularly promising substrate material because it features a high chemical stability and biocompatibility, making it ideal for biomedical and biosensing applications. However, a fundamental requirement for using SiC in biosensing applications is the ability to immobilize tailored molecular and biomolecular layers on the semiconductor surface. Organophosphonate chemistry is an important alternative to silanization for applications where hydrolytically stable functional interfaces are required. Because organophosphonate SAM (SAMP) attachment is insensitive to water contamination and often yields highly ordered monolayers with minimal risk of multilayer formation, superior film properties at the nanoscale level may be attained [1]. Recently, we demonstrated covalent functionalization of n-type 6H-SiC with organonot;phosphonates [2]. Structural and chemical properties of these monolayers were investigated through contact angle measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), contact potential difference (CPD), and Fourier transformation infrared spectroscopy (FTIR), revealing covalent bonding of the phosphonates to both (0001)- and (000-1)-oriented 6H-SiC crystal faces. Here we describe the potential of hydroxy-terminated SAMPs for the tailored biofunctionalization of (0001) 6H-SiC surfaces. In particular, we have focused our work on the covalent immobilization of peptide nucleic acid (PNA) oligonucleotides, which are receptors for DNA hybridization. [1] A. Cattani-Scholz, K.-C. Liao, A. Bora, A. Pathak, M. Krautloher, B. Nickel, J. Schwartz, M. Tornow, G. Abstreiter, Angew. Chem. Ed. Int. Ed. Engl. 2011, 37, A11-A16. [2] M. Auernhammer, S. J. Schoell, M. Sachsenhauser, K.-C. Liao, J. Schwartz, I. D. Sharp, A. Cattani Scholz, Appl. Phys. Lett. 2012, 100, 101601.

We have developed a new method for fabrication of living multicellular structures that could show how colonial organisms evolved in nature and be used in tissue engineering. These structures, termed cellosomes, were made by polyelectrolyte mediated assembly of yeast cells on to templates of aragonite (rod-shaped) and calcite (rhombohedral) microcrystals.[1] The templates were pre-coated with magnetite nanoparticles so they could be manipulated with an external magnetic field. We used this to extract the cell-coated templates by magnetic separation which is very useful for selective sorting and separation of the cellosomes from the excess of single cells. The templates were dissolved with EDTA to give rod- and rhombohedral-shaped, hollow 3D cellosomes. We also pioneered a similar technique for templating of air micro-bubbles with yeast cells to produce spherical cellosomes.[2] We analyzed the arrangement of cells in the obtained cellosome by SEM. Viability tests showed that the yeast cells were still active in these assemblies and remained viable for more than two weeks.[2,3] These cellosomes resemble primitive multicellular organisms like Volvox to a certain degree, so we could speculate that nature has used a similar assembly mechanism in evolution. This cellosome assembly could be applied with stem cells and opens new possibilities for novel ways of engineering tissues where their shape can be directed by the shape of the micro-template. Our method also works for fabrication of living cellosomes of various shapes and from different types of cells, to produce symbiotic colonies of cells which is the next step in the design of "artificial" living multicellular organism.[3] We also report a related technique for polyelectrolyte mediated magnetization of living cells which leads to deposition of an integrated layer of magnetite nanoparticles on the surfaces of yeast cells.[4] We show that these “magnetic yeast” cells preserve their viability and can be manipulated by external magnetic field. We demonstrate the usefulness of these materials by fabricating magnetic yeast cells that have been genetically modified to express their Green Fluorescent Protein (GFP) gene whenever the cells repair damaged DNA. We report a simple microfluidic device in which these cells were directed by external magnetic field and used to simultaneously detect genotoxicity and cytotoxicity.[5] References [1] R.F. Fakhrullin, V.N. Paunov, Chem. Commun., 2009, 2511. [2] M.-L. Brandy, O. J. Cayre, R. F. Fakhrullin, O. D. Velev, V. N. Paunov, Soft Matter, 2010, 6, 3494. [3] R. F. Fakhrullin, M.-L. Brandy, O. J. Cayre, O. D. Velev, V. N. Paunov, PCCP, 2010, 12, 11912. [4] R.F. Fakhrullin, J. García-Alonso, V.N. Paunov, Soft Matter, 2010, 6, 391. [5] J. García-Alonso, R.F. Fakhrullin, V.N. Paunov, Biosensors and Bioelectronics 2010, 25, 1816.

RNA nanotechnology has emerged as a new field and brought vitality to the area of therapeutics (Guo P, The emerging field of RNA nanotechnology, Nature Nanotechnology, 2010). RNA can be manipulated with simplicity characteristic of DNA, while possessing versatile structure and diverse function similar to proteins. Loops and tertiary architecture can serve as mounting dovetails or wedges for self-assembly to eliminate the requirement of external linking dowels. Unique features in transcription, termination, self-assembly, self-processing, and acid-resistance enable in vivo production of nanoparticles harboring aptamer, siRNA, ribozyme, riboswitch, or other regulators for therapy, detection, regulation, and intracellular computation. The unique property of noncanonical base-pairing and stacking enables RNA to fold into well-defined structures for constructing nanoparticles with special functionalities. Bacteriophage phi29 DNA packaging motor is geared by a ring consisting of six packaging RNA (pRNA) molecules. pRNA is able to form a multimeric complex via the interaction of two reengineered interlocking loops. This unique feature makes it an ideal polyvalent vehicle for nanomachine fabrication, pathogen detection, and delivery of siRNA or other therapeutics. This talk will describe methods of using pRNA as a building block for the construction of RNA dimers, trimers and hexamers as nanoparticles in medical applications. Methods for industrial-scale production of large and stable RNA nanoparticles will be introduced. The unique favorable PK (pharmokinetics) profile with a half life (T_1/2) of 6-10 hrs compared to 0.25 hrs of conventional 2&’-F siRNA, and advantageous in vivo features such as non-toxicity, non-induction of interferons or non-stimulating of cytokine response in animals will also be presented. References: [1] Guo P (2010). The emerging field of RNA nanotechnology. Nat Nanotechnol 5:833-42. [2] Shu D, Shu Y, Haque F, Abdelmawla S, Guo P (2011). Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nat Nanotechnol 6:658-67. [3] Shu Y, Cinier M, Fox SR, Ben-Johnathan N, Guo P (2011). Assembly of therapeutic pRNA-siRNA nanoparticles using bipartite approach. Mol Ther 19:1304-11. [4] Abdelmawla S, Guo S, Zhang L, Pulukuri SM, Patankar P, Conley P, Trebley J, Guo P, Li QX (2011). Pharmacological characterization of chemically synthesized monomeric phi29 pRNA nanoparticles for systemic delivery. Mol Ther 19:1312-22. [5] Liu J, Guo S, Cinier M, Shlyakhtenko LS, Shu Y, Chen C, Shen G, Guo P (2011). Fabrication of stable and RNase-resistant RNA nanoparticles active in gearing the nanomotors for viral DNA packaging. ACS Nano 5:237-46.

High preferment soft functional materials consisting of interconnecting network become increasingly important in both sciences and technologies. It has been shown that in many cases, crystal networks of the hybrid structure give rise to much more superior properties than singles crystals themselves. For instance, the special crystal network structure of amino acids in spider silk leads to the tensile strength several magnitude higher than single chains of amino acids. Due to these facts, our interests are shifted from the control of single crystals, such as size and shape of single crystals to the engineering the crystal network. In this contribution, new understandings on the formation kinetics of crystal network, and the between the network structure and the properties of the systems will be presented. This represents a new direction in the field of crystal growth and crystal engineering. It can also be visualized that in the 21st century, the engineering of crystal networks will become one of the most active directions in materials sciences. In this talk, I will introduce the latest development in the kinetics of fiber network formation, the correlation between the structures of biological functional materials and the in use properties, and the application to nanoengineering. This includes the engineering of nano phase and ultra-functional soft-materials.

There is considerable interest in producing mineralized materials using biomimetic methods due to their low energy costs and potential to create biominerals more similar to those found in vivo, including a significant organic component. The alternate soaking process (ASP) is a rapid method of forming biomineral coatings at room temperature and pressure. Here, the alternate soaking process is modified to include molten protein at small concentrations in the soaking solutions in order to co-precipitate mineral-protein composites. Samples are investigated with Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive x-ray spectroscopy and nanoindentation. It is demonstrated that the resultant volume fraction of protein in the composite can be controlled by adjusting the concentration of protein in the soaking solutions. Calcium carbonate-gelatin composites that contain a substantial mineral volume fraction (>90%) are deposited at a rate of approximately 50µm in 2 hours on de-mineralized eggshell membrane and glass and the CaCO3 polymorph present is dependent on the substrate for mineralization. This modified alternate soaking process opens up possibilities for rapidly creating biomimetic mineral-protein composites that more closely mimic the composition and structure-properties relationships of composites found in natural mineralized materials.

Novel electron microscopy based imaging techniques allow to investigate in-situ the dynamics of the mineral formation in solution from an early stage on, giving inside into fundamental aspects of the crystal growth. This enables a direct imaging of clusters from the nanometer range to macroscopic crystals. Both scanning electron microscopy (SEM) as well as scanning transmission electron microscopy (STEM) have been employed to study the precipitation from supersaturated solutions of calcium and carbonate ions in the absence and in the presence of organic polymers found to substantially affect the onset of precipitation as well as the size distribution and shape evolution of the resulting crystals. The imaging is thereby performed through thin SiN membranes separating the microscopy vacuum from the liquid phase. The atmospheric SEM (ASEM) reveals the presence of an amorphous phase, which is enhanced by organic polymers such as polyaspartic acid. The surrounding of the growing crystals show 4-6 µm wide depletion regions of the amorphous calcium carbonate (ACC) underlining the important role of the dissolution dynamics of ACC for the ion transport to the growth sites. The observed depletion layer width and crystal growth rates are consistent with the solution for the transport equation using the equilibrium constant for the ACC dissolution and the diffusion coefficient found in literature [1,2]. First STEM investigations using a three port liquid cell holder indicate that STEM in comparison to TEM is very well suited for the study of the precipitation process since it provides improved contrast and the possibility to obtain nanometer resolution even through micrometer thick films of water. A variation of spacer thicknesses and hence fluid layer thicknesses in a range from 150 nm to 5 µm shows the impact of confinement on the precipitation via the limitation of ion transport in such a system. For a spacer thickness of 500 nm it was observed that 20 - 30 nm large particles were spontaneously formed after introducing a supersaturated solution of carbonate and calcium ions. These clusters tend to form “superclusters” of several micrometer size. It is yet unclear if such an agglomeration of carbonate nanoclusters provides a precursor phase for the crystal formation. However, these observations highlight the prominent role of confinement on the precipitation, an important aspect of mineralization in biological systems. [1] L. Bre#269;evicacute; and A.E. Nielsen, J. Cryst. Growth 98, 504 (1989). [2] Y.H. Li et al., Geochim. Cosmochim. Acta 38, 703 (1974).

In living organisms, minerals are often created within a confined space under the control of organic matter. The resultant structures are organic-inorganic hybrid materials possessing crystals of well-defined sizes, polymorphs and shapes, which can emerge their intriguing properties. Inspired by such awesome systems, we developed a novel route to control both mineralization and polymerization inside a nanoreactor based on water-in-oil (W/O) miniemulsion system to create organic-inorganic hybrid nanoparticles [1]. In W/O miniemulsion system, nanodroplets containing calcium ions and 2-hydroxylethylmethacrylate (HEMA) were mixed with nanodroplets containing carbonate ions through the fusion and fission process to produce nano-sized calcium carbonate (nano-CaCO3). We could produce nano-CaCO3 particles with a spherical or rod-like shape and amorphous ones. Then, subsequent polymerization was carried out to encapsulate nano-CaCO3 inside the polymeric nanoparticles at a desired stage of crystal growth. The shape and structure of nano-CaCO3 depended on incubation time and initiator concentration and they remained the same even after 1 month of ageing. Therefore, we concluded that poly(HEMA) inhibits crystal growth inside the nanodroplet, so that the size and structure of nano-CaCO3 can be preserved inside hybrid nanoparticles. We have applied this system for creation of fluorescent hybrid nanoparticles with diverse emitting colours. Preparation of fluorescent nanocrystals (ZnS:Mn2+) and their encapsulation inside polymeric nanoparticles were performed in the same manner as described above. We could control the fluorescent properties of nanocrystals by tuning the effects of surface capping polymers (polymethacrylic acid; PMAc) and the doping concentration of Mn2+. The resultant hybrid nanoparticles exhibited a strong light emitting under UV irradiation. The resultant hybrid nanoparticles were then spin-coated to form transparent hybrid nanofilms. The nanofilms prepared from ZnS-encapsulated and ZnS:Mn2+-encapsulated nanoparticles showed a blue and an orange emission, respectively. Furthermore, we developed a simple and facile route in making fluorescent colours. By mixing of hybrid nanoparticle of ZnS (blue) and those of ZnS:Mn2+ (orange) at their different volume ratios, the output colour of the nanofilm could be controlled from blue to orange. We expect that the multicolor tuning of hybrid nanofilms would offer a potential way in fabricating light emitting diode (LED) for displays and lights. In summary, we believe that our developed nanoreactor system will open up a path for creation of functional hybrid nanomaterials by enabling us to control the interaction between polymers and nanocrystals. [1] Y.Fukui, K. Fujimoto, J. Mater. Chem., 2012, 22, 3493-3499

Macromolecules are often incorporated into single crystals in the course of biomineralization. In this way, mechanical properties of the crystals are greatly enhanced over those of their pure synthetic counterparts. For example, proteins and cellular tissue networks are incorporated within sea urchin spines and tooth plates, which are made of calcite single crystals, improving their fracture toughness. Mimicking these biominerals, synthetic calcite single crystals with incorporated carboxylated block copolymer (PSPMA30-PDPA47) micelles showed greater hardness than pure calcite single crystals. To determine the micelle incorporation mechanism at the near molecular level and subsequently gain insight into the process of incorporation of biomacromolecules into biogenic minerals, we used two types of negatively charged micelles of the aforementioned carboxylated PSPMA30-PDPA47 and sulfonated block copolymer, SBA: PHPMA30-PDPA47 and observed their incorporation process into growing calcite single crystals by in situ atomic force microscopy (AFM). We found that both micelles directly adsorbed from solution to the atomic steps and observed no or little micelle adsorption to the terraces. Whereas the PSPMA30-PDPA47 micelles adsorbed to both acute and obtuse steps with similar affinities, the SBA: PHPMA30-PDPA47 micelle had a much greater affinity for the acute than the obtuse steps. The sites to which both types of micelles adsorbed were not influenced by solution supersaturations. Steps advanced past the adsorbed micelles with little or no inhibition and the continual passage of steps encapsulated the micelles into the calcite single crystal. More detailed investigation done with the PSPMA30-PDPA47 micelle showed that steps passing by the adsorbed micelles compressed the micelles causing them to be elongated perpendicular to the growing face. While the micelles were encapsulated into the calcite single crystal by the continual passage of steps, incorporation was accompanied by the formation of cavities at the locations where the micelles were incorporated. These cavities were eventually filled when growth occurred at high supersaturation. The extent of micelle incorporation into the crystals increased with supersaturation for both types of the micelles, simply due to the increase in step density with supersaturation. Companion experiments on mica surfaces showed that the negatively charged PSPMA30-PDPA47 micelles formed at the high pH of the growth experiments did not adsorb to negative (bare) mica, but adsorbed to positive (polylysine-treated) mica. This suggests that the calcite step along with the solution-calcium ions play a key role in establishing electrostatic adhesion of the micelles to the steps.

Zinc Oxide (ZnO) is a direct bandgap semiconductor with a large exciton binding energy and a high electron mobility that has proven useful for electronic and optoelectronic device applications. Bio-directed synthesis of ZnO is a promising technique capable of morphological control under generally mild, low temperature conditions. Specifically, there have been numerous reports of peptide-directed synthesis of ZnO using free peptides and peptides bound to a planar surface. Although successful in creating nanoscale ZnO materials with unique morphologies, these peptide configurations lack a high degree of order and hierarchical structure. Because the organization and proximity of peptide binding sites during biomineralization are expected to strongly influence the properties of the resulting inorganic material, a well-ordered template is preferable. In this work, the M13 virus is used as a three-dimensional scaffold to control the density and arrangement of binding peptides. The major coat of this filamentous virus is composed of thousands of highly ordered, densely packed proteins. Using bioconjugation, peptides can adopt the extremely organized structure of the p8 major coat protein through covalent bond formation. Furthermore, the density or coverage of the peptide on the viral surface can be varied to control the closeness of the binding sites and explore cooperative effects during nucleation and growth. In these studies, sulfo-SMCC, a bifunctional linker was used to chemically graft a previously reported ZnO-binding peptide to the M13 major coat protein. The NHS-ester of sulfo-SMCC is amine reactive and couples to both the exposed lysine residue and the protein N-terminus, thus allowing up to two ZnO-binding peptides to be bound to each of the thousands of copies of the p8 protein located along the length of the virus. MALDI-TOF mass spectroscopy and SDS-PAGE were used to confirm covalent bonding of the binding peptides to the template and identify single and double peptide modifications. The density of the binding peptide on the virus was systematically varied from 5% to full coverage by controlling the peptide concentration available for reaction. The bioconjugated templates were biomineralized using a Zn(OH)2 precursor solution. TEM was used to determine nanocrystal size and size distribution, crystal structure, and morphology of the mineralized templates in relation to the coverage of the ZnO-binding peptide. In addition, material composition and crystallinity were investigated using EDX spectroscopy and electron diffraction, respectively. Photoluminescence and absorption measurements were used to correlate the optical properties of the mineralized materials to the nucleation site density of the template. The assembly of ZnO nanostructures on M13 virus allows specific control and fine tailoring over material structure and properties. This method of synthesis can be extended for mineralization of other functional inorganic materials.

In our experimental studies of peptide ZnO interactions using the peptide G12 (GLHVMHKVAPPR) previously identified by Phage display, we have shown that modification of ZnO crystal morphology occurs by an adsorption-growth inhibition mechanism with the peptide influencing growth of the mineral phase along both the a-axis and the c-axis. In order to explore the role played by individual amino acids within the sequence we have performed an alanine mutant screening study and identified sequences that very differently affect crystal morphology. The observed differences in behaviour can be understood by considering both Zn(II) ions/ ZnO surface- peptide binding and the inherent solution behaviour of the peptides themselves, including self assembly. A model to explain the observed sequence specific effects will be presented based on detailed experimental and computational simulation studies.

Non-collagenous proteins, known as NCPs, are able to inhibit hydroxyapatite (HA) formation in living organisms. The polar and charged amino acids (AAs), which are highly expressed in NCPs, are known to be responsible for the inhibitory effect of NCPs. These AAs can inhibit HA formation by chelating calcium and phosphate ions in solution, or by binding to the nascent nuclei of HA and restricting its further growth. Many studies have been conducted to investigate the inhibitory effect of AAs on HA formation; however, these studies are mostly focused on the AAs/ions or AAs/HA interactions at the precipitation (nucleation and growth) stage while the calcium and phosphate complexation by AAs can initiate on earlier stages when the stock reactants&’ solutions are prepared and stored to be mixed later for HA precipitation. In this work, we investigated the effect of aging time (3 days vs. 0 day) of calcium and phosphate reactants&’ solutions, containing either arginine (Arg) or glutamic acid (Glu), on HA precipitation. We found out that HA precipitation time was significantly increased with the aging time of reactants&’ solutions in the presence of either of Arg or Glu. However, the precipitation of control reactants&’ solutions without any AAs was not significantly influenced by their aging time. Here, we performed a series of analyses, such as X-ray photo spectroscopy (XPS) and fluorescence correlation spectroscopy (FCS) to investigate the formation of AAs complexes, and interoperated our results in terms of formation of pre-nucleation clusters between AAs and calcium and phosphate ions, which needed to dissociate and evolve into HA pre-nucleation clusters before HA can precipitate. This resulted in HA precipitation being delayed more strongly with the aging time of reactants&’ solutions containing the AAs, either Arg or Glu.

Magnetite (Fe3O4) is a widespread magnetic iron oxide encountered in both geological and biomineralizing systems (1), which also has many technological applications, e.g. in ferrofluids, inks, magnetic data storage materials and as contrast agents in magnetic resonance imaging (2, 3). As its magnetic properties depend largely on the size and shape of the crystals, control over crystal morphology is an important aspect in the application of magnetite nanoparticles, both in biology and synthetic systems. Indeed, in nature organisms such as magnetotactic bacteria demonstrate a precise control over the magnetite crystal morphology (4-7), resulting in uniform and monodisperse nanoparticles. The magnetite formation in these bacteria is believed to occur through the co-precipitation of Fe(II) and Fe(III) ions (7), which is also the most widely applied synthetic route in industry (2, 3). Synthetic strategies to magnetite with controlled size and shape exist (2, 3), but involve high temperatures and rather harsh chemical conditions. However, synthesis via co-precipitation generally yields poor control over the morphology and therefore over the magnetic properties of the obtained crystals. Here we demonstrate biomimetic control over the size and shape of magnetite nanocrystals, but also over their organization in solution and their magnetic properties. We employ amino acids-based polymers to direct the formation of magnetite in aqueous media at room temperature. With these we have found that - in line with the proteins used by magnetotactic bacteria (5-7) - acidic amino acid monomers are the most effective in affecting the crystal morphology. By changing the composition of the polymers we can tune the morphology as well as the magnetic properties of these nanoparticles. Further, the polymers immobilize on the surface of the magnetite and consequently improve its dispersibility, allowing the nanocrystals to organize into long strings in solution. (1) R. M. Cornell, U. Schwertmann, The Iron Oxides: Structure, Properties, Reactions, Occurrences and Uses, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2003. (2) A.-H. Lu, E. L. Salabas, F. Schüth, Angew. Chem. Int. Ed. 2007, 46, 1222. (3) S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. V. Elst, R. N. Muller, Chem. Rev. 2008, 108, 2064. (4) B. Devouard, M. Posfai, X. Hua, D. A. Bazylinski, R. B. Frankel, P. R. Buseck, Am. Mineral. 1998, 83, 1387. (5) A. Arakaki, J. Webb, T. Matsunaga, J. Biol. Chem. 2003, 278, 8745. (6) A. Scheffel, A. Gardes, K. Grunberg, G. Wanner, D. Schuler, J. Bacteriol. 2008, 190, 377. (7) D. Faivre, D. Schuler, Chem. Rev. 2008, 108, 4875.

The interface between organisms and materials is of fundamental interest to the study of the function and formation of biominerals. Bacteria produce a vast range of mineral deposits within the earth and are involved in the degradation of many materials. These organisms produce complex organic scaffolds made of extra-cellular polymeric substance (EPS) to bind to material surfaces. Experimental studies have demonstrated that a major differentiating factor in cell attachment behaviour is the macromolecular chemical signature of biomolecules within the cell wall [1]. Studies have demonstrated that extracellular DNA (eDNA) is a major component of the EPS and in some cases crucial to attachment [2]. Understanding the mechanisms of eDNA attachment is therefore necessary to understand biofilm development which consists of a complex biological scaffold interacting with a mineral system. We have explored the mechanisms of attachment using molecular dynamics simulations which provide atomic-scale detail to analyse the interactions. We consider how eDNA binds at amorphous silica surfaces which provides an analogue to many geological and experimental systems. We use a force-field that combines the long-range electrostatic contribution and short-range forces. A range of different solvated cations is simulated to enable us to look at their effect on both the space-charge layer and on localised bonding. We discuss the implications of these results for bacterial attachment and the building of DNA based scaffolds on mineral surfaces comparing them with current experimental work where possible. [1] M. Li, J. Xu, M. Romero-Gonzalez, S.A. Banwart, W.E. Huang Current Opinions in Biotechnology (2011) 23(1), 56-63 [2] J.S. Andrews, S.A. Rolfe, W.E. Huang, J.D. Scholes, S.A. Banwart, Environmental Microbiology (2010) 12(9), 2496-2507

The self-assembly of bio-organic molecules into nanostructures is an attractive route to fabricate functional materials. For example, diphenylalanine (Phe-Phe, FF), an aromatic dipeptide consisting of two covalently linked phenylalanine units, can form various nanostructures such as nanotubes, nanowires, and nanospheres under different processing conditions. FF-based nanostructures can readily self-assemble in a simple way and possess the functional flexibility and molecular recognition capability suitable for a wide range of applications, such as biosensors, imaging, guest encapsulation, and nanofabrication. Herein, we report on the development of light-harvesting peptide nanotubes that integrate photosynthetic units, thus mimicking natural photosynthesis. The light-harvesting peptide nanotubes were synthesized by the self-assembly of FF and porphyrin. Porphyrins are macrocyclic compounds that include the chlorophyll molecules found in light-harvesting photosystems of green plants. We further incorporated platinum nanoparticles (nPt) on the surface of the FF/porphyrin nanotubes by means of self-metallization. Similar to quinone and ferredoxin, which act as an electron separator and a mediator in natural photosynthetic systems, respectively, nPt was introduced here in order to efficiently separate and transfer the exited electrons from porphyrin to an electron mediator (i.e., M=[Cp*Rh(bpy)H2O]2+, Cp*=C5Me5, bpy=2,2prime;-bipyridine). M can facilitate the selective and efficient regeneration of nicotinamide cofactors. We found that light-harvesting peptide nanotubes synthesized in this way are able to harvest solar energy, thereby regenerating NAD(P)H for the production of fine chemicals by means of redox enzymes in a manner similar to natural photosynthesis, in which photochemically regenerated NAD(P)H is consumed by enzymatic reduction reactions in the Calvin cycle. Our Recent Publications Related to This Presentation: J. H. Kim, M. Lee, J. S. Lee, C. B. Park. Angewandte Chemie Int. Ed. 124: 532-535 (2012) J. H. Kim, S. H. Lee, J. S. Lee, M. Lee, C. B. Park. Chem. Commun. 47: 10227-10229 (2011)

We developed new autonomous biochemical motors by integrating metal-organic framework (MOF) and diphenylalanine (DPA). The DPA is well-known as an important peptide motif in the Alzheimer&’s β-amyloid polypeptide and its robust self-assembling nature is used to power the driving motion in water. The MOF used in this study to store peptides and trigger re-assembly is [Cu₂L₂ted]_n where L is 1,4-benzenedicarboxylate. This MOF has a highly ordered pore array of coordination framework with the porous size of 0.75 nm and the release of guest peptides can be induced by partially breaking MOF at the interface with EDTA. When the hybrid DPA-MOF particle is dropped onto an aqueous solution containing EDTA, the release of DPA builds surface tension gradient around the particle, resulting in the motion of the DPA-MOF particle toward the higher surface tension direction. The translational motion of this peptide-MOF system is 30 times faster than existing gel motor systems because the robust assembling nature of peptides enables reconfiguring their assemblies at the water interface, which is efficiently converted to fuel energy. This peptide-MOF motor particle could fuel driving macroscale plastic boat, working as an engine of the boat. This demonstration opens the new application of MOF and reconfigurable molecular self-assembly and it may evolve into the smart autonomous motor that mimic bacteria to swim and harvest target chemicals by integrating recognition units. It may further develop to smart machines with features that can change characteristically by sensing and adapting to new environments.

Proteins are surprisingly good solid-state electronic conductors. This holds also for proteins without any known biological electron transfer function. How do they do it? To answer this question we measure solid-state electron transport (ETp) across proteins that are “dry” (only tightly bound water, to retain the conformation, still present). We compare results for the electron transfer (ET) protein, Azurin (Az), the proton-pumping membrane protein Bacteriorhodopsin (bR), and for Human and Bovine Serum Albumin (HSA and BSA). Clear differences between these proteins are seen, which preserve their structure in the solid state measurement configuration. Importantly for future bioelectronics, the results are sensitive to protein modification, e.g., removing or disconnecting the retinal in bR and removing or replacing the Cu redox centre in Az. THESE COFACTORS CAN THUS BE VIEWED AS NATURAL DOPANTS FOR PROTEINS. Insight in the ETp mechanism comes from temperature-dependent studies. Az shows 40-360K temperature-independent ETp across its 3.5 nm long axis, until its denaturation temperature, indicative of tunneling. Cu removal, replacement (by Zn) or deuteration changes this to thermally activated ETp. This suggests hopping and involvement of the amide back-bone in the ETp. The latter, which rhymes with indications from ETp experiments on oligopeptide and simulations of ET in proteins, opens the way for modeling what otherwise is an awfully complex system. Below 200K all proteins and their variants show temperature-independent ETp. WE CAN FURTHERMORE MAKE A TOTALLY ELECTRICALLY INACTIVE PROTEIN, HSA, INTO AN EFFICIENT ETp MEDIUM BY DOPING IT WITH A NATURAL POLY-ENE. Putting our data in perspective by comparing them to all known protein ETp data in the literature, we conclude that, in general, proteins are well described as "DOPABLE" MOLECULAR WIRES *collaboration with M. Sheves, I. Pecht, Work of L. Sepunaru, N. Amdursky, W. Li, N. Friedman, I. Ron, Y. Jin; all from the Weizmann Inst.; Support from the Minerva foundation (Munich).

Membrane proteins represent an interesting and promising extension of the bionanoelectronic toolkit because of the many important functions that they perform in the living cells. Nanoelectronics with membrane proteins requires a versatile biocompatible matrix that can preserve the protein functionality and yet can efficiently couple the protein to the device. We accomplish this task by using hierarchical assembly of lipid molecules and membrane proteins into a nanowire transistor to create a fully integrated bionanoelectronic device. We will discuss our efforts to make devices that translate photoactivated proton transport events into electrical signals using proteorhodopsin proton pumps. We will also focus on the challenges that are encountered during the formation of these structures, and the strategies for overcoming these challenges and improving device performance.

Cellulose is one of the most abundant carbon resources, and the material is degraded by cellulolytic enzymes, called cellulases. Cellulases are generally modular proteins with independent catalytic and cellulose-binding domain (CBD) modules, and in some bacteria, catalytic modules are non-covalently assembled on a scaffold protein with CBD to form a giant protein complex, called a cellulosome, which efficiently degrades water-insoluble hard materials. In this study, we independently prepare a catalytic module and CBD by recombinant means, and we heterogeneously cluster them on inorganic nanoparticle for the construction of artificial cellulosomes. Heteroclustering of the catalytic module with CBD results in significant improvements in the enzyme&’s degradation activity for water-insoluble substrates; especially, the increase of CBD valency in the cluster structure critically enhances the catalytic activity by improving the affinity for substrates, and clustering with multiple CBDs on CdSe nanoparticles generates a several-fold increase in the production of reducing sugars relative to that of the native free enzyme. The multivalent design of substrate-binding domain on clustered cellulases is important for the construction of the artificial cellulosome, and the nanoparticles are an effective scaffold for increasing the valence of CBD in clustered cellulases. Here, we propose a new design for artificial cellulosomes with multiple CBDs on non-cellulosome-derived scaffold structures.

Natural cellulase based multicatalytic enzyme complex such as Cellulosome uses cooperativity to drastically improved biological activities, which have inspired researchers to develop artificial systems with similar or higher efficiency. However, till now synthesis of such enzyme complexes using commercial individual cellulase enzymes have failed to achieve higher efficiency. In the present study, we tried to understand the molecular mechanism of artificial enzyme complexes by correlating its surface adsorption and diffusion behavior with its enzymatic activity. We observed that by specifically controlling the different experimental condition and material properties, multicatalytic enzyme complex with higher efficiency can be achieved. First, commercial cellulase enzyme mixture was purified to obtain Cellobiohydrolase I (CBHI) and Endoglucosidase II (EGII) and then enzyme complex was synthesized by conjugating CBHI/EGII on polymer coated magnetic particles using EDC/NHS reaction. Magnetic particles have a core-shell type structure where the shell is made up of loosely cross-linked polymer which contains large concentration of -COOH groups. Significant change in surface adsorption of cellulase enzyme was observed on complex formation as it shows much lower adsorption rate and percentage adsorption which can be attributed to orders of lower concentration of binding species. As per our expectation, cellulose binding domain of CBHI which is the most important component for higher efficiency of free CBHI plays insignificant role in case of enzyme complexes. The enzymatic activity of cellulase shows a cross-over behavior on complex formation i.e. at higher enzyme concentration, the enzymatic activity of complex form is lower but at lower enzyme concentration, complex enzymes shows much higher activity. The higher activity of complex enzymes at lower concentration can be attributed to enzyme localization which promotes cooperativity between enzymes. These behaviors were similar in case of both CBHI and EGII with EGII showing better enzymatic activity on complex formation which can be attributed to different mode of catalysis of two enzymes. We also observed considerable effect of complex size (particle size) and polymer type (scaffold) on the enzymatic activity. Thus, it can be said that mode of action of cellulase enzyme complex is much different than freely floating individual cellulase enzyme and hence experimental condition and material (scaffold) properties need to be optimized to achieve enzyme complex with higher efficiency.

Cephalopods, such as the cuttlefish Sepia officinalis, are capable of manipulating their total body color and patterning by varying the state and local distribution of their dermal pigments to reflect, absorb and transmit light. The pigment-containing chromatophore is responsible for altering dermal coloration; however, the optical properties of individual pigment granules within the chromatophore are unknown. We hypothesized that the nanostructure of pigment granules localized within the cuttlefish chromatophore determines the granules&’ spectral response to absorption and scattering of light. Thus, the nanostructure is a critical aspect of the active coloration. To test this hypothesis, pigment granules isolated from the S. officinalis chromatophore were assembled into isotropic thin films and analyzed using photoluminescence and reflectance spectroscopy. Light scattering arising from the isotropic, nanostructured arrangement of pigment granules both increased color contrast upon reflection and enhanced absorbance. In addition, optical characterization of isolated pigment granules revealed a broad photoluminescence peak centered at ~700 nm, a property that has not been reported for cephalopod pigments. Thus, it appears that pigment granules are both photoluminescent and efficiently absorb and scatter light. The interplay of the granules within different chromatophores produce the dynamic range of colors and patterns exhibited by S. officinalis. Insights gained from this remarkable, nano-biophotonic system can provide important guidelines for the design of new dynamic optical displays and devices, engineered from nanoscale components.

Biological nanocomposites such as bone, dentin, enamel and nacre, exhibit outstanding mechanical properties including elastic modulus, strength and fracture toughness. The smallest building blocks in biological composites are generally on the nanoscale, e.g. 15-20 nm thick needle-like crystals in tooth enamel; less than hundred nanometers of mineralized collagen fibrils in bone; a few hundred nanometers aragonite bricks in nacre. Understanding the mechanical properties of these basic building blocks at the nanoscale is not only essential to explain the macroscopic biophysics of different tissues, but also helpful for the development of novel nanocomposites. In this study, nanoindentation experiments were conducted inside a scanning electron microscope using an in situ nanomechanical test device (PI 85 PicoIndenter®, Hysitron, Inc.). Two nanocomposites, biomimetic mineralized collagen fibrils and bovine cortical bone, were prepared and dispersed on Si substrates for in situ experiments. Biomimetic mineralized collagen fibrils are composed of a bundle of subfibrils that are 10 nm thick and 50 to 150 nm long. For the individual mineralized fibrils, the elastic modulus was determined to be in the range from 20 GPa to 50 GPa. These values are similar to the reported results for bovine bone. The indentation imprints on fibrils indicate an anisotropic structure originating from the aligned subfibrils.

Within the extracellular matrix (ECM), mechanically active proteins are critical for maintaining the structural integrity and spatial organization of cells and tissues. In vivo one such protein, fibronectin (FN), exists in its globular conformation before cellular traction forces unfold the molecule to expose cryptic FN-FN binding sites required for fibrillogenesis. In vitro however, production of 3D FN fiber scaffolds remains a challenge. We hypothesized shear forces induced during Rotary Jet-Spinning (RJS) may be used to unfold globular FN, exposing protein-protein binding sites, to fabricate FN nanofibers with mechanical and physiological integrity. To test this hypothesis, shear forces on fluids within the RJS were first modeled using Poiseuille flow. Using our model of Poiseuille flow in RJS, we predicted FN fibrillogenesis during spinning and confirmed fibrillogenesis occured with experimental results quantifying FN polymerization into fibers. The insolubility of the produced FN nanofibers in physiological media is direct evidence of shear-induced fibrillogenesis in vitro in beta sheet containing proteins. The effect of shear forces on FN molecules was then studied by measuring protein conformation using fluorescence resonance energy transfer (FRET). Results suggest that FN is globular when injected into the reservoir and after spinning FN molecules are in an extended conformation, further evidence of shear unfolding inducing fibrillogenesis. We show evidence mechanically and chemically that fiber properties can be modulated by shear. Thus, we observe that shear forces induced via RJS are strong enough to unfold FN molecules to induce fibrillogenesis for rapid, cell-free production of beta sheet protein scaffolds.

Biomolecular unit cells are solid materials that incorporate two compartments that contain aqueous or gel droplets encased in lipid monolayers. The droplets in each compartment are surrounded by a hydrophobic fluid (e.g., Hexadecane), and are positioned on built-in silver-sliver chloride (Ag/AgCl) fixed electrodes. A lipid bilayer interface is then formed by bringing the aqueous droplets into contact. Full encapsulation of the droplet interface bilayer is achieved with a low-density Polyurethane film that is poured on top of the oil surrounding the droplets. The encapsulation of the bilayers provides increased portability over the exposed networks by significantly enhancing droplet stability. On the other hand it provides the ability to test the biomolecular unit cells in different fluid environments. The biomolecular unit cell is a useful platform for testing the properties of transmembrane channels and pores. Single channel measurements of Alamethicin peptides inserted into the interfacial lipid bilayers demonstrate that the biomolecules retain their stimuli-responsive properties when reconstituted within the biomolecular unit cell. Furthermore, durability tests show that bilayer membrane can last days or weeks before failure. These tests prove the ability of the biomolecular unit cell to serve as a platform to test the protein activity and a variety of biomolecules. We have also demonstrated the ability to test novel combinations of hydrophilic and hydrophobic fluids within the unit cell. Results show that it is possible to consistently and repeatedly form lipid bilayers on droplets of ionic liquid solutions. The characterization tests demonstrate that the stability and electrical properties are not affected. However, the conductance levels in gating events of the Alamethicin peptide are affected by some ionic liquids. The biomolecular unit cell is also used as an embodiment for hair like sensors in an effort to develop and characterize artificial hair cell that resembles the stereocilia of the human ear. The sensor is tested in both air and water environment, by subjecting it to air flow across the hair the first case and pulse-like water flows in the second case. The hair is supposed to transmit vibrations to the unit cell which in turns transmits it to the bilayers interface. The results show an electrical response from the bilayer interface due to the hair movement. On the other hand, the results show that by changing the physical parameters of the hair sensor, such as hair length, we are able to alter the response of the sensor. It is also shown that the sensitivity of the sensor is reliant on the size of the lipid bilayer.

Technological innovation at the pre-clinical stage during cardiovascular drug development is becoming increasingly critical because of spiraling costs of clinical trials^[1] and high occurrence of unforeseen cardiac toxicity leading to drug development delays and market withdrawal^[2]. Hence, there is a compelling need for new in vitro pre-clinical screening tools for better predicting cardiotoxicity. We sought to design a higher throughput “heart on a chip” which utilizes a commercial laser engraver as an engineering tool to fabricate sub millimeter sized thin film cantilevers in soft elastomers. Laser cutting of cantilevers instead of cutting by hand confers the ability to fabricate much smaller films (1mm X 300µm), of standard and reproducible dimensions, and in a batch process which is amenable to an assembly line type fabrication. We are able to produce up to 50 technical replicates from a chip with a cell requirement of 1 million cardiac myocytes and as a result this design significantly advances the throughput of our Muscular Thin Film (MTF) technology^[3] for building a heart on a chip. We also present the design of a one channel fluidic device completely built out of autoclaveable and non-absorbent materials which incorporates various features required for an optical cardiac contractility assay: metallic base which fits on a heating element for temperature control, transparent top for recording cantilever deformation and embedded electrodes for electrical field stimulation of the tissue. Accurate contractility data collection and quantitative tissue structural evaluation is carried out for cardiac microtissues engineered on the surface of each thin film. In an effort to validate our fluidic higher throughput tool, we test the effect of isoproterenol (a positive inotropic agent) on cardiac contractility at dosages ranging from 0.1nM to 100µM. We find a close match of pD2 values obtained from our in vitro tool (= 7.15) and from ex vivo muscle strip experiments (=6.77). The higher throughput chip has applications in testing of cardiac tissues built from rare/expensive healthy and diseased cell sources (such as primary human cardiomyocytes and stem cells) and the fluidic device enables drug testing studies for both chronic and acute exposure, and for integration with other organ mimics. References: [1] E. L. Eisenstein et al., Am Heart J, 2005, 149, 482-488. [2] J. Morganroth, J Electrocard, 2004, 37, 25-29. [3] A. Grosberg et al., Lab Chip, 2011, 11, 4165-4173.