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 p