AVS2004 Session BI+NS-TuM: The Nano-Bio Interface

Tuesday, November 16, 2004 8:20 AM in Room 210D

Tuesday Morning

Start	Invited?	Item
8:20 AM	Invited	BI+NS-TuM-1 Programmed Macromolecular Synthesis D.A. Tirrell (Caltech) We have developed three approaches to the synthesis of proteins and protein-like macromolecules containing novel amino acids. In the first approach, we replace every copy of one of the natural amino acids by an analogue, in effect building proteins from an altered set of twenty starting materials. This approach is most useful when one is interested in changing the overall physical properties of the protein, or in de novo design of protein-based biomaterials. A second method, which has also been implemented successfully by Schultz and coworkers, allows site-specific incorporation of a single copy of an amino acid analogue in response to a stop codon. Such methods are useful in probing protein structure and function. The third approach, developed most recently, uses mutant transfer RNAs to break the degeneracy of the genetic code, and offers the prospect of a protein chemistry based on a substantially expanded set of amino acid building blocks. This lecture will describe the most important elements of each of these strategies as well as some thoughts on the design of wholly artificial proteins with potential application in biotechnology and materials science.
9:00 AM	Invited	BI+NS-TuM-3 The Art of Mechano-Transduction within the Extracellular Matrix V. Vogel (Swiss Federal Institute of Technology (ETH), Switzerland) While engineered matrices allow asking well defined questions of how cells interact and respond to their environment, it remains unclear whether a minimal set of cues exists by which synthetic matrices can be engineered that mimics biological matrices in their essential functions. Here we address how mechanical force can alter the conformation of extracellular matrix proteins and consequently regulate the display of the protein's functional states. The function of cells is tightly controlled by their interaction with the surrounding extracellular matrix to which they are coupled via the transmembrane integrins. Using intramolecular fluorescence resonance energy transfer (FRET), we studied the extent to which fibronectin is stretched and partially unfolded by the traction forces generated by fibroblasts in 2d and 3d matrices. We then derive structural models of the unfolding pathways of ECM proteins by computational techniques (steered molecular dynamics simulations), and gain insight how tension applied to extracellular matrix proteins affects the exposure of their molecular recognition sites. The consequences of our findings to the field of biomaterials and tissue engineering will be discussed. V. Vogel, G. Baneyx, The tissue engineering puzzle: a molecular perspective, Annual Review Biomed. Eng., 5 (2003) 441-463. G. Baneyx, L. Baugh, V. Vogel, Co-existing conformations of fibronectin imaged in cell culture by fluorescence resonance energy transfer, Proc. Natl. Acad. Sci. USA, 98 (2001) 14464-14468. G. Baneyx, L. Baugh, V. Vogel, Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension, Proc. Natl. Acad. Sci. USA, 99 (2002) 5139-5143. D. Craig, M. Gao, K. Schulten, V. Vogel, Structural insights how sequence variations tune the mechanical stability of fibronectin type III modules, Structure, 12 (2004) 21-30.
9:40 AM		BI+NS-TuM-5 Capture and Release of Proteins on the Nanoscale by Surface-Confined Biomolecular Switches J. Hyun (Seoul National University, Korea); W.K. Lee, N. Nath, A. Chilkoti, S. Zauscher (Duke University) In this paper we describe the fabrication and characterization of stimulus-responsive elastin-like polypeptide (ELP) nanostructures grafted onto ï·-substituted thiolates that were patterned onto gold surfaces by dip-pen nanolithography (DPN). We exploited the hydrophilic-hydrophobic phase transition of ELP in response to a change in ionic strength as a switch in order to reversibly immobilize a thioredoxin-ELP fusion protein onto the ELP nanopattern above the lower critical solution temperature. We demonstrated the biological activity of the Trx-ELP nanoarray by binding an anti-thioredoxin monoclonal antibody. Furthermore, we showed that the resulting Trx-ELP/anti Trx-mAb complex could be released below the LCST. Our research demonstrates proof-of-principle that "smart," surface-confined biomolecular switches can be built at the nanoscale. Our method of fabricating switchable surfaces is attractive because it is entirely modular and generic, in that it only requires an ELP-modified or patterned surface and a protein that can be appended with an ELP tag. ELP synthesis is easily achieved through genetic engineering techniques. The nanoscale miniaturization of on-chip separation and the presentation and triggered release of the captured proteins made possible by this methodology should be integrable into nanoscale bioanalytical devices that are currently under development.
10:00 AM		BI+NS-TuM-6 Micro- and Nanopatterns of DNA-Tagged Vesicles B. Städler, D. Falconnet (Laboratory for Surface Science and Technology, Switzerland); F Höök, I Pfeiffer (Chalmers University of Technology, Sweden); H Solak (Paul Scherrer Institute, Switzerland); J. Vörös (Laboratory for Surface Science and Technology, Switzerland) A new approach for the creation of vesicular micro-and nanoarrays is presented based on a novel patterning approach termed Molecular Assembly Patterning by Lift-off (MAPL) in combination with the immobilization of DNA-tagged intact vesicles. This technique is shown to be a promising platform for future studies of enzyme and membrane protein activity in a controlled, native nanoenvironment. Fabrication of DNA microarrays by spotting is state-of-the-art today. This arraying technology, however, cannot be directly applied to membrane-based microarrays because the contact with the ambient environment damages the membranes. Our approach starts with conventional single stranded DNA arrays, which are subsequently converted into a membrane protein array by using phospholipidic vesicles tagged with the complementary DNAs. These functionalized vesicles specifically couple to the surface through hybridization of the DNA strands. The MAPL process was used to provide a surface with a background resistant to the nonspecific adsorption of vesicles and active spots (diameter between 1 and 200 µm) for the immobilization of the single stranded DNAs. The surface chemistry of the active spots and background consisted of biotinylated PEG and non-functionalized PEG, respectively. Complexes of biotin-terminated DNA and neutrAvidin, preformed in solution, were immobilized to the biotinylated, active spots. POPC vesicles tagged with complementary cholesterol-terminated DNA could then be specifically coupled to the surface through the hybridization of the DNA strands. Quartz crystal microbalance and optical waveguide technique were used to monitor in situ and optimize the multistep surface modification process. The micropatterns of DNA-tagged, fluorescently labeled vesicles were investigated by fluorescence microscopy. X-ray Interference Lithography was successfully used to downscale the patterning process to the nanometer scale in order to produce single vesicle arrays.
10:20 AM		BI+NS-TuM-7 Label-Free Biosensor Based on the Surface Plasmon Resonance of Gold Nanoparticles S.M. Marinakos, N. Nath, A. Chilkoti (Duke University) The optical properties of gold nanoparticles immobilized on a surface were used in a label-free biosensing scheme. The sensing modality is based on the change in the local refractive index associated with receptor-ligand binding at the particle surface which shifts the surface plasmon resonance (SPR) peak in the absorbance spectra of the nanoparticles. In previous work, we have shown that solid, spherical gold nanoparticles with a size in the range of 13-50 nm could be self-assembled on amine-functionalized glass. These chemisorbed nanoparticles were then functionalized with a biotin derivative. We showed that this scheme enabled single wavelength monitoring of streptavidin binding at the surface by single wavelength measurements of the change in intensity that was caused by binding of streptavidin at the nanoparticle-solution interface. In this study, we extend these measurements to anisotropic gold nanorods, in an effort to further improve the analytical sensitivity and detection limits of this label-free transmission optical sensor. Results will be presented that compare streptavidin-biotin binding with sensors fabricated from gold nanorods with previous results on spherical gold nanoparticles.
10:40 AM	Invited	BI+NS-TuM-8 Activation of Integrin Function by Nanopatterned Adhesive Interfaces J.P. Spatz, M. Arnold (University of Heidelberg, Germany) To study the function behind molecular arrangement of single integrins in cell adhesion, we designed a hexagonally close-packed rigid template of cell adhesive gold nano-dots coated with cyclic RGDfK peptide by lithographic means of diblock copolymer self-assembly. The diameter of the adhesive dots is <8nm, which allows the binding of one integrin per dot. These dots are positioned with high precision at 28, 58, 73 and 85 nm spacing at interfaces. A separation of >= 73nm between the adhesive dots results in limited cell attachment and spreading and dramatically reduces the formation of focal adhesion and actin stress fibers. We attribute these cellular responses to restricted integrin clustering rather than insufficient number of ligand molecules in cell-matrix interface since "omicro-nanopatterned" substrates consisting of alternating fields with dense and no nano-dots support cell adhesion. We propose that the range between 58-73 nm is a universal length scale for integrin clustering and activation, since these properties are shared by a variety of cultured cells.
11:20 AM		BI+NS-TuM-10 Lifetime of Biomolecules in Hybrid Nanodevices: The Aging Process of Motor Protein-based Molecular Shuttles H. Hess (University of Washington); C. Brunner (ETH Zurich, Switzerland); K.-H. Ernst (EMPA Duebendorf, Switzerland); V. Vogel (University of Washington and ETH Zurich, Switzerland) Prolonging the lifetime of biomolecules in their functional states is critical for applications where biomolecules are integrated into synthetic materials or nanodevices. A simplified molecular shuttle system, which consists of fluorescently labeled microtubules propelled by kinesin motor proteins bound to the surface of a flow cell, served here as a model system for such a hybrid device. In this system, the functional decay can easily be assayed by utilizing optical microscopy to detect motility and disintegration of microtubules (MTs). We found that the lifetimes of these hybrid systems were mainly limited by the stability of MTs, rather than of kinesin. To determine the biocompatibility of polymers widely used in microfabrication, we assembled flow cells with glass bottom surfaces and covers fabricated from glass, poly(urethane) (PU), poly(methyl-metacrylate) (PMMA), poly(dimethylsiloxane) (PDMS), and ethylene-vinyl alcohol copolymer (EVOH). Without illumination, only PU had a substantial negative impact on MT stability, while PMMA, PDMS and EVOH showed stabilities comparable to glass. Under the influence of light, however, the MTs degraded rapidly on PDMS or PMMA. A similar effect was observed on glass if oxygen scavengers were not added to the medium. Strong bleaching of the fluorophores was again only found on the polymer substrates and photobleaching coincided with an accelerated depolymerization of the MTs. The presented data provide a benchmark for the lifetime of motor protein-based bionanodevices which utilize glass as the primary synthetic material, and test the impact of a variety of polymer materials on the longevity of microtubules, the most fragile biological structure in the device. This study demonstrates that our definition of biocompatibility evolves, as we progress towards architectures engineered on a molecular level, which integrate multimeric proteins and protein assemblies.
11:40 AM		BI+NS-TuM-11 Analysis of Collision Events of Self-Propelled Biomolecular Shuttles Carrying Cargo B.C. Bunker, A.K. Boal, S.B. Rivera, G.D. Bachand (Sandia National Laboratories) Collision events between cargo carrying biotinylated microtubules (MTs) laden with 0.56 mm diameter streptavidin coated polystyrene beads (SBs) while being transported across kinesin coated surfaces were observed. Six distinct actions resulted from such collisions: no interaction, SB transfer between MTs, one MT deforming as a result of the collision, co-joining of the two MTs through mutual attachment to the SB, the SB being dislodged from the MT, or one of the MTs being severed. Interactions were studied both as a function of percent biotin-tubulin used to prepare the MTs and temperature. While biotin percent was observed to have a negligible effect of the percent chance of the various outcomes, heating the system from 24ËsC to 30ËsC decreased the likelihood of a SB transfer event while increasing the rate of MT bending and dragging events. Two important factors are proposed to determine the outcome of these collisions: the geometry of the collision event and the nature of the binding site that the SB is originally attached to.