Symposium Organizers
Alberto Saiani University of Manchester
Joel Collier University of Chicago
Athene Donald University of Cambridge
William Murphy University of Wisconsin-Madison
Laurent Jeannin Solvay SA
NN1: Peptide Amphiphile and Conjugate: Self-Assembly and Applications
Session Chairs
Sarah Heilshorn
Alberto Saiani
Thursday PM, April 08, 2010
Room 3020 (Moscone West)
10:00 AM - **NN1.1
Multivalent Polymer-peptide Conjugates in the Assembly of Hybrid Biomaterials.
Kristi Kiick 1
1 Department of Materials Science and Engineering , University of Delaware, Newark, Delaware, United States
Show AbstractMacromolecular structures that are capable of selectively and efficiently engaging cellular targets offer important approaches for mediating biological events and in the development of responsive materials. We have employed a combination of biosynthetic tools, bioconjugation strategies, and biomimetic assembly in the design of new materials for these purposes. The use of this combination of strategies has permitted us to investigate the impact of multivalent polymer architecture on materials properties in multiple areas. In one area, the assembly of multivalent polymer conjugates with proteins has been used in the formation of hydrogels. The release of growth factors from these materials, in response to their receptors, provides a novel mechanism for targeted delivery via delivery-mediated erosion. Adhesion and proliferation of select cell types with these materials can be modulated on the basis of mechanical and chemical cues; direction of cell phenotype for specific applications is also possible.New modular polypeptides capable of binding to relevant polysaccharides have also been developed to show both excellent mechanical properties and cellular responsiveness via these principles. Furthermore, manipulation of the assembly of polymer-modified multivalent polypeptides has also yielded routes to new assembled soluble and fibrillar structures with applications in the display of nanoparticles and ligands. These approaches will therefore continue to be useful for understanding cellular interactions with materials and to develop hydrogels with controlled properties useful for biomaterials applications.
10:30 AM - NN1.2
Peptide Amphiphiles for Tumor Targeting and Therapy.
Matthew Black 1 , Mark Kastantin 1 , Dimitris Missirlis 1 , Matthew Tirrell 1 2
1 Chemical Engineering , University of California, Santa Barbara, Santa Barbara, California, United States, 2 Bioengineering, University of California, Berkeley, Berkeley, California, United States
Show AbstractSelf-assembled peptide-displaying micelles can be used to combine targeting, imaging, and therapeutic biomolecules and deliver them efficiently to tissues in vivo. These micelles are formed in aqueous solution using peptide-amphiphiles that consist of a biofunctional peptide as the hydrophilic head group and either a single-chain fatty acid or a double-chain lipid as the hydrophobic group, often separated by a polyethylene glycol (PEG) or other spacer to drive self-assembly in aqueous solutions. Mixing different monomers leads to multifunctional mixed micelles with precise control over number and ratio of functionalities without the need for orthogonal chemical reactions. We have demonstrated that these micelles are useful for in vivo disease targeting and delivery of therapeutic and imaging payloads. Despite their versatility and ease of production, little is known about micelle behavior in vitro or in vivo and what fundamental principles may link their structure and composition to their ability to perform desired therapeutic and diagnostic tasks. In this work, the effect of micelle stability and composition on the behavior of the micelles has been investigated for tumor targeting and therapy. By attaching and varying the length of a hydrophobic tail to the apoptosis inducing peptide, (KLAKLAK)2, the efficacy of the peptide can be increased by up two orders of magnitude. Increasing the length and number of hydrophobic tails was shown to significantly slow micelle break up. Incorporating (KLAKLAK)2 into mixed micelles containing a targeting and internalizing peptide, LyP-1, has also been shown to increase its efficacy in vitro. Current work is aimed at further optimizing how micelle composition, stability, and shape affect the efficacy of therapeutic micelles. In addition to advancing a specific strategy useful for cancer treatment, the insights contained in this work will help provide a blueprint for future design of therapeutic and diagnostic micelles.
10:45 AM - NN1.3
Bioactive Three-dimensional Synthetic Matrices: Peptide Amphiphiles, Nanostructured Gels, and Tissue Engineering.
Won H. Suh 1 2 , Katie Megley 1 , Tomoko Shimada 3 , Matthew Tirrell 1
1 Bioengineering, University of California, Berkeley, Berkeley, California, United States, 2 Materials Research Laboratory, University of California, Santa Barbara, Santa Barbara, California, United States, 3 Advanced Medical Center, Asahi Kasei Corporation, Fuji Japan
Show AbstractAn alanine rich peptide sequence of WA4KA4KA4KA (W3K) when appended with a hydrocarbon tail will transform into three-dimensional fibrous worm-like micelles with prolonged standing or with the introduction of shear force in aqueous media. Its variants including bioactive sequences such as RGD will also undergo phase switching between spherical micelles and worm-like micelles. We will present data from analyses conducted via circular dichroism, rheometry, microscopy, and cell culture experiments. We are in the process of developing defined synthetic matrix systems that utilize C16-W3K PAs for human stem cell engineering under the context of regenerative medicine.
11:30 AM - **NN1.4
Peptide Nanostructures for Regenerative Medicine.
Samuel Stupp 1
1 , Northwestern University, Evanston, Illinois, United States
Show AbstractBioactive nanostructures molecularly crafted to signal cells or deliver genes and proteins have great potential in the regeneration of organs and tissues. The supramolecular chemistry of such nanostructures should allow them to interact specifically with cell receptors or intracellular targets. Our laboratory has developed a broad class of peptide amphiphilies that are programmed to self-assemble into nanoscale filaments with the capacity to display signals to cells and bind specific proteins. This lecture will also discuss hierarchical structures based on these peptide amphiphilies and biopolymers with potential for multiple biomedical functions.
12:00 PM - NN1.5
The Emergent Behaviour of Peptide-Lumiphore Conjugates following Self-assembly into Nanofibers.
Kevin Channon 2 , Glyn Devlin 3 , Cait MacPhee 1
2 School of Chemistry, The University of Bristol, Bristol United Kingdom, 3 Department of Biochemistry & Molecular Biology, Monash University, Melbourne, Victoria, Australia, 1 School of Physics and Astronomy, The University of Edinburgh, Edinburgh United Kingdom
Show AbstractSmall peptides offer an attractive starting point for the development of self-assembling materials, since they are relatively simple to produce and can be tailored to provide an expansive range of chemical functionality. Proteins and polypeptides readily self-assemble into multimolecular fibrillar architectures via non-specific hydrogen-bonding interactions. These fibrils are stable in a wide range of physio-chemical environments that are not ordinarily tolerated by protein-based structures, making them an attractive starting-point for the development of peptide-based self-assembling bionanomaterials. We describe the formation of self-assembling nano-scale fibrillar aggregates from a hybrid system comprising a short polypeptide conjugated to the fluorophore fluorene. We demonstrate that peptide self-assembly into fibrils drives the co-assembly of a “cargo” species and that it is the polypeptide backbone that dictates the structure of the resulting aggregate, rather than self-assembly of the large hydrophobic moiety. We further show that this process can be extended to drive the co assembly of two independent luminescent moieties and that the resulting complex performs a light-harvesting function.
12:15 PM - NN1.6
Biomimetic Design of Peptide-based Polymers having Hierarchical Structures for Advanced Properties.
Zhibin Guan 1 , Aaron Kushner 1 , Ting-Bin Yu 1 , Yulin Chen 1 , Gregory Williams 1
1 Chemistry, University of California, Irvine, California, United States
Show AbstractFollowing Nature’s strategies, one research thrust in our group is to program weak molecular forces into synthetic macromolecules including peptide-based polymers to guide the formation of high order and hierarchical structures. In this presentation, I will discuss a number of biomimetic polymer designs using various biopolymers as models. In the first example, by mimicking skeletal muscle protein titin, we have designed multidomain polymers having tandem arrays of modules folded by well-defined hydrogen bonds. In the second example, using spider silk and beat-amyloid fibers as models, we have created synthetic peptide-based polymers that fold into extensive beta-sheets and self assemble into nanofibrils. In the third example, by following the modular structure of elastin proteins, we have developed efficient “click’ polymerization of short peptides to form polymers with excellent elasticity. Finally, mimicking the hierarchical structures of biocomposites, we are using organic molecular recognition motifs to direct the assembly of inorganic nano-building blocks into hierarchical nanocomposites. Drawing various inspirations from Nature, we aim at programming subtle non-covalent molecular forces to create a plethora of biomimetic materials showing dynamic, responsive, shape-memory, and self-healing properties. To elucidate the molecular origin for material properties and build fundamental structure-property correlations, we are also investigating their materials properties at various length scales: from single molecule to bulk level.
12:30 PM - **NN1.7
Self-assembly of Amyloid Peptide Fragments and PEG Polymer/Peptide Hybrids.
Ian Hamley 1
1 Chemistry, University of Reading, Reading, Berkshire, United Kingdom
Show AbstractThere has been great interest recently in the fibrillisation of peptides, especially the amyloid beta (Aβ) peptide which is involved in diseases such as Alzheimer’s. We have recently commenced a study of the self-assembly of peptides and peptide copolymers based on a fragment KLVFF, corresponding to the core region of Aβ(16-20). Aβ self-assembly is driven by inter-molecular β-sheet self-assembly into fibrils. A primary objective of our work is to identify fragments that bind to amyloid fibrils and disrupt fibrillisation (aggregation inhibitors based on self-recognition elements). We are also interested in peptides and peptide/polymer conjugates as hydro- and organo-gelators.I will present results on the self-assembly of peptides including sequence KLVFF, hydrophobic variants and PEGylated diblock copolymers of these peptides. We have also studied the self-assembly of several model Fmoc-tripeptide hydrogelators. Self-assembly is studied in water for hydrophilic peptides and peptide copolymers and in organic solvents for hydrophobic peptides. Gelation at higher concentration is also discussed. Peptides AAKLVAA and AAKLVFF have been the subject of detailed studies of self-assembly into nanotubes and fibrils, depending on solvent. Very recently we been investigating several β- and γ-amino acid modified peptides, extending at the N terminus of KLVFF. Self-assembly and cytotoxicity experiments will be discussed. We are currently examining the binding of these peptides to the amyloid β peptide Aβ(1-42), as part of a project to develop aggregation inhibitors, which may be useful in the treatment of amyloid disease. In addition, we have found that PEGylated βAβAKLVFF forms spherical micelles in aqueous solution (the peptide on its own forms helically twisted fibrils), pointing to the ability to modulate the self-assembled structure by introduction of amphiphilicity. The enzymatic cleavage of the peptide from PEG chain (at a phenylalanine residue) is presently under investigation, with the aim of creating an enzyme-responsive self-assembling system (enzyme induced transition from spherical micelles to peptide fibrils).A model amphiphile comprising tetraphenylalanine conjugated to PEG5000 has been investigated, and a critical aggregation concentration has been identified. This relates to hydrophobic or β-stacking interactions of the phenylalanine units, β-sheets only forming at much higher concentration. For the PEGylated KLVFF-based fragments, a fascinating range of self-assembled structures are being uncovered including fibrils, lyotropic liquid crystal phases in concentrated solution and microphase-separated structures in the melt and dry states.
NN2: Peptide Self-Assembly and Applications I
Session Chairs
Joel Collier
Laurent Jeannin
Thursday PM, April 08, 2010
Room 3020 (Moscone West)
2:30 PM - NN2.1
A Twist on Polymer-Peptide Hybrid Design Yields ``Sticky” Supramolecular Cones.
Yan Geng 1
1 Chemistry, University of Georgia, Athens, Georgia, United States
Show AbstractAmphiphilicity defines one of the most fundamental self-assembly principle for polymer-peptide hybrids and has long found use in constructing discrete mesostructures for nanotechnology and biomedical applications. Self-assembly from traditional amphiphiles with the two distinct hydrophobic-hydrophilic segments, however, is generally limited to highly symmetric spherical and cylinder structures. Herein, we break the traditional amphiphile design by linking a few ionic peptides along the hydrophobic segment of an amphiphilic block copolymer, and we report that such twist on polymer-peptide design yields unprecedented angular conical self-assembly geometry with asymmetry apex and base. The supramolecular cones are formed via nucleation and growth-from-the-base self-assembly pathway, and they demonstrate an interesting “stickiness” property at the base that enables base-to-base adhesion, leading to higher-hierarchy strings of the cones. The apex angle, length, as well as the “stickiness” of the supramolecular cones can all be tuned by chemical manipulations. We also reveal the role of hydrophobic effect and peptide hydrogen bonding in this unique cone self-assembly mechanism. The asymmetry between the base and the lateral sides of such supramolecular cones can also be explored as Janus particles for pickering emulsion applications.
2:45 PM - NN2.2
Lamellar Self-assembly and Crystal Polymorph Selection by a Single Protein Fragment.
Rebecca Metzler 1 , Tyler Churchill 1 , John Evans 2 , Christopher Killian 1 3 , Narayan Appathurai 4 , Susan Coppersmith 1 , Pupa Gilbert 1
1 Physics, University of Wisconsin-Madison, Madison, Wisconsin, United States, 2 Center for Biomolecular Materials Spectroscopy, Laboratory for Chemical Physics, New York University, New York, New York, United States, 3 Molecular and Cell Biology, University of California, Berkeley, California, United States, 4 , Synchrotron Radiation Center, Stoughton, Wisconsin, United States
Show AbstractThe formation of inorganic phases by living organisms, biomineralization, is an important and widespread phenomenon that attracts the attention of scientists from a variety of disciplines. The development of biomineralized systems requires participation of macromolecular components, such as proteins, which chemically and physically interact with minerals to determine crystal polymorph, habit, and morphology. Hence, the role of proteins in biomineral formation must be crucial. The key to exploiting the materials construction strategies employed by biomineralizing organisms, such as nacre-forming ones, is to understand the interactions that occur at the organic-mineral interface (OMI) and the function of key biomineral proteins.We, along with several others, have taken the approach of creating model biomineral systems in which a limited number of molecular participants interact under controlled conditions. These in vitro systems provide a model for the role of individual proteins, allowing us to examine the OMI and function of individual proteins. In the study presented here, we address questions dealing with peptide interaction and function of a single 30-amino acid peptide, n16N. The n16N peptide is sequenced after the mineral binding portion of the nacre protein n16-1 from Pinctada fucata. n16-1 is one of three proteins from the n16 protein family extracted from the water-insoluble matrix of the nacreous layer in the bivalve Pinctada fucata. Using x-ray absorption near edge structure (XANES) spectromicroscopy, x-ray photoemission electron microscopy (X-PEEM), and scanning electron microscopy (SEM), we revealed that n16N induces the nucleation of aragonite, even in calcite growth conditions. This is of particular interest because aragonite is thermodynamically less stable compared with calcite (both are CaCO3 polymorphs) and, yet, aragonite appears in many mollusk shells and corals. The aragonite polymorph-selection function of the n16N peptide in a synthetic growth solution is evident in our experiments. We also revealed that n16N self-assembles into regularly spaced lamellar structure, similar to natural nacre and dramatically different than the control calcite crystals grown in the absence of peptide.Many other proteins are present in natural nacre, thus, the functions exhibited by n16N here may be shared by other proteins. We cannot claim that n16 is the only protein with this function, but we can claim with certainty that the function exists, and a simple 30-amino acid peptide is sufficient to perform lamellar self-assembly of aragonite.
3:00 PM - **NN2.3
Biocatalytic Induction of Supramolecular Order and Function.
Rein Ulijn 1
1 , University of Strathclyde, Glasgow United Kingdom
Show AbstractThe ability to reproducibly access structures that do not represent global thermodynamic equilibrium is a major challenge in biomolecular self-assembly. This is of relevance to supramolecular materials for (opto-) electronics where emergent functionality is a direct consequence of molecular order. It is also of relevance for self-assembled hydrogels for cell culture applications, where kinetically induced stiffness dictates cell fate. We will demonstrate that catalytically controlled self-assembly of aromatic peptide amphiphiles gives rise to kinetically induced across the molecular, nano- and microstructure scales- with faster reactions giving rise to fewer defects. The approach is based on enzyme controlled activation and amplification of self-assembling peptide derivatives, where a direct positive correlation between catalyst concentrations and molecular order is observed. We will propose that this observation can be explained by catalysts (enzymes) working in concert within nucleation clusters, locally controlling the amplification rate and fidelity of structure formation. We show that our approach can be exploited to control pi-orbital overlap thereby facilitating intermolecular pi-pi communication. This approach may therefore give rise to supramolecular wires with kinetically induced electro-conductivity. In addition, the approach allows for construction of chemically identical hydrogels with varying modulus. We will present our recent work on induced stem cell differentiation using this approach.
3:30 PM - NN2.4
Binding, Molecular Recognition, and Supramolecular Assembly of Solid Binding Peptides on Solid Surfaces: A Multi-scale Perspective.
Christopher So 1 , Ersin Oren 1 , John Evans 2 , Candan Tamerler 1 3 , Paul Mulheran 4 , Mehmet Sarikaya 1
1 Materials Science and Engineering, University of Washington, Seattle, Washington, United States, 2 Laboratory for Chemical Physics, New York University, New York, New York, United States, 3 Molecular Biology and Genetics, Istanbul Technical University, Istanbul Turkey, 4 Chemical Engineering, University of Strathclyde, Glasgow United Kingdom
Show AbstractThe understanding of biomineralization and realization of biology-inspired materials technologies depends on understanding the nature of the chemical and physical interactions between proteins and biominerals or synthetically made inorganic materials. However, little is known about the molecular structure of these peptides/proteins and their specific recognition mechanisms onto their counterpart inorganic surfaces. To interrogate these processes we combine atomistic techniques such as nuclear magnetic resonance (NMR) and simulated annealing molecular dynamics (SA/MD) with molecule and cluster length-scale methods such as high resolution atomic force microscopy (AFM) and coarse-grained Monte Carlo modeling (KMC) for a multi-scale perspective of peptide adsorption to solid surfaces. We confirmed the intrinsic disorder of a genetically engineered gold binding peptide, 3rGBP1 ([MHGKTQATSGTIQS]3), and identified putative Au docking sites where surface-exposed side chains align with both the <110> and <211> Miller indices of the Au lattice. Further, using unique morphological measures applied to time-lapsed AFM studies, we find that the observed spatial nucleation and growth mechanisms of 3rGBP1 onto an Au(111) surface requires the mobility of clusters as well as monomers of peptides across the solid surface as described by ad-hoc KMC simulations borrowed from metal/semiconducting atomic deposition theory. The research was supported by Genetically Engineered Materials Science and Engineering Center (GEMSEC), an NSF-MRSEC, and an NSF-BioMat grant at the UW.
3:45 PM - NN2.5
Characterizing the Self-assembly of a Natural Cyclic Lipodepsipeptide in Solution With NMR Diffusion and Heteronuclear Relaxation Measurements.
Davy Sinnaeve 1 , Bruno Kieffer 2 , José Martins 1
1 Organic Chemistry, Ghent University, Gent Belgium, 2 IGBMC Biomolecular NMR group, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch France
Show AbstractPseudodesmin A[1] is a small natural cyclic lipodepsipeptide (CLP) of 9 amino acids that belongs to a class of compounds that has the capacity to form ion pores in cellular membranes. This constitutes the basis of its biological activity. Pore formation requires the molecule to self-assemble into larger structures within the apolar membrane environment so as to be capable to traverse the bilayer. Using solution NMR spectroscopy, we have found in apolar organic solvents such as chloroform that pseudodesmin A forms large supramolecular structures which can be linked to the ion-pore forming capability[2]. The methodology used exploits the capacity of NMR to address both translational and rotational diffusion in solution by using diffusion NMR and relaxation studies respectively. Using translational diffusion NMR measurements, we show that the self-assembly is indefinite and that at high concentrations the hydrodynamic radius increases over a factor of 5 compared to the monomer state. The solution structure of an individual pseudodesmin A unit was determined and found to be a small amphipatic left handed α-helix with its C-terminal end covalently connected with the middle of the helix by a three residue loop. Importantly, this structure is preserved in the supramolecular assembly. A model for the self-assembly was proposed, which involves stacking interactions between the complementary ends of helices, while the hydrophilic side of the molecules pack together to minimize the hydrophilic contact surface, effectively creating a hydrophilic tunnel capable of spanning the membrane.To validate our proposed model, a novel approach was used, consisting of heteronuclear 13Cα relaxation (T1, T2, 1H-{13C}-nOe, relaxation dispersion) NMR measurements performed at different concentrations under self-assembling conditions. The NMR relaxation parameters are sensitive to the rotational diffusion tensor of the supramolecular structure and thus to its size, anisotropy and direction of the anisotropy. It was found that the anisotropy of the self-assembled structure increased with the peptide concentration and that the direction of growth is parallel to the helix axis, in agreement with our proposed model. Our approach provides a new method to characterize self-assembling peptides in solution using NMR spectroscopy, provided solvent conditions are identified that promote self-assembly. In addition, the information obtained should in principle allow to guide the elucidation of the molecular details of the supramolecular structure using 1H-1H distance information obtained from intermolecular rOe contacts.[1]Sinnaeve et al, Tetrahedron 65 (2009) 4173-4181, DOI: 10.1016/j.tet.2009.03.045[2]Sinnaeve et al, Chem Eur J, in press, DOI: 10.1002/chem.200901885
4:30 PM - **NN2.6
Short Synthetic Self-assembling Peptide-based Nanomaterials: Tubes, Vesicles, Fibers and Pores.
Arindam Banerjee 1
1 Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata, West Bengal, India
Show AbstractMolecular self-assembly plays a pivotal role to make various nanostructures using the ‘bottom-up’ approach. Judicious choice of synthetic oligopeptides can be properly utilized to construct different nanostructures including nanotubes, nanovelices, nanofibers and others using self-assembly. Water soluble short peptide based nanostuctural transformations from tubes to vesicles have been observed and these transformations are either pH-sensitive [1] or concentration dependent [2]. Self-assembling short peptide based nanovesicles can be envisaged as a carrier for delivering drugs and other biologically important molecules [2]. Dipeptides containing N-terminally located omega-amino acid and C-terminally located alpha-amino acid residues are self-assembled to form nanotubes in water and these tubes are stable against heat up to 80°C, proteolytic degradation and over a wide range of pH values [3,4]. Some of these tubes have been used as templates for fabricating dipeptide capped Au nanoparticles on surfaces of these tubes [4] to make organic-inorganic hybrid nanomaterials. Short peptide based hydrogel nanofibers have been used as templates for fabricating luminescent CdS nanoparticles on these nanofibers and optoelectronic property of CdS nanoparticles can be tuned through fabrication [5]. Porous materials have been obtained from self-assembling cyclic peptide based compounds and dipeptide capped gold nanoparticles can be deposited on the surface of these pores to form organic-inorganic hybrid nanomaterials [6]. Recently, water soluble synthetic dipeptide based biodegradable nanoporous materials have been obtained implying the creation of a new type of nanoporous materials that are environmentally friendly [7]. References:1.P. P. Bose, A. Das, R. Hegde, N. Shamala, A. Banerjee Chem. Mater. 2007, 19, 6150-6157.2.J. Naskar, A. Banerjee Chem.-Asian J. 2009 (accepted). 3.S. Guha, M.G.B. Drew, A.Banerjee Chem. Mater. 2008, 20, 2282–2290.4.S. Guha , A. Banerjee Adv. Func. Mater. 2009, 19,1949–1961.5.G. Palui, J. Nanda, S. Ray, A. Banerjee Chem.-Eur. J., 2009,15, 6902 – 6909.6.S. Guha , A. Banerjee Macromol. Chem. Phys. 2009, 210, 1422–1432.7.S. Guha, T. Chakraborty, A. Banerjee Green Chem. 2009, 11, 1139-1145.
5:00 PM - NN2.7
Self-assembly and Gelation Properties of Novel Peptides for Biomedical Applications.
Jie Gao 1 , Aline Miller 2 , Julie Gough 1 , Alberto Saiani 1
1 School of Materials, University of Manchester, Manchester United Kingdom, 2 School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester United Kingdom
Show AbstractMolecular self-assembly is a powerful tool for the preparation of molecular materials with a wide variety of properties. The increasing interest in the self-assembly of peptides is mostly centered on the relationship between their conformation and function. It is now well known that short peptides can self-assemble to form β-sheet rich fibres that become entangled to form hydrogels and these show promise for use in the tissue engineering field[1]. Consequently many studies have focussed on the self-assembly behavior of such peptides and their influence on fibre, mechanical and biological properties. Here we will focus on establishing design rules for the effect of ionic complementary peptide primary sequence on hydrogel mechanical properties, and consequently cell behavior.More specifically four octa-peptides have been synthesized thus far, with purity ≥ 85%; they are FEFEFKFK, FEFKFEFK, VEVEVKVK, VEVKVEVK. The phase diagram of each peptide has been mapped out as a function of temperature, pH and salt concentration with each forming a transparent hydrogel above a critical peptide concentration through the association of β-sheet rich fibres. Oscillatory rheology confirmed the presence of a gel and showed the elastic modulus can be tuned between 0.001 and 10 kPa depending on peptide concentration, pH and salt type and concentration. Such results will be presented here and general design rules will be discussed. Furthermore we have gone on to show that our peptides self-assemble in the presence of cell culture media and we will discuss how the different processing routes, and properties of our hydrogel network, influence cell behavior. 1. Stuart Kyle AA, Eileen Ingham and Michael J. McPherson. Trends in Biotechnology 2009;27(7).
5:15 PM - NN2.8
Towards Structure Prediction for Low Molecular Weight Hydrogelators.
Dave Adams 1 , Lin Chen 1 , Kyle Morris 2 , Louise Serpell 2 , John Bacsa 1 , Graeme Day 3
1 Chemistry, University of Liverpool, Liverpool United Kingdom, 2 Chemistry and Biochemistry, University of Sussex, Brighton United Kingdom, 3 Chemistry, University of Cambridge, Cambridge United Kingdom
Show AbstractThere is currently significant interest in the assembly of low molecular weight molecules to give gels. Gels are formed via the formation of a fibrous network, resulting from the assembly of the gelator molecules through uni-directional non-covalent forces such as hydrogen-bonding, π-π stacking and van der Waals interactions. This approach has been shown to be successful for a number of very different systems. The enormous structural diversity coupled with different triggers by which assembly occurs (for example pH, temperature, enzymatic action) means that there are limited correlations between the molecular structure and the intermolecular interactions leading to assembly.17,18 Further, there are limited correlations between the assembled fiber structures and the material properties of the resulting gels. Overarching both these issues is the lack of predictability in the design of the gelators. Whilst many gelators are known, serendipity still plays a role in their discovery. It can therefore be seen that a method predicting both the packing of potential hydrogelators and whether hydrogelation will occur would revolutionize this field. Here, we describe our work towards such a goal. We have examined the self-assembly behavior of two chemically and structurally similar functionalized dipeptides, one of which is found to be a gelator, the other forms a crystalline solid. We have employed computational methods to explore the crystal energy landscapes of the two molecules in an attempt to explain their contrasting behavior and to predict the likely molecular arrangement in the gel.
5:30 PM - **NN2.9
Controlling Assembly at the Nano-scale: From Therapy to Devices.
Ehud Gazit 1
1 , Tel Aviv University, Tel Aviv Israel
Show AbstractThe precise and scalable control of biomolecular self-assembly is essential for the integration of bionanotechnology into real world applications and devices. This is in line with the well-known central role of aromatic-stacking interactions in self-assembly processes. Following this notion, we demonstrated that the diphenylalanine recognition motif of the Alzheimer’s beta-amyloid polypeptide self-assembles into ordered peptide nanotubes with a remarkable persistence length. Our model recently gained directed support from high-resolution X-ray and electron diffraction and solid-state NMR structures of amyloid fibrils as well as parameter-free models and molecular dynamics studies. Applications of these nano-assemblies include ultra-sensitive biosensors, energy-storage devices, and metallic nanowires.Reches, M, & Gazit, E. (2003) Casting Metal Nanowires Within Discrete Self-Assembled Peptide Nanotubes. Science 300, 625-627.Reches, M., and Gazit, E. (2006) Controlled Patterning of Aligned Self-Assembled Peptide Nanotubes. Nature Nanotechnol.1, 195-200.Adler-Abramovich et al. (2009)Self-assembled arrays of peptide nanotubes by vapour deposition. Nature Nanotechnol.(in press).
NN3: Poster Session
Session Chairs
Friday AM, April 09, 2010
Salon Level (Marriott)
9:00 PM - NN3.1
Nanofibers and Nanotubes Built With Versatile Amyloid Beta Peptide Fragments.
Valeria Castelletto 1 , Ian Hamley 1 , Peter Harris 2 , Celen Cenker 3 , Ulf Olsson 3
1 School of Chemistry, University of Reading, Reading United Kingdom, 2 Centre for Advanced Microscopy, University of Reading, Reading United Kingdom, 3 Physical Chemistry 1, Lund University, Lund Sweden
Show AbstractThe amyloid beta (Ab) peptide is a major cause of Alzheimer’s disease. We study the influence of specific residues on the self-assembly of the core fragment of the Ab peptide, namely KLVFF. It is believed that short peptides containing the sequence KLVFF can influence the Ab self-assembly process, and therefore might be used as a tool to cure Alzheimer’s disease. KLVFF is not soluble in water. We generated a family of modified water soluble KLVFF fragments, by adding a two amino acids AA sequence [1]. In this work we focus on two water soluble modified KLVFF fragments (AAKLVFF and bAbAKLVFF).AAKLVFF can be dissolved in water and in methanol. We found that AAKLVFF exhibits distinct structures of twisted fibrils in water, or nanotubes in methanol [2]. For intermediate water/methanol compositions, these structures are replaced by wide filamentous tapes. The fibrils, the tapes and the nanotubes are made by b-strands of peptide aligned perpendicular to the main axis of the self assembled structure. These results are interpreted in the light of recent results on the effect of competitive hydrogen bonding on self-assembly in soft materials in water/methanol mixtures.The self assembly of AAKLVFF in water was further studied. We induced variations in the surface charge of AAKLVFF fibres, by adding NaCl and changing the pH of the solution. We have previously proved that bAbAKLVFF self-assembles in helically twisted ribbons in water [3]. Therefore charge screening experiments were also performed on bAbAKLVFF, in an attempt to understand the influence of the beta-alanine residues in the self-assembling process. It was found that, upon addition of NaCl and increasing the pH of the solution, AAKLVFF fibres change into fibrous nanotapes while bAbAKLVFF fibres change into nanotubes. [1] I. W. Hamley, M. J. Krysmann, V. Castelletto, et al. Adv. Mat., 20, 4394 (2008) V. Castelletto, I. W. Hamley, Biophys. Chem. 141, 169 (2009) [2] V. Castelletto, I. W. Hamley, J. P.F. Harris et al. J. Phys. Chem. B, 113, 9978 (2009) . V. Castelletto, I. W. Hamley, P.J.F. Harris Biophys. Chem. 138, 29 (2008)[3] V.Castelletto, I.W.Hamley, R.A.Hule et al. Angew. Chem. 48, 2317 (2009)
9:00 PM - NN3.10
Mechanical Stability and Transport Properties Calculations for Peptide Nanostructures from Molecular Dynamics Simulations.
Jennifer Carvajal 1 , Tahir Cagin 1 2
1 Chemical Engineering, Texas A&M University, College Station , Texas, United States, 2 Materials Science and Engineering , Texas A&M University, College Station , Texas, United States
Show AbstractSelf-assembled peptide nanostructures represents new possibilities in the area ofBiotechnology and Biomedicine due to their wide set of applications, ranging from molecular Bioelectronics to Drug Delivery. We have chosen peptide nanotubes as peptide nanostructure model, these peptide nanotubes have been experimentally reported to self-assemble by a process based on β-sheetlike interactions and hydrogen bonding between macro cyclic D,L-peptides. Cyclic peptide nanotubes have a lot of remarkable features, which provide it many aspects applications, such as size-selective sensor, transmembrane-ion channel and drug delivery agents. We have used molecular dynamics simulation techniques to study conformational properties of peptide nanotubes; such as structural stability, thermodynamic properties and transport properties. The transport properties calculations were performed using NAMD2.5 and the CHARMM force field, under constant temperature (300K) and constant pressure (1.013 bars) conditions. After the equilibration of the system, self-diffusion coefficient, dipole correlation functions and hydrogen bond probabilities were calculated. Different diameters of the peptide nanotubes nanotubes were studied and compared with equivalent carbon nanotubes (CNTs). The transport of water within cyclic peptide nanotubes was found favored compared with the carbon nanotubes channels, due the more hydrophilic character of the hollow tubular zones of the peptide nanotubes. Thermo-mechanical stability studies on these materials are also important and crucial for the design of their applications. We have used a homogeneous deformation method combined with the finite elasticity theory and molecular dynamics simulations (MD) for the calculation of second order anisotropic elastic constants for a membrane model made up of self-assembled cyclic peptide nanotubes. We have computed the values of all anisotropic elastic and engineering Young’s modulus constants at 300 K; yield behavior was observed in z direction for large strain values. Furthermore, we report calculated heat capacity, thermal expansion coefficient and isothermal compressibility of the peptide nanostructure under study. Because of the potential application of these peptide-based structures, the study of their properties and mechanism of formation by theoretical and experimental methods will contribute to the rational design of novel materials and devices.
9:00 PM - NN3.11
Self-reporting and Actuation of Peptide Nanotube Polymers.
John Kulp 1 , Thomas Clark 1
1 Chemistry, Naval Research Laboratory, Washington , District of Columbia, United States
Show AbstractProteins and peptides, utilizing molecular self-assembly, provide a variety of functional nanostructures and attractive materials. For example, synthetic peptides self-assemble into well-ordered structures such as nanotubes; the assembly is directed through amide bonds within the backbone. These structures find use in a variety of biotechnology applications including electronics, sensors, ion channels, and catalysis. We will present design, modeling, synthesis, and characterization of a cyclic beta-tripeptide-based polymer that is designed to be self-reporting, sensitive to mechanical state or environment, and capable of tunability or actuation, changes in properties induced by external stimuli such as pH and electrostatics.
9:00 PM - NN3.12
Clathrin Assemblies as Biotemplates.
Jules VanDersarl 1 , Nick Melosh 1
1 Materials Science, Stanford University, Stanford, California, United States
Show AbstractNature presents us with an amazing variety of self-assembling nanoscale architectures. Recently, several methods have been developed to interface biological structures with inorganic materials, using the biological molecules as templates to fabricate nanowires and nanospheres with unprecedented order and regularity, however, these are generally 0D or 1D systems that require non-trivial assembly processes and charge-transport hopping mechanisms for practical applications. We propose using the versatile protein clathrin as a biotemplate at the molecular level to create 2D and 3D conducting nanostructures, desirable structures for energy related fields such as battery electrodes and solar cells. Clathrin adopts a triskelion (three-armed) structure that can self-assemble in vitro into both 2D sheets and various 3D shapes depending on environmental conditions. We present our research into these clathrin structures in vitro. We investigate the assembly process, properties of final assemblies, and progress in creating conducing nanostructures from these quaternary protein structures.
9:00 PM - NN3.13
Structure-Property Studies of Biomimetic Complex Coacervates by Magnetic Resonance Methods.
Julia Ortony 1 2 , Dong Soo Hwang 2 , J. Herbert Waite 2 , Song-I Han 1 2
1 Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, California, United States, 2 Materials Research Laboratory, University of California Santa Barbara, Santa Barbara, California, United States
Show AbstractComplex coacervation occurs when oppositely charged polyelectrolytes associate to form dense liquid droplets of about a micron diameter, suspended in solution. The coacervate droplets have unique physical properties such as low interfacial tension, high density, and the ability to be modulated by pH. The low interfacial tension of these systems and the liquid character of their internal structure allow them to spread readily over essentially all surfaces. This behavior endows them with clear function such as in the field of biomedical adhesives, while presenting substantial, intrinsic challenges for their physical characterization in situ. We study complex coacervates assembled from recombinant mussel proteins that are engineered to exhibit both strong adhesive and cohesive properties under biological conditions, and we circumvent the challenges of in situ characterization by using non-invasive magnetic resonance techniques. Our implementation of these techniques is made possible through new instrumental and methodological developments for the measurements of interfacial and surface hydration water dynamics of polymers and their assemblies. We employ site-specific dynamic nuclear polarization spectroscopy (DNP) in conjunction with electron spin resonance spectroscopy (ESR) to simultaneously determine the solvent dynamics and the polymer segment mobilities of our biomimetic systems. With these techniques, we elucidate key dynamic properties of complex coacervates in situ and gain an understanding of the relationships between these dynamic properties and the observed rheological behavior. With our studies we are able to provide a basis by which to design biocompatible complex coacervates for targeted applications.
9:00 PM - NN3.14
Self-assembled Biomimetic Matrices for Skin Regeneration.
Daniela Ferreira 1 2 , Alexandra Marques 1 2 , Rui Reis 1 2 , Helena Azevedo 1 2
1 3B's Research Group, University of Minho, Guimarães Portugal, 2 Institute for Biotechnology and Bioengineering, University of Minho, Guimarães Portugal
Show AbstractNowadays, wound healing presents a major clinical problem considering the millions of individuals worldwide with potential difficulties in healing wounds because of diabetes. We believe that materials technology can assist in the regeneration of human tissues but it will need to include design at the molecular level for interactions with cells. The ideal design of synthetic matrices for tissue regeneration should mimic, at least in part, the architecture and function of the extracellular matrix, to provide support and signalling cues that can elicit a specific cellular behaviour. Towards this challenge, we have selected natural macromolecules because they are either components of or have properties similar to the native extracellular matrix. Hyaluronic acid or hyaluronan (HA) is a high-molecular mass polysaccharide ubiquitously distributed in the extracellular matrix. HA is an immense polyanion which is soluble in water up to relatively high concentrations, and results in solutions which possess a highly visco-elastic nature. The special physiochemical and biological properties of HA and its non-immunogenic nature have made this biopolymer a particularly useful substance for skin tissue engineering. We intend to develop new biomimetic matrices for skin tissue engineering in which biological activity is intrinsic to the material. Peptides are of great interest for this purpose due to their demonstrated bioactivity and can be used to influence cell behavior. In addition, they provide very interesting supramolecular chemistry to build complex materials. This study reports the development of new biomimetic matrices in which functions emerge as a result of self-assembly of bioactive peptide molecules in presence of the biopolymer hyaluronic acid. An array of advanced spectroscopy and microscopy techniques has been used for characterizing these self-assembling matrices. These studies have been complemented by testing in vitro the hybrid matrices with relevant cells (e.g. primary human fibroblasts). The self-assembled matrices can support the adhesion of skin cells with the typical fibroblastic morphology.
9:00 PM - NN3.15
Particle Separation Using Inorganic Binding Peptides Based on Phage Display Biopanning.
Chih-Wei Liao 1 , Laurie Gower 1
1 Materials Science & Engineering, University of Florida, Gainesville, Florida, United States
Show AbstractIn the mining industry, flotation is the most common method used to separate particles of interest from an ore mixture. Flotation is a physico-chemical separation process that utilizes the difference in surface properties between valuable minerals and unwanted gangue minerals. In this method, surfactants are used to facilitate the attachment of mineral particles to air bubbles, such that the mineral particles of interest can be collected from the froth on top of the flotation bath. However, if two or more mineral particles have similar surface characters (such as polarity), they can not be effectively separated. In order to overcome this limitation, we are examining the feasibility of using a biotechnology approach for particle-based applications that may benefit from the high degree of specificity achieved by molecular recognition between peptides and inorganic surfaces. More specifically, a biopanning approach based on phage display is being used to screen for peptides that have high binding affinity and selectivity for particles of interest. Phage display is a combinatorial approach that utilizes a commercially available library of phage (virus) particles that have DNA encoding short peptide inserts into the protein ‘tails’ (pIII) of the phage, to pan for inorganic binding peptides. The objective here is pan for peptides which preferentially bind to francolite. Francolite, a mineral used in fertilizer, is a fluoridated apatite containing carbonate groups substituting for some of the phosphate groups: (Ca, Mg, Sr, Na)10(PO4, SO4, CO3)F2-3. It is notoriously difficult to separate francolite from dolomite because of the close similarity between their physicochemical properties. If high specificity can be achieved with the biopanning approach, these inorganic binding peptides have the potential to be tailored by coupling with a hydrophobic tail to form surfactants, where one end could provide specific binding affinity to the mineral particles of interest, and the other end could insert in air bubbles to provide flotation. Based on this design, we hope to demonstrate a new approach to enhance the efficiency and yield during mineral beneficiation processes.
9:00 PM - NN3.16
Bio-functionalization of Implant Materials Using Inorganic Binding Peptides.
Mustafa Gungormus 1 , Dmitriy Khatayevich 1 , Christopher So 1 , Sibel Cetinel 2 , Candan Tamerler 1 2 , Mehmet Sarikaya 1 2
1 Genetically Engineered Materials Science and Engineering Center, Materials Science and Engineering, University of Washington, Seattle, Washington, United States, 2 Molecular Biology and Genetics, Istanbul Technical University, Istanbul Turkey
Show AbstractUncontrolled interactions between synthetic materials and human body are a major concern in implants and tissue engineering. The most successful approaches to circumvent this issue involve the modification of the implant or scaffold surface with various functional molecules, such as anti-fouling polymers or cell growth factors. To date, such techniques have relied on surface immobilization methods that are often applicable only to a limited range of materials and require the presence of specific functional groups, synthetic pathways, or biologically hostile environments. In this study we use peptide motifs that have been selected to bind to gold, platinum and quartz to modify surfaces with Poly(ethylene glycol) anti-fouling polymer and RGD integrin-binding sequence. Solid binding peptides have several advantages over conventional molecular immobilization techniques; they perform binding in aqueous environment, do not require any biologically hostile environmentsto function, are specific to their substrates, and could be conjugated to carry various active entities. We successfully imparted cell-resistant properties to gold and platinum using 3GBP1 and PtBP1 peptide motifs, respectively, in conjunction with PEG. The surfaces performed as favorably as those functionalized through thiol chemistry. We also created SiO2 surfaces with enhanced cyto-compatibility using the quartz binding peptide fused with the RGD motif. The results obtained from the different approaches followed in this study, i.e. targeted and directed assembly, on-resin and in solution conjugation; show that the peptide-based surface modification is an adaptable platform to the requirements that may arise because of the nature of the surface to be modified, the biological molecule to be immobilized or the peptide itself. Given the ever increasing number of peptides identified for various types of materials, and the ability to make them material specific, peptide-based immobilization is a promising platform for biologically safe and simple modification of multi-material medical devices. This project is supported by GEMSEC, an NSF-MRSEc at the University of Washington, and NSf-BioMater, IRES, and TUBITAK (TR).
9:00 PM - NN3.17
Designing Synthetic, Modular Peptide Antigen Delivery Systems Using Self-assembling Peptide Amphiphiles.
Amanda Trent 1 , Matthew Black 1 , Matthew Tirrell 1 2
1 , University of California, Santa Barbara, Santa Barbara, California, United States, 2 , University of California, Berkeley, Berkeley, California, United States
Show AbstractWith the goal of exploring strategies for the rational design of immunotherapeutics, we propose using peptide amphiphile (PA) molecules as building blocks for novel peptide antigen delivery systems. PAs can form versatile macromolecular constructs called protein analogous micelles (PAMs) that combine the biofunctionality of peptides with a controllable self-assembly character obtained by synthetic chemical conjugation of peptide head groups with hydrophobic hydrocarbon tails (i.e. a fatty acid). In aqueous conditions, the hydrophobic effect drives PAs to self assemble into hierarchical structures, including spherical, cylindrical and worm-like micelles. The shape and structure of PAMs can be manipulated via both the structure of the individual PA molecule and the interactions among the peptide headgroups. Alternatively, the hydrophobic tails allow PAs to be incorporated into vesicle bilayers in which the peptide head groups are displayed on the vesicle surface.PAs confer a number of attractive properties for peptide-based immunotherapy, including the multivalent presentation of peptide antigens, the enhanced peptide secondary structure often seen upon aggregation into PAMs, the flexibility to form various micellar shapes (or for the PAs to be inserted into vesicles), and the potential of the hydrophobic tails to act as adjuvants. Moreover, the nanoscale dimensions of PAMs and PA-functionalized vesicles make it possible for these antigen delivery vehicles to efficiently enter the lymphatic system and be transported to regional draining lymph nodes. Several projects are ongoing that aim to assess the feasibility of using PAs for immunotherapy. Characterization of properties such as size, shape, and secondary structure will be presented for a number of PA-based designs, including those incorporating amyloid-β peptides involved in Alzheimer’s disease and peptides from the M protein of the group A streptococcus bacterium. Additionally, we are looking at creating multifunctional delivery systems that contain not only antigenic peptides, but also cell-targeting ligands and Toll-like receptor (TLR) agonists as adjuvants. Preliminary data demonstrates synergistic up-regulation of inflammatory cytokines by antigen presenting cells when stimulated by liposomes simultaneously displaying the TLR agonists monophosphoryl lipid A and tri-palmitoyl-S-glyceryl cysteine.
9:00 PM - NN3.18
Development of a Virus-like Particle Platform that Integrates Phage Display and Targeted Delivery Capabilities.
Carlee Ashley 1 , Walker Wharton 2 , David Peabody 2 , C. Jeffrey Brinker 1 2
1 Chemical Engineering, University of New Mexico, Albuquerque, New Mexico, United States, 2 Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, New Mexico, United States
Show AbstractAcute lymphoblastic leukemia (ALL), a disease characterized by the uncontrolled proliferation of malignant lymphocytes leading to the suppression of normal hematopoiesis, is the most frequently diagnosed cancer in children. Current therapies result in the induction of long-term remission in 80% of pediatric ALL patients. Death from relapsed ALL remains the second leading cause of mortality in children, however. Additionally, children who enter remission suffer from significant short- and long-term complications due to the side effects of cytotoxic therapies. Therefore, new generations of therapies are required both to enhance survival and improve quality of life. To this end, we have developed a virus-like particle (VLP) platform that integrates phage display and targeted delivery capabilities within a single particle. VLPs of the MS2 bacteriophage self-assemble from 180 identical copies of coat protein, each of which is highly tolerant of a wide-variety of peptide insertions Each MS2 VLP selectively encapsidates the mRNA that templates its synthesis, providing the genotype-phenotype linkage necessary for affinity selection. Furthermore, MS2 VLPs can be constructed entirely in-vitro, enabling the development of highly complex libraries and making it possible to automate the library construction and affinity selection processes. We have utilized microarray analysis of leukemic blast samples obtained from 207 ALL patients enrolled in Children’s Oncology Group (COG) trial 9906 to identify CD99 and CRLF2 as ideal targets for affinity selection experiments. The expression of either CD99 or CRLF2 alone is predictive of a markedly poor outcome, and only refractory pediatric ALL express both CD99 and CRLF2 in combination. We have employed pediatric ALL cells, NALM-6 parental cells stably transformed with either CD99 or CRLF2, and CD99 or CRLF2 fused to latex beads via a GST linkage as targets in initial affinity selections experiments. We found that five rounds of selection were sufficient to yield a single, high affinity sequence family. We also performed selections at 37°C to identify peptides with a high affinity for CD99 or CRLF2 that also induce endocytosis of the VLP carrier within the target cell. MS2 VLPs naturally self-assemble in the presence of RNA, a feature that we exploit to rapidly and specifically load siRNA as well as RNA-modified drugs and protein toxins within the interior volume of the 28-nm capsid. We have successfully encapsidated and delivered prenisolone, vincristine and daunomycin (the chemotherapeutic agents of choice in pediatric leukemias), AG490, a general JAK inhibitor whose clinical use is limited by side effects, siRNA cocktails that silence expression of cyclin A, cyclin B, and anti-apoptotic genes (e.g. bcl-x), and ricin A chain. Preliminary experiments indicate that internalization of as few as two VLPs containing ricin A chain is sufficient to induce cell death.
9:00 PM - NN3.19
Structural and Mechanical Properties of Alzheimer's Aβ(1-40) Amyloid Fibrils: From Angstrom to Micrometer Scales.
Markus Buehler 1 , Raffaella Paparcone 1 , Zhiping Xu 1
1 Laboratory for Atomistic and Molecular Mechanics, Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractAmyloid fibrils are highly ordered protein aggregates associated with several pathological processes such as prion propagation and Alzheimer’s disease. A key issue in amyloid science is the understanding of the mechanical properties of amyloid fibrils and fibers, in order to quantify biomechanical interactions with surrounding tissues and to identify mechanobiological mechanisms associated with changes of material properties as amyloid fibrils grow from nanoscale to microscale structures. Here we report a series of computational studies using atomistic simulation, elastic network modeling and finite element simulation, utilized to elucidate the mechanical properties of Alzheimer's Aβ(1-40) amyloid fibrils as a function of the length of the protein filament for both 2-fold and 3-fold symmetric amyloid fibrils. We calculate elastic constants associated with the torsional, bending and tensile deformation as a function of the length of the amyloid fibril, for fibril lengths ranging from nanometers to micrometers. The resulting Young's moduli are found to be consistent with available experimental measurements obtained from long amyloid fibrils and predicted to be in the range of 20-30 GPa. Our results show that Aβ(1-40) amyloid fibrils feature a remarkable structural stability and mechanical rigidity for fibrils in excess of several hundreds of nanometers in length. However, local instabilities that emerge at the ends of short fibrils on the order of tens of nanometers reduce their stability and contribute to their disassociation under extreme mechanical or chemical conditions. Moreover, we find that amyloids with lengths shorter than the periodicity of their helical pitch, typically between 90 and 130 nm, feature significant size effects of their bending stiffness due the anisotropy in the fibril's cross section. Further studies are presented that elucidate a strong coupling between compression and twisting deformation modes, resulting from the characteristic structure of amyloid fibrils. A detailed comparison between amyloid fibrils with different symmetries will be discussed. Our studies reveal the importance of size effects in understanding the mechanical properties of amyloid fibrils, an issue of great importance for the comparison between experimental and simulation results and for the understanding of the biological mechanisms during the growth of ectopic amyloid materials. We conclude the presentation with a discussion on the use of amyloids as a new class of biological self-assembled nanomaterials for a variety of applications.
9:00 PM - NN3.2
Microrheology of Low Molecular Weight Hydrogelators.
Anders Aufderhorst-Roberts 1 , Athene Donald 1
1 Sector for Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge United Kingdom
Show AbstractThe self-assembly of organic molecules into hydrogel networks has significant potential in contributing to the development of novel stimuli-responsive biomaterials. The effectiveness of such materials is determined by their responsiveness to external stimuli and the tuneability of their characteristics, in particular, their rheological properties. In designing such materials a better understanding of the rules which determine how molecular structure affects physical properties is desired.Peptides coupled to the FMOC moiety have been identified as a useful model to study such design rules. This is due to the simplicity of their molecular structure and their ability to self-assemble in water into fibrous networks [1].Particle tracking microrheology (PTM) is a method for measuring the viscoelastic properties of soft materials, such as hydrogels, on the micron scale. Viscoelastic properties are inferred by monitoring the time evolution of the Brownian motion of embedded micron sized probe particles and by analysis of the resulting particle tracks [2].In this study, the gelation behaviour of FMOC-Tyrosine is examined. Self-supporting hydrogels are observed to form at low pH. The viscoelastic behaviours of the networks at varying pH are then characterised using PTM. A significant challenge in designing such an experiment is controlling the gelation kinetics such that they are slow enough to allow the gel point to be studied, as the peptide is observed to form a weak gel at relatively short times, which becomes more rigid with decreasing pH. Of particular interest is whether such a system exhibits the phenomenon of “dynamic scaling”, in which the gel network structure shows self similarity around the gel point. Not only would this shed light on the fundamentals of low molecular weight hydrogel formation but it would also allow a number of additional methods of analysis associated with PTM to be used. In particular the method of time-cure superposition allows the point of gelation to be determined by the superposition of the mean-squared displacement curves of the embedded particles [3].[1] A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R. F. Collins, M. L. Turner, A. Saiani and R. V. Ulijn. “Fmoc-diphenylalanine self assembles to a hydrogel via a novel architecture based on pi-pi interlocked beta-sheets.” Advanced Materials, 20(1), 37–41, 2007.[2] T. G. Mason and D. A. Weitz. “Optical measurements of frequency-dependent linear viscoelastic moduli of complex fluids.” Phys. Rev. Lett., 74,1250 – 1253, 1995.[3] T. H. Larsen and E M. Furst. Microrheology of the liquid-solid transition during gelation. Phys Rev Lett, 100(14), 146001, 2008.
9:00 PM - NN3.4
The Self-assembly Mechanism for a Naphthalene-Dipeptide Leading to Hydrogelation.
Dave Adams 1 , Lin Chen 1 , Kyle Morris 2 , Andrea Laybourn 1 , Matthew Hicks 3 , Alison Rodger 3 , Louise Serpell 2
1 Chemistry, University of Liverpool, Liverpool United Kingdom, 2 Chemistry and Biochemistry, University of Sussex, Brighton United Kingdom, 3 Chemistry, University of Warwick, Coventry United Kingdom
Show AbstractSuitably functionalized dipeptides have been shown to be effective hydrogelators. The design of the hydrogelators and the mechanism by which hydrogelation occurs are both currently not well understood. Here, we have examined the assembly of a naphthalene-dipeptide in detail to understand the process leading to the formation of hydrogels. We have utilized the hydrolysis of glucono-δ-lactone to gluconic acid as a means of adjusting the pH in a naphthalene-alanylvaline solution allowing the specific targeting of the final pH. This method allows the assembly process to be characterized. We have used a number of techniques (fluorescence, TEM, X-ray fibre diffraction, IR, circular and linear dichroism and rheology) to probe the assembly at different length scales. We show that the assembly process translates directly onto the mechanical properties of the hydrogel, allowing a detailed understanding of the hydrogelation process.
9:00 PM - NN3.5
Synthesis and Characterization of Well-defined Responsive Peptide-Polymer Conjugates.
Jean-Baptiste Guilbaud 1 , Aline Miller 1 , Alberto Saiani 2
1 Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester United Kingdom, 2 School of materials, The University of Manchester, Manchester United Kingdom
Show AbstractThe increasing utility of polymer-protein conjugates in drug delivery, regenerative medicine, biotechnology and nanotechnology has driven the need for generating homogeneous and well defined biohybrid materials. Of importance for such applications are stimuli responsive materials which present the ability to reversibly alter their structure and physico-chemical properties when subjected to an external stimulus such as temperature or pH making them suitable candidates for application in drug delivery. Herein we focus on the design of temperature responsive peptide-polymer conjugates. To synthesise such conjugates, two strategies can be used: (1) synthesis of the polymer from a peptide macroinitiator and (2) synthesis of the polymer with defined architecture and molecular weight with subsequent attachment to the peptide at a specific site.In this work, we used ionic complementary oligopeptides containing the alternating hydrophobic/hydrophilic and charged/non-charged amino acids F, E and K. Octapeptides containing these amino acids are known to self-assemble into β-sheet rich fibrillar networks. These octapeptides were linked to poly-N-isopropylacrylamides (pNIPAAm), a polymer displaying temperature dependent phase transitions in aqueous solution from a hydrophilic to hydrophobic state, which were synthesised with narrow polydispersity and defined molecular weight via Atom Transfer Radical Polymerisation (ATRP). Peptide chain ends were chemically functionalised and used as the macroinitiator for the synthesis of ATRP pNIPAAM. Furthermore, we have also demonstrated that sufficient coupling can occur directly between end-functionalised polymer and peptide. Such methods open up the possibility of synthesising a diverse range of polymer-peptide conjugates with defined architecture. In turn, controlling the structure and properties of such conjugates (molecular weight of the polymer, polymer/peptide ratio in the conjugate) can enable fine tuning of their physical properties and therefore their stimuli responsiveness, both of which will be discussed here.
9:00 PM - NN3.6
Ordered Human Recombinant Type I Collagen Scaffolds for Regenerative Medicine.
Amit Yaari 1 , Or Dgani 2 , Hagit Amitai 2 , Ron Avrahami 3 , Eyal Zussman 3 , Oded Shoseyov 1
1 The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture and the Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agricultural, Food and Environment Quality Sciences. The Hebrew university of Jerusalem, Rehovot Israel, 2 , CollPlant Ltd., Nes Ziona Israel, 3 Department of Mechanical Engineering, Technion—Israel Institute of Technology, Haifa Israel
Show AbstractType I collagen is a key component in load bearing tissues (ligaments, fascia, bones), giving them tensile strength and elasticity. The unique mechanical properties of these tissues are dependent on the highly ordered and hierarchical organization of the collagen fibers from the nanometer scale and upwards. Recently, we have developed human recombinant type I collagen in transgenic tobacco plants [1]. There are significant advantages for the use of monomeric collagen based scaffolds for tissue repair because of its purity, biocompatibility and biodegradability, but so far there was only a limited success in creating structures that would have the required strength. This is partly attributed to a lesser degree of order achieved so far in reconstituted collagen fibers.Above a threshold concentration, acid soluble collagen exhibits liquid crystalline properties, displaying nematic and cholesteric mesophases. In this work, we utilize collagens unique liquid crystalline and self assembly properties to create ordered scaffolds from recombinant human type I collagen for tissue replacement. Collagen Sheets:By applying shear force to a thin layer of liquid crystalline collagen, we are able to induce nematic order in the collagen rod-like molecules. Drying of the thin film elevates the collagen concentration, inducing a gradual transition into a precholesteric mesophase. The thin sheets(10 microns) are then neutralized to induce fibrillogenesis (a sol-gel transition) and crosslinked, resulting in a neatly ordered array of aligned collagen fibers. The sheets thus obtained have numerous applications in regenerative medicine.Collagen Fibers:We have developed a method for electrospinning of pure collagen. Liquid crystalline collagen displays high shear viscosity but low elongational viscosity, which makes it difficult to spin into fibers. In order to overcome the problem, a core-shell electro-spinning technique is used. The core is liquid crystalline collagen, and the shell is a solution of high molecular weight PEO (polyethylene oxide) dissolved in water and ethanol, which has excellent spinability. During the spinning process, the shell “pulls” the core and molds it into a thin fiber. The shear forces applied in the process align the collagen monomers into a precholesteric order. The fibers are then immersed in an aqueous neutralizing buffer solution, to induce fibrillogenesis and to wash off the shell material that dissolves in water solution. We are left with a pure fibrillated collagen fiber, with a diameter of ~ 5 micrometers, that can readily be knitted or woven into sheets or ropes for tendon and ligament repair.[1] Stein, H., Dgany O., Wilensky M., Tsafrir Y. , Rosenthal M., Amir R., Avraham T., Ofir K., Yayon A. and Soseyov O. (2009) Production of bioactive, post-translationally modified, heterotrimeric, human recombinant type-I collagen in transgenic tobacco. Biomacromolecules. 10(9):2640-5.
9:00 PM - NN3.7
3D Responsive Hydrogel Scaffolds from Self-assembling Polymer-Peptide Conjugates.
Antons Maslovskis 1 2 , Nicola Tirelli 3 , Alberto Saiani 4 , Aline Miller 1 2
1 Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester United Kingdom, 2 School of Chemical Engineering & Analytical Science, The University of Manchester, Manchester United Kingdom, 3 School of Pharmacy, The University of Manchester, Manchester United Kingdom, 4 School of Materials, The University of Manchester, Manchester United Kingdom
Show AbstractIn recent years, stimuli-responsive and self-assembling materials have been studied widely due to their potential applications in drug delivery and tissue engineering [1]. One strategy to produce such materials has been to combine stimuli-responsive synthetic polymers with self-assembling proteins or peptides [2]. Such systems are attractive for biomaterials applications as they combine the controlled mechanical, thermal and electronic properties of polymers with the functionality of designed bioactive groups. This work focuses on the design and self-assembly of a doubly thermo-responsive polymer-peptide conjugate and its mixtures with pure peptide. The prototypical thermo-responsive polymer poly(N-isopropylacrylamide) (PNIPAAM) has been chosen since its lower critical solution temperature (LCST) is observed in water around body temperature, i.e. 37 °C [3]. The ionic oligo-peptide containing the alternating hydrophobic/hydrophilic and charged/uncharged amino acids of phenylalanine (F), glutamic acid (E) and lysine (K) was selected as it is well known to form a beta-sheet rich fibrillar network at low concentrations (~17 mg mL-1) [4]. Consequently the conjugate PNIPAAM-FEFEFKFK has been synthesised here, using the method developed by Stoica et al. [5], and its phase behaviour mapped out as a function of temperature for a range of conjugate:pure peptide ratios (from 1:0 to 1:200). Visual and micro differential scanning calorimetric studies show that such mixtures exhibit a double thermoresponsiveness: an LCST transition ~32 °C and a hydrogel melting at ~ 60-65 °C, depending on the conjugate:peptide ratio. Oscillatory rheology confirmed that a macroscopic hydrogel formed when above the critical gelation concentration of peptide and a combination of electron microscopy and small angle neutron scattering revealed the morphological changes occurring within the hydrogel as a function of temperature. Such results will be discussed here and a model for the self-assembly of the polymer-peptide conjugate:pure peptide mixtures will be proposed. These materials form an interesting class of new biomaterials that could find application in the formation of ‘smart’ scaffolds for tissue engineering and drug delivery vehicles.References 1 Mano J.F., Adv. Eng. Materials, 2008, 10, 515;2 Lutz J.F., Borner H.G., Prog. Polym. Sci., 2008, 33, 1.3 Heskins M., Guillet J.E., J. Macromol. Sci., Chem., 1968, A2, 1441.4 Saiani A. et al., Soft Matter, 2009, 5, 193.5 Stoica F. et al., Chem. Commun., 2008, 4433.
9:00 PM - NN3.8
Strong Piezoelectricity in Bioinspired Peptide Nanotubes.
Andrei Kholkin 1 , Nadav Amdursky 2 , Igor Bdikin 3 , Alejandro Heredia 1 , Ehud Gazit 2 , Gil Rosenman 4
1 Department of Ceramics and Glass Engineering, CICECO, University of Aveiro, Aveiro Portugal, 2 Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv Israel, 3 Department of Mechanical Engineering, TEMA, University of Aveiro, Aveiro Portugal, 4 School of Electrical Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv Israel
Show AbstractRecent success in developing piezoelectric nanotubes has shown their great potential [1], but most inorganic piezoelectrics are intrinsically incompatible with biological environment and can be even harmful. Hence, organic [2] and bio-organic materials having significant piezoactivity are indispensible to revolutionize the piezoMEMS. However, as for today, only weak piezoelectric effects in bio-organic materials have been observed [3]. Therefore, the search for micro- and nanoscale piezoelectric materials has drawn much attention in the last decades the search for bio-inspired materials exhibiting strong and robust piezoelectricity is a task of utmost importance. We report our finding of piezoactivity in bio-inspired peptide nanotubes (PNT) made by a self-assembly process of small diphenylalanine, NH2-Phe-Phe-COOH (FF), peptide monomers by atomic force microscopy (AFM). Following the topography acquisition, the AFM was switched to the piezoresponse force microscopy (PFM) regime in which the conducting tip is scanned in contact mode while an ac voltage (Vac) is applied between the tip and Au electrode. In these conditions, we could measure both out-of-plane (OOP) and in-plane (IP) polarization components. Anomalously strong shear piezoelectric activity in FF PNTs, indicating electric polarization directed along the tube axis was found. Comparison with well-known ferroelectrics LiNbO3 and Pb(Zr,Ti)O3 yield sufficiently high piezoelectric coefficient values of about 70-100 pm/V for d15 (shear response) and ~7 pm/V for d33 (longitudinal response). PNTs demonstrate linear deformation without irreversible degradation in a broad range of driving voltages. The results show that peptide nanotubes are promising for micromechanical and MEMS applications.1. F. D. Morrison et al, Rev. Adv. Mat. Sci. 2003, 4, 114-122.2. S. Horiuchi, Y. Tokura, Nature Mater. 2008, 7, 357-366.3. M. H. Shamos, L. Lavine, Nature 1967, 213, 267-269.
9:00 PM - NN3.9
Self-assembly of Lipid-peptide Conjugates as Nanoparticle Scaffolds for Plasmon-enhanced Absorption and Circular Dichroism.
Jared Crasto 1
1 Nanostructured and Biological Materials Branch, Air Force Research Laboratories, Centerville, Ohio, United States
Show AbstractThe directed synthesis and assembly of nanoparticles is necessary when creating materials with tunable properties (optical, mechanical, catalytic). Biomolecules such as peptides and nucleic acids have been explored as scaffolds for the directed synthesis and assembly of nanomaterials as well as in providing inspiration for materials and structures. In our study, we have exploited the self-assembly of amphiphilic molecules and the biotemplating activity of small peptides in the design of lipid-peptide conjugates used in the synthesis and assembly of different inorganic nanostructures. The resulting structures were assembled as chains and used to template metal nanoparticles. The assembled metal nanoparticles exhibit plasmon enhanced absorption and circular dichroism effects. We will present results on the assembly and optical characterization of the metal nanoparticle supramolecular structures.
Symposium Organizers
Alberto Saiani University of Manchester
Joel Collier University of Chicago
Athene Donald University of Cambridge
William Murphy University of Wisconsin-Madison
Laurent Jeannin Solvay SA
NN4: Protein Self-Assembly and Applications
Session Chairs
Friday AM, April 09, 2010
Room 3020 (Moscone West)
10:00 AM - **NN4.1
Engineering of Spider Silk Proteins and Processing into Novel Materials.
Thomas Scheibel 1
1 Biomaterials, Universität Bayreuth, Bayreuth Germany
Show AbstractBiopolymers reflect one fascinating class of hierarchically structured substances. Living organisms employ these substances for a whole variety of applications including protection and stabilization among others. The interplay between the polymeric building blocks is intriguing, and for long researchers tried to unravel the underlying concepts. The precise interplay between the individual components yields the extraordinary material properties (especially concerning mechanical strength). One well-known example is spider silk with superior mechanical properties such as strength and toughness. During 400 million years of evolution spiders became outstanding silk producers. In contrast to insects, such as caterpillars of the mulberry moth Bombyx mori (commonly known as silkworms), spiders can produce different silks – orb web spiders even up to seven different ones. Orb web spiders can precisely control their production and application. Most spider silks are used for building the web, which reflects an optimized trap for flying prey. In order to analyze the structure-function relationship of the underlying spider silk proteins, we have developed a bio-inspired system using bacteria as production hosts which produce silk proteins mimicking the natural spider silks. Besides the protein fabrication, we analyzed fiber formation in detail and unraveled several important aspects involved in silk assembly. Based on this knowledge, we have developed a spinning technique to produce spider silk threads closely resembling natural silk fibers.Further, we employ the bio-inspired silk proteins also in other application forms such as hydrogels, spheres or films. Our bio-inspired approach serves as a basis for new materials in a variety of medical, biological, or chemical applications.
10:30 AM - NN4.2
Development and Investigation of Novel Nanomaterials Based on Protein Fibers.
Christoph Meier 1 , Tuomas Knowles 1 , Mark Welland 1
1 Nanoscience Centre, University of Cambridge, Cambridge United Kingdom
Show AbstractIn addition to their implication in neurodegenerative diseases such as Alzheimer's and Parkinson's, a lesser known aspect of amyloid protein nanofibers is their nature as high performance biomaterials.[1] The strength of these self-assembled and highly ordered nanostructures is comparable to that of steel and their mechanical stiffness is comparable to that of silk.[2] Nature has found ways to exploit these properties and functional amyloid fibers adopt important roles in biosynthetic pathways and as structural components in lower organisms.[3] Some proteins with fibrillization propensities are readily available, cheap and from sustainable resources; their ex vitro self-assembly into fibrillar aggregates can be controlled by choosing appropriate denaturation conditions. However, the ability to fine-tune the functional properties of such natural protein nanofibers and the ability to process the fibrillar aggregates into macroscopic nanomaterials are key issues on the way towards nanotechnological solutions.Herein we discuss how the chemical and physical properties of amyloid nanofibers can be exploited to develop novel protein based nanomaterials. Using noncovalent interactions, amyloid nanofibers can be spun into macroscopic fibers with diameters from tens to hundreds of µm, exhibiting tensile strengths comparable to that of oil-based fibers and elastic moduli comparable to that of dragline silk.The mechanical stability of protein nanofibers is mainly governed by their beta-sheet backbone, whereas their chemical properties are mainly defined by the functional groups of the peptide side chains. In this context, the chemical functionalization of the protein building blocks of non-artificial amyloidic nanostructures constitutes an amenable way to control their functional properties. We demonstrate the introduction of functional groups to the surface of the nanofibers and the effects of the modification on the chemical properties. For instance, in contrast to the pristine nanofibers the functionalized nanofibers possess the ability to interact with other nanometer sized components.[1] T. P. J. Knowles, A. W. Fitzpatrick, S. Meehan, H. R. Mott, M. Vendruscolo, C. M. Dobson and M.E. Welland. Role of intermolecular forces in defining material properties of protein nanofibrils. Science 318 (5858):1900-1903, 2007.[2] J. F. Smith, T. P. J. Knowles, C. M. Dobson, C. E. MacPhee and M. E. Welland. Characterization of the nanoscale properties of individual amyloid fibrils. P. Natl. Acad. Sci. USA 103 (43):15806-15811, 2006.[3] D. M. Fowler, A. V. Koulov, W. E. Balch and J. W. Kelly. Functional amyloid--from bacteria to humans. Trends Biochem. Sci. 32 (5):217-224, 2007.
10:45 AM - NN4.3
Fabrication and Characterization of Protein Nanofiber Composites.
Tomas Oppenheim 1 , Tuomas Knowles 1 , Stephanie Lacour 1 , Mark Welland 1
1 Nanoscience Centre, University of Cambridge, Cambridge United Kingdom
Show AbstractWe have developed a multi-functional protein nanofibre composite based on protein fibrils and silicone elastomer. Insulin fibrils and lysozome fibrils are obtained from lyophilized bovine insulin and lyophilized hen egg white lysozome, respectively, by dilution in a mild acidic solution. The fibrils are typically 2-20nm in diameter and up to several microns long. The “raw” silicone elastomer (Sylgard 184, Dow Corning) is prepared in a 10:1 weight ratio. The fibrils and the elastomer may be mixed in order to form a 10% in weight fibril-silicone composite or parallel silicone micro-channels (120μm x 500μm x 10mm) may be filled with the fibril hydrogel to form an anisotropic structure. We have characterised the mechanical properties of both composites using a Q800 DMA extensiometer. The “raw” silicone has an elastic modulus of 1MPa; the composite filled with 10wt% lysozyme fibrils displays a maximum modulus of 7.5 MPa. This is three times stiffer than a carbon nanotube-elastomer composite prepared with the same composition. Furthermore we found that by patterning the silicone-lysozome fibrils composite into channels, a mechanically anisotropic composite can be produced. We found that the storage modulus along the length of the composite was three times stiffer than that in the direction perpendicular to the channels.
11:30 AM - **NN4.4
Designing Protein Based Hydrogels for Regenerative Medicine.
Aline Miller 1
1 Manchester Interdisciplinary Biocentre and School of Chemical Engineering & Analytical Science, University of Manchester, Manchester United Kingdom
Show AbstractHydrogels have recently attracted much interest in the biomaterials sector because of their ability to entrap large quantities of water or biological fluids. This high water content mimics the natural living environment which gives them excellent biocompatibility. Moreover, their porous microstructure gives them good permeability while the 3-dimensional network provides mechanical support. Over the past few decades many synthetic hydrogels have been developed and used extensively in biomedical applications. Recent research, however, has focused on creating hydrogels from natural materials including peptides and proteins. Here we will focus on creating, controlling and understanding the self-assembly of a wide range of non-related proteins into novel thermo-reversible fibrillar hydrogels under physiological conditions simply by adding the reductant dithiothreitol (DTT). Proteins include beta-lactoglobulin, ovalbimum, lysozyme and bovine serum albimum; all contain an increasing number of disulfide bridges that are disrupted by the reductant DTT. Such disruption destabilises the native state of the protein and exposes hydrophobic groups that encourage organised aggregation leading to the formation of self-supporting beta-sheet rich fibrillar hydrogels. The potential to control and manipulate the gel properties, including mechanical strength and structure (fibre diameter and mesh size of hydrogel) has been explored by varying the protein (consequently the number of disulfide bridges), protein concentration, reductant concentration and ionic strength of the matrix. Our results will show that hydrogelation via reductive conditions is another generic process of protein self-assembly, that differs to the self-assembly behaviour of hydrogels formed via low pH and high temperature. Similarities and differences between protein systems and hydrogel fabrication route will be highlighted. Furthermore we will present both our 2- and 3-dimensional cell culture experiments that show the gel matrix promotes both fibroblast and chondrocyte cell spreading, attachment and proliferation; indicating our hydrogels gels are biocompatible and they can provide a viable support for different types of cells.
12:00 PM - NN4.5
Construction of Functional Protein Assemblies from Component Proteins of Bacteriophage T4.
Takafumi Ueno 1 , Tomomi Koshiyama 1 , Yoshihito Watanabe 2
1 Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto Japan, 2 Department of Chemistry, Nagoya University, Nagoya Japan
Show AbstractProtein assemblies provide a wide variety of nano-size architectures. Conjugation of protein assemblies with organic and inorganic materials provides an attractive strategy for development of catalysts, imaging reagents and drug delivery systems in nano-biotechnology. For instance, spherical- and tube-like protein assemblies a have been utilized as powerful platforms for the arrangement of synthetic molecules in protein cages. For the preparation of improved integrated protein systems, organization of multiple functionalities on a single protein assembly is needed. We have therefore exploited a heteroprotein assembly isolated from bacteriophage T4.[1-3] The assembly is designated (gp27-gp5)3 and consists of a (gp27)3 bio-nanocup and a (gp5)3 bio-nanotube. We have constructed a catalytic nanoreactor using the bio-nanocup which has the capacity to accommodate Fe(III) protoporphyrins by covalent linkage to cysteinyl thiols. We have also demonstrated site-specific conjugation of two different types of fluorescence molecules on and within the gp27 trimer bio-nanocup using a heteromeric assembly reaction of three gp27 monomers with (gp5)3 to construct an energy transfer system. Since these methods enables construction of effective nanoreactors, We present our recent results for construction of multifunctional biomaterials using homo- and heterolytic protein assembly consisting of the component proteins. [1] Org. Biomol. Chem. 7, 2649-2654 (2009), [2] Small 4, 50-54 (2008), [3] Angew. Chem. Int. Ed. 45, 4508-4512 (2006).
12:15 PM - NN4.6
Controlling the Self-assembly of Tobacco Mosaic Virus Proteins.
Michael Bruckman 1 , Heather McDowell 1 , Carissa Soto 1 , Jinny Liu 1 , Elizabeth Mobley 1 , Christopher Spillmann 1 , Banahalli Ratna 1
1 , Naval Research Laboratory, Washington, District of Columbia, United States
Show AbstractThe phase diagram of the tobacco mosaic virus (TMV) coat protein assembly in buffered solutions was established nearly 40 years ago by A. Klug et. al. They demonstrated that in a narrow window of ionic strength and pH conditions, disks form in excess with a diameter of 18 nm, a thickness of 4.7 nm and a center hole with a diameter of 4 nm. Upon lowering the pH, the disks change their structure to a lock-washer type assembly before polymerization into long rods. Here, the incorporation of a His6 tag at the C-terminus of the coat protein for expression in and purification from E. Coli. has lead to a shift in equilibrium lines surrounding the disk structure in the phase diagram of TMV. In addition to using transmission electron microscopy to characterize these structures, atomic force microscopy was used to visualize large surface areas. Circular dichroism and differential scanning calorimetry were used to study the stability of the disks at a variety of conditions. Increasing the stability of disks designed from modified TMV coat proteins can lead to applications in optics and sensing.
12:30 PM - **NN4.7
Protein and Nanoparticle Self-assembly in Natural Biominerals.
Pupa Gilbert 1 , Christopher Killian 1 , Rebecca Metzler 1 , Tyler Churchill 1 , Yutao Gong 1 , Susan Coppersmith 1
1 Physics, University of Wisconsin, Madison, Wisconsin, United States
Show AbstractMollusk shell nacre, sea urchin spicules and sea urchin teeth use protein self-assembly to aggregate amorphous calcium carbonate nanoparticles, and subsequently switch on their crystallization, resulting in centimeter-scale biominerals, in which the nano-scale crystalline particles are highly co-oriented. Nanometer-resolution imaging (1, 2) of these biominerals shows the high degree of co-orientation (3-6), and reveals some of the mechanisms leading to co-orientation (4, 6).1. RA Metzler, M Abrecht, RM Olabisi, D Ariosa, CJ Johnson, BH Frazer, SN Coppersmith, and PUPA Gilbert, Architecture of columnar nacre, and implications for its formation mechanism, Phys Rev Lett 98, 268102 (2007).2. RA Metzler, D Zhou, M Abrecht, J-W Chiou, J Guo, D Ariosa, SN Coppersmith, and PUPA Gilbert, Polarization-dependent imaging contrast in abalone shells, Phys Rev B 77, 064110-1/9 (2008).3. Y Politi, RA Metzler, M Abrecht, B Gilbert, FH Wilt, I Sagi, L Addadi, S Weiner, and PUPA Gilbert, Transformatin mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule, Procs Natl Acad Sci USA 105, 17362-6 (2008).4. PUPA Gilbert, RA Metzler, D Zhou, A Scholl, A Doran, A Young, M Kunz, N Tamura, and SN Coppersmith, Gradual Ordering in Red Abalone Nacre, J Am Chem Soc 130, 17519-27 (2008).5. YR Ma, B Aichmayer, O Paris, P Fratzl, A Meibom, RA Metzler, Y Politi, L Addadi, PUPA Gilbert, and S Weiner, The grinding tip of the sea urchin tooth exhibits exquisite control over calcite crystal orientation and Mg distribution, Procs Natl Acad Sci USA 106, 6048-53 (2009).6. CE Killian, RA Metzler, YT Gong, IC Olson, J Aizenberg, Y Politi, L Addadi, S Weiner, FH Wilt, A Scholl, A Young, SN Coppersmith, and PUPA Gilbert, The mechanism of calcite co-orientation in the sea urchin tooth, J Am Chem Soc under review (2009).
NN5: Peptide Self-Assembly and Applications II
Session Chairs
Cait MacPhee
William Murphy
Friday PM, April 09, 2010
Room 3020 (Moscone West)
2:30 PM - NN5.1
Crystallization of S-layer on Supported Lipid-Bilayers (SLB): The Importance of Amorphous to Crystalline Transition and Self-catalyzed Growth in 2D Limited by Folding.
Sungwook Chung 1 2 , Seong-Ho Shin 1 3 4 , Stephen Whitelam 1 3 , Carolyn Bertozzi 1 3 4 , Jim DeYoreo 1 3
1 Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 3 Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 4 Department of Chemistry, University of California, Berkeley, Berkeley, California, United States
Show AbstractSelf-assembled protein architectures have shown a range of interesting structural motifs such as particles, ribbons, fibers, and sheets. Their functions range from selective transport, structural scaffolding, mineral templating and propagation of or protection from pathogenesis. Although the molecular structures and interactions of the isolated individual proteins determine their governing interactions, these functions emerge from the nanoscale organization that stems from self-assembly. Surface layer proteins (S-layers), which forms the outermost cell envelop in many strains of bacterial systems, presents a unique example of nanoscale organization of self-assembled protein architecture. While the quaternary folding structure arising our of self-assembly is crucial to S-layer function, the assembly process and its detailed mechanism has been poorly understood. Here we report results using in situ AFM to follow 2D crystallization of monomeric SbpA of Lysinibacillus sphaericus on SLBs at the molecular-scale. We show that the initial assembly process begins with the unstructured, adsorbed monomers, which form a mobile phase on SLBs. These then condensed into amorphous clusters, which undergo a phase transition to ordered 2D clusters of 2 to 15 folded tetramers. The ordered clusters then enter a growth phase in which new tetramers form from unstructured monomers exclusively at unoccupied lattice sites along the cluster edges, implying the new tetramer formation is auto-catalytic. The final size of the crystalline domains is strongly dependent on the formation temperature, which appears to control the density of amorphous nuclei. Analysis of the growth dynamics leads to a quantitative model in which the main rate limiting parameter is the probability of tetramer creation. The estimated energy barrier of ca. 51 KJ/mol for this process is much less than expected from scaling laws for folding of isolated proteins, providing an energetic rationale for the auto-catalytic nature of assembly. Finally, we present results from dynamic Monte Carlo simulations that show how the combination of non-specific interactions and directional bonds characteristic of many proteins lead to non-classical assembly pathways, such as the one observed here involving formation of amorphous clusters followed by relaxation to the ordered state. The results predict that the final degree of order depends strongly on the relative strengths of the two interactions with high crystallinity being obtained for an optimum ratio of directional to non-specific bond strengths. This work was performed at the Molecular foundry, Lawrence Berkeley National Laboratory, with support from the Office of Science, Office of Basic Energy Science, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
2:45 PM - NN5.2
Peptides as Molecular Building Blocks for Materials Assembly and Synthesis.
Candan Tamerler 1 2 , Mehmet Sarikaya 1
1 Materials Science & Engineering, University of Washington, Seattle, Washington, United States, 2 Molecular Biology & Genetics, Istanbul Technical University, Istanbul Turkey
Show AbstractBiocombinatorially selected peptides with inorganic surface binding ability have opened up new ways towards fabrication, assembly and architectural design of hybrid materials, all at ambient and environmentally friendly conditions. Achieving the capability to closely manipulate behavior of peptides to fully control material assembly would be a giant leap towards realizing nanometer-scale building blocks in which the peptides and their molecular recognition characteristics are tailored using molecular biology and genetics with respect to their inorganic or synthetic counterparts such as nanoparticles, quantum dots, molecular- or nano-wires or synthetic molecular systems towards tailored nanofunctions. We utilize evolutionary engineering methods in the design of peptides that are specific to materials surfaces. Here, we will explain how we select and tailor the peptides with high affinity and material selectivity, summarize our design parameters for controlled peptide-based self assembly nanohybbrid systems. We next provide examples on their use as molecular linkers, couplers and growth modifiers for synthesis of materials, morphogenesis, and assembly. For example, the peptides are used as catalyzers in the controlled formation of inorganic nanoparticles; molecular linkers in the targeted and co-assembly of proteins for advanced functionality; in directed enzyme immobilization through genetic fusion, molecular surface-functionalizers in the formation of molecular films and scaffolds. The presentation will provide an overview of the approaches carried out in our collaborative groups, with highlights of the recent developments and future prospects. The project supported by an NSF-MRSEC at the UW, NSF Biomater and NIH programs, and MOBGAM-ITU, TR-SPO, TUBITAK-NSF. -C. Tamerler and M. Sarikaya, ACS Nano, 3, 1606-1615(2009); -T. Kacar, M. Zin, A. K-Y. Jen, H. Ma, M. Sarikaya, C. Tamerler, Bioengineering and Biotechnology, 103, 696-705 (2009); C. -Tamerler and M. Sarikaya, MRS Bulletin, Guest Editors on, 33, 504 (2008)
3:00 PM - **NN5.3
Self-assembled Beta-Hairpin Peptides-responsive Gels and Templates for Hybrid Materials.
Darrin Pochan 1
1 Materials Science and Eng, university of Delaware, Newark, Delaware, United States
Show AbstractThe local nano- and overall network structure, and resultant viscoelastic and cell-level biological properties, of hydrogels that are formed via β-hairpin self-assembly will be presented. These peptide hydrogels are potentially excellet scaffolds for tissue repair and regeneration due to inherent cytocompatibility, porous morphology, and shear-thinning but instant recovery viscoelastic properties. The 20 amino acid parent peptide MAX1 (H2N-VKVKVKVKVDPPTKVKVKVKV-CONH2), has been shown to fold and self-assemble into a rigid hydrogel based on environmental cues such as pH, salt, and temperature including physiological conditions. The hydrogel is composed of a network of fibrils that are 3 nm wide and heavily branched and entangled with no covalent crosslinking required for gel stiffness. By controlling hydrogel self-assembly kinetics, one dictates the ultimate stiffness of the resultant network and the kinetics through which gelation occurs.Importantly, once formed into a solid, self-supporting gel the network can be disrupted by the introduction of a shear stress. The system can shear thin but immediately reheal to preshear stiffness on the cessation of the shear stress. This shear thinning behavior of these physical networks makes them interesting candidates for injectable delivery in vivo where no post injection chemistry is required to set up the network. Initially, 2D cultures of several cell lines including progenitor osteoblasts proved that the hydrogel is nontoxic and sustains cellular attachment with or without serum proteins without altering the physical properties of the hydrogel. The cell-material interaction is normal in 2-D and so was extended into 3D by cell encapsulation. Cells were observed to remain viable in 3-D culture for extended periods of time. Peptides for folding and self-assembly, self-assembly characterization, gel material properties, shear thinning and rehealing, and cell-level biological properties of these peptide hydrogels will be discussed.In addition, the peptide fibrils can be used to template the growth of inorganic materials as well as the assembly of inorganic nanoparticles. The study of the hybrid assembly process with the above mentioned beta-hairpin peptides as well as with new polypeptides will be discussed.
3:30 PM - NN5.4
Designing Enzyme-triggered Hydrogels from Self-assembling Peptides.
Jean-Baptiste Guilbaud 1 , Aline Miller 1 , Alberto Saiani 2
1 Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester United Kingdom, 2 School of Materials, The University of Manchester, Manchester United Kingdom
Show AbstractIn recent years, self-assembly and stimuli-responsive materials have received considerable attention due to their potential applications in drug delivery, bio-sensing or regenerative medicine. In this context the choice of building block is crucial to allow the design of structures with controlled geometry and properties. While such supramolecular structures are traditionally fabricated from high molecular weight amphiphilic polymers, peptides have emerged as alternative building blocks due to their propensity to self-assemble into ordered supramolecular architectures. Being able to trigger the self-assembly of these molecules by an external stimuli such as pH, ionic strength, light or enzyme is attracting increasing attention as a route to control the fabrication of novel soft-solid biomaterials. Such a fabrication route is particularly attractive for 3D tissue engineering applications offering the opportunity to incorporate cells into a 3D network by triggering the sol-gel transition.Herein we focus on the enzymatic triggered gelation of ionic oligopeptides containing the alternating hydrophobic/hydrophilic and charged/non-charged amino acids F, E and K. Octapeptides containing these amino acids are known to self-assemble into β-sheet rich fibrillar networks at low concentrations (15-20 mg ml-1 depending on the sequence). Our aim was to generate self-assembling oligopeptides from soluble, short precursor peptides (namely FEFK) simply by adding enzyme, thus inducing a sol-gel transition. Thermolysin, a protease enzyme known to specifically catalyze the hydrolysis of peptide bonds containing hydrophobic amino acids (F), was selected as it has been shown that proteases can be encouraged to work in reverse hydrolysis when the reaction product is thermodynamically stabilized relative to its precursors. Solutions of FEFK were prepared by dissolving the tetrapeptide in water and adjusting the pH to 7 prior to the addition of thermolysin. At low tetrapeptide concentrations (≤ 50 mg mL−1), the sample remained in the liquid state. At high concentrations (≥ 60 mg mL−1), clear, self-supporting gels formed; the higher the concentration, the faster the gelation. The resulting systems have been characterised using MALDI-TOF MS, HPLC, TEM, oscillatory rheometry, FTIR and SAXS/SANS. This revealed that a dynamic library of peptides formed over time, with octapeptides forming preferentially. Once formed the octapeptide sequences assembled into β-sheet rich nanofibers that subsequently entangled triggering the sol-gel transition. This method opens up the possibility of producing hydrogels from a diverse range of short peptides with no harsh chemicals and with only water as the by-product.
3:45 PM - NN5.5
Conductivity of Peptide Nanotube Networks via Enzyme Triggered Self-assembly.
Haixia Xu 1 , Ping Xiao 1 , Ian Kinloch 1 , Rein Ulijn 2
1 The School fo Materials, The University of Manchester, Manchester United Kingdom, 2 West CHEM/Department of Pure and Applied Chemistry, The University of Strathclyde, Glasgow United Kingdom
Show AbstractThe design of organic conductive nanowires prepared by molecular self-assembly of oligopeptides coupled to aromatic components is an exciting challenge for next generation electronic devices. For effective charge transport, it is imperative that precisely controlled, tunable geometries with very few defects can be accessed. Exploring conductivity of nanotubes by enzyme-instructed molecular self-assembly will help understand and control the development of organic electronic materials which have been employed in the study of biological systems due to the prevalence of electrical signals.1 Here, we demonstrate that the use of enzymatic reactions to convert precursors into hydrogelators that self-assemble into nanotubes with Beta-sheets under aqueous conditions.2 One system of aromatic short peptide derivatives has been illustrated is that of fluorenylmethoxycarbonyl-L-leucine-L-leucine-L-leucine (Fmoc-L3),3 gives rise to nanotubular networks by Pi-stacking interactions in between Fmoc groups which is analyzed both from molecular dynamics simulations and wide angle x-ray scattering (WAXS). We used a combination of spectroscopy, transmission electron microscopy (TEM), Cryo-TEM, and atomic force microscopy (AFM) to characterize the structures of the gel. A complex impedance plot (or Cole-Cole plot) was obtained to analyze the charge transport properties of dried networks based on peptide nanotubes. The nanotubes formed by enzyme-triggered self-assembly with out-diameter and inner-diameter of around 25 nm and 9 nm, which are in agreement with the proposed model, presented here exhibit structural stability and significant electronic conductivity in vacuum. The impedance spectrum characteristics show electroconductivity of 1.5 × 10-2 s/m in air, 6 × 10-5 s/m in vacuum (pressure: 1.03 mbar) at room temperature. We describe that this kind of new Pi-Beta nanostructure peptides nanomaterials has reproducibility and uniformly on electronic conductivity in vacuum, which could be applied to interfacing biology with electronic devices resulting from Pi-Pi stacked Fmoc with amino acids within the nanotubes.References1. D. A. Stone, L. Hsu, S. I. Stupp, Soft Matter, 2009, 5, 1990-1993.2. A. M. Smith, R. J. Williams, C. Tang, P. Coppo. R. F. Collins, M. L. Turner, A. Saiani, R. V. Ulijn, Advanced Materials, 2008, 20, 37. 3. A. K. Das, R. F. Collins, R. V. Ulijn, Advanced materials, small, 2008, 4, 279.
4:30 PM - **NN5.6
Self-assembling Peptides: Biomimetic Fabrication and Application.
Xiaojun Zhao 1
1 Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan Univ, Chengdu, Sichuan Province, China
Show AbstractTwo complementary strategies can be employed in the fabrication of molecular biomaterials. In the ‘top-down’ approach, biomaterials are generated by stripping down a complex entity into its component parts. This contrasts with the ‘bottom-up’ approach, in which materials are assembled molecule by molecule and in some cases even atom by atom to produce novel supramolecular architectures. The latter approach is likely to become an integral part of nanomaterials manufacture and requires a deep understanding of individual molecular building blocks, their structures, assembling properties and dynamic behaviors. Two key elements in molecular fabrication are chemical complementarity and structural compatibility, both of which confer the weak and noncovalent interactions that bind building blocks together during self-assembly. Significant advances have been achieved at the interface of biology and materials science, including the fabrication of nanofiber materials for tissue engineering and regenerative medicine. Molecular fabrications of nanobiomateirals have fostered diverse scientific discoveries and technological innovations.
5:00 PM - NN5.7
Self-assembled Peptide Microgels for Cell Encapsulation.
Ye Tian 1 2 , Jason Devgun 1 , Joel Collier 1
1 Department of Surgery, University of Chicago, Chicago, Illinois, United States, 2 Biomedical Engineering, Illinois Institute of Technology, Chicago, Illinois, United States
Show AbstractOne of the advantages of biomaterials created by peptide self-assembly is that their gelation can be triggered in any container to provide objects of defined shape and size. In this work, we induced the self-assembly of fibrillizing peptides in the aqueous phase of water-in-oil emulsions to produce spherical microgels with controlled sizes and compositions, a process which was cytocompatible and capable of encapsulating cells with high viability levels. Micron-scale gels, particles, and beads are increasingly utilized for biomedical applications such as cell delivery and controlled therapeutic release, but in many cases these gels are produced using covalent chemistries, solvents, temperatures, and other conditions that are not highly cytocompatible. Here the extreme salt-sensitivity of beta-sheet fibrillization enabled the rapid production of cell-laden microgels in physiologically benign conditions. Peptides utilized included the previously reported self-assembling Q11 peptide (QQKFQFQFEQQ), RGD-Q11 (GGRGDSGGGQQKFQFQFEQQ) and a fluorescent nitrobenzoxadiazole (NBD)-RGD-Q11 derivative. Peptides were dissolved in water and mixed with USP mineral oil using either a rotor/stator homogenizer or a paddle-type mixer with variable mixing times and shear rates. Triggering of self-assembly was achieved by adding phosphate buffered saline (PBS) to the stirred homogenate. The spherical microgels were collected by extraction in PBS, where they were stable for at least several weeks. Microgels were stained either with Congo red or through the incorporation of NBD-RGD-Q11, and microgel dimensions and quantities were measured using epifluorescence microscopy. Average particle size could be controlled on the order of 1-100 microns by adjusting the blade type, blade speed, and a post-gelation shear step, and polydispersity indexes as low as 1.22 were achieved. To encapsulate cells, NIH 3T3 fibroblasts were suspended in pre-assembled peptide solutions containing 10% sucrose for osmotic balancing. Cell/peptide mixtures were then emulsified as described above, and the cell-laden microgels were collected in culture medium. Cell survival was quantified using calcein AM and ethidium homodimer, and viabilities greater than 80% were achieved and maintained for at least three days. Finally, formed microgels could be suspended in un-assembled peptide solutions and overlaid with PBS to produce microgels of one peptide composition embedded in a matrix having a different peptide composition. In this way, 3D cultures with spatially defined distributions of cells and matrix components could be created. Such materials will be useful for the construction of precise 3D cultures for investigative biology, and also for applications in regenerative medicine.
5:15 PM - NN5.8
Using Peptide Hetero-assembly to Trigger Physical Gelation and Cell Encapsulation.
Andreina Parisi-Amon 1 , Cheryl Wong Po Foo 2 , Ji Seok Lee 2 , Widya Mulyasasmita 1 , Sarah Heilshorn 2 1
1 Bioengineering, Stanford University, Stanford, California, United States, 2 Materials Science and Engineering, Stanford University, Stanford, California, United States
Show AbstractPhysical hydrogels, characterized by transient crosslinks and shear-thinning behavior, are ideal vehicles for minimally invasive cell transplantation. Most "smart" hydrogels rely on non-physiological environmental changes to enable fine-tuning of the sol to gel phase transition and the resulting viscoelastic properties. Furthermore, use of these hydrogels for cell encapsulation often requires momentarily exposing cells to low temperature or pH, which can be irreversibly detrimental to encapsulated cells and proteins as well as difficult to reproducibly control in a clinical setting.In response, we utilized a molecular recognition strategy to design MITCH (Mixture-Induced, Two-Component Hydrogels) that hetero-assemble upon mixing at constant physiological conditions via hydrogen bonding. MITCH consist of two recombinantly engineered block-co-poly-peptides containing repeats of WW and proline-rich peptide domains interspersed with random coil segments. Applying hydrogel percolation theory, we tailored two design variables, the domain association energy and the domain repeat frequency, to synthesize hydrogels with tunable viscoelasticity. Our results demonstrate that design choices at the molecular-level can predictably alter the hetero-assembled network structure and hence hydrogel rheology.The synthesis of these precisely designed polymers was achieved using recombinant protein technology to encode each primary sequence in an exact modular genetic construct. Circular dichroism was employed to verify that the association domains properly fold when fused to hydrophilic random coils on their C- and N-termini. Separately, the individual block-co-poly-peptides exhibit Newtonian fluid behavior and cannot self-assemble; however, upon simple mixing, the polymers hetero-assemble via hydrogen bonding to form a percolating network at constant physiological pH, temperature, and ionic strength. Gels form within ~10 seconds, are shear-thinning and injectable, and re-form after removal of shear to their original viscoelasticity. The gels exhibit plateau storage moduli, G’, in the range of 10-100 Pa, similar to common biological hydrogels such as Matrigel. Rheology measurements demonstrate that tuning the domain association energy and the domain repeat frequency provides a direct link between molecular-level design of the polymeric network and macroscopic material properties.Assembly by specific molecular-recognition renders the viscoelasticity of MITCH unaffected by media compositions, ensuring reproducibility across cell-culture systems. Human endothelial cells, rat mesenchymal stem cells, and rat neural stem cells have been successfully cultured in 3D MITCH gels. Furthermore, MITCH materials promote the growth and differentiation of rat neural stem cells into glial and neuronal phenotypes, which adopt a 3D-branched morphology, with neurites often extending over hundreds of microns.
5:30 PM - **NN5.9
Rational Peptide Design in Nanoscience and Synthetic Biology.
Zahra Mahmoud 1
1 School of Chemistry, University of Bristol, Bristol United Kingdom
Show AbstractThe rational design of peptides that fold and self-assemble into nano-to-micron structured materials is an exciting challenge. Such efforts test and extend our understanding of sequence-to-structure relationships in proteins, and potentially provide materials for applications in the areas of bionanotechnology and synthetic biology.Over the past decade, rules for the folding and assembly of one particular protein-structure motif—the α-helical coiled coil—have advanced sufficiently to allow the de novo design of peptides that fold to prescribed structures (1). As such, they present excellent starting points for building complex objects and materials that span the nano-to-micron scales from the bottom up. In this talk, I shall briefly outline the rules for the folding and assembly of coiled coil motifs and illustrate how we have applied these rules in the rational de novo design of fibrous biomaterials (2), peptide-based switches (3) and discrete nanoscale objects. A major part of the talk will focus on the engineering of coiled-coils to form hydrogels with >99% water content (4), and their functionalisation as scaffolds to direct cell growth and differentiation. Using a two-peptide hydrogel system gives us considerable control over assembly, additional advantages are that it is proteinogenic and engineerable. In the second part of the talk I will focus on potential application areas of de novo design in synthetic biology (5) including generation of discrete, closed peptide nanostructures by controlling coiled-coil oligomerization states.1.Woolfson, D.N., The design of coiled-coil structures and assemblies. Adv Prot Chem, 2005. 70: 79-112.2.Woolfson, D.N. & M.G. Ryadnov, Peptide-based fibrous biomaterials: some things old, new and borrowed. Curr Opin Chem Biol, 2006. 10: 559-567.3.Cerasoli, E., B.K. Sharpe, & D.N. Woolfson, ZiCo: A peptide designed to switch folded state upon binding zinc. J Am Chem Soc, 2005. 127: 15008-15009.4.Banwell, E.F. et al., Rational design and application of responsive α-helical peptide hydrogels. Nature Mater 2009. 8, 596-600.5.Bromley, EHC et al. Designed α-Helical Tectons for Constructing Multicomponent Synthetic Biological Systems. J. Am. Chem. Soc. 2009. 131, 928-930.