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Inspired by Biology: From Molecules to Materials to Machines 4 Probes and Tools for Biomolecular Materials Research This chapter describes experimental and computational tools that promise to extend knowledge and understanding of biology and biomacromolecular materials in the next decade and beyond. Structural, biochemical, and physiological studies relate the motions and dynamics of these materials to particular cellular functions. A further motivation for these studies is the expectation that the process of learning how work is performed at the very small subcellular scales should pave the way for the development of functional biomolecular materials in future nanotechnological applications. Biological systems consist of collections of interacting molecules (proteins, carbohydrates, lipids, and nucleic acids) that give rise to a variety of supramolecular complexes with hierarchical structures spanning sizes from angstroms to micrometers. For example, the mechanical and structural properties of filamentous (F)-actin, one of the three components of the cytoskeleton in eukaryotic cells, is an area of intense research. F-actin further assembles to form either bundles, or loosely packed, two-dimensional and three-dimensional network structures in cells. The distinct functions resulting from these highly regulated structures interacting with other biomolecules and motors include cell shape and mechanical stability, cell adhesion and motility, and cell cytokinesis, the splitting of the cell body into two daughter cells during division. Special techniques are required to span spatial and temporal ranges appropriate to the functions of these networks. While macromolecular crystallography has revolutionized our understanding of protein structure and function, the technique requires high-quality crystal samples. However, many proteins (notably membrane proteins) are difficult to crystallize, and the molecular basis of cellular function usually involves interactions
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Inspired by Biology: From Molecules to Materials to Machines among more complex and highly disordered biological complexes (for example, the immunological and neuronal synapses that mediate cell-cell communication). Elucidation of the molecular details of the structure and dynamics of such noncrystalline supramolecular complexes requires continual advancement in the development of techniques to solve these biological problems. The techniques should also apply equally well to the broader class of man-made biomolecular materials. The new experimental probes of biomolecular materials are expected to have a major impact on future science and technology, including imaging methods based on novel optical and electron microscopic techniques, new synchrotrons, and X-ray free electron lasers, which provide ultrashort and extremely intense pulses. There are significant synergistic advances in the development of instrumentation to probe the physical, chemical, structural, and dynamical behavior of individual molecules, so-called “single-molecule biophysics.” It must be emphasized that biomolecular materials are naturally complex, often containing many components, inhomogeneous characteristics, and fluctuations. These features make it difficult to intuit principles from experimental observations alone. Thus it is important to combine experimental techniques that provide crucial observations with powerful numerical simulations and insightful analytical modeling to elucidate the mechanisms underlying biomolecular processes. Theory and computation can be critical to the discovery and design process because they can be used to predict the consequences of different mechanistic hypotheses, interpret experimental results, and examine alternative design motifs for biomaterials. Some of the potential advances to be achieved from next-generation experimental tools and computational methods inspire research efforts. Imagine that one could … View cells in atomic to molecular detail and at a time resolution of milliseconds, appropriate to observe the events during neuronal synaptic transmission or chromosome replication. Measure the forces and motions of nanobiomachines directly and on the millisecond to microsecond timescale. Determine the sequence of an individual DNA molecule, providing the ultimate sensitivity for forensic purposes or diagnosing inherited disease. Understand how biological machines conquer the chaotic and crowded conditions of the cell. Use principles of biological recognition to design new molecular interactions ab initio. Predict how molecules with a specific sequence of monomers will adopt a specific conformation and self-assemble into precise supramolecular structures.
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Inspired by Biology: From Molecules to Materials to Machines Use cell-mimetic cooperativity to design biosensors that can detect minute amounts of hazardous substances without noise-induced false positives. Describe systems far from equilibrium with a rigorous theoretical framework. THREE-DIMENSIONAL ELECTRON MICROSCOPY Electron microscopy imaging is an indispensable research tool of modern materials science, biology, and biomolecular materials. As compared to nuclear magnetic resonance (NMR) spectroscopy or macromolecular crystallography, which are used primarily for elucidating the structure of single protein molecules at angstrom resolution, three-dimensional cryo-electron microscopy (cryo-EM) is emerging as a powerful tool for capturing images, albeit at lower resolution, of directed- or self-assembled collections of biological molecules, complexes, and machines that function in concert. The most widely used method of three-dimensional electron imaging is cryo-electron tomography (cryo-ET), which consists of three-dimensional reconstruction of an object from a series of tilt projections. In an alternative method, referred to as single-particle cryo-EM, a three-dimensional reconstructed image is obtained from the superposition of a very large number (20,000 to nearly 300,000) of particle images. The latter method is facilitated if the particle (e.g., an assembled molecular machine or a viral capsid) is monodisperse, so that all of the two-dimensional projected images used in the reconstruction can be assumed to be images of a single particle viewed from different orientations. On the other hand, to image the interior architecture of cells, where no two cells are ever identical, requires cryo-ET. Figure 4.1 shows an example of a three-dimensional reconstruction of an in vitro assembled infectious P22 bacteriophage virion, a large asymmetric complex at 17 Å resolution, from the superposition of 26,442 particle images. The biology of bacteriophage P22, which infects Salmonella enterica, and its molecular-level mechanisms of action have been studied extensively by scientists worldwide as a model for virus assembly. In particular, studies are beginning to elucidate the noncovalent virus assembly pathway and the mechanism by which the double-stranded (ds) DNA genome is actively packaged followed by reinfection pathways and release of viral genome. The elucidation of the P22 structure at this resolution has revealed a likely mechanism for the late stages of dsDNA packaging and the sequence-independent switch, which senses that the virion has reached the correct physical packing density and signals termination of the genome packaging process. Cryo-ET appears poised to become a key tool for creating three-dimensional images of directed assembly within inner cell structures in the next decade and beyond. In particular, cryo-ET is expected to be important for elucidating the spatial and temporal (via imaging of vitrified cells as a function of time during a
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Inspired by Biology: From Molecules to Materials to Machines FIGURE 4.1 The structure of an assembled infectious P22 bacteriophage virion as revealed by three-dimensional cryo-EM at 17 Å resolution. The coat protein is shown in blue. The tail machinery complex, composed of multiple copies of four gene products (mustard, green, yellow, pink) exhibits 6-fold and 12-fold symmetry and is located at a single 5-fold vertex of the capsid. SOURCE: G.C. Lander, L. Tang, S.R. Casjens, E.B. Gilcrease, P. Prevelige, A. Poliakov, C.S. Potter, B. Carragher, and J.E. Johnson, “The structure of an infectious P22 virion shows the signal for headful DNA packaging,” Science 312:1791 (2006). particular cell function) distribution of supramolecular assemblies of biological complexes, molecular machines, and motors. Figure 4.2 shows images of the actin cytoskeleton in the slime mold Dictyostelium discoideum using state-of-the-art cryo-ET. In order to appreciate the profound benefits that may be expected from cryo-ET as it becomes more readily available, one has only to think about the medical era before and after the availability of computed tomography (CT) scanners and
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Inspired by Biology: From Molecules to Materials to Machines FIGURE 4.2 (A) Transmission electron micrograph of a peripheral region of the vitrified slime mold Dictyostelium discoideum. The thickness of the cell in this region was between 200 nm and 350 nm. (B) The cell-cytoskeletal architecture in a section of the slime mold visualized by three-dimensional reconstructions from cryo-ETs. The three-dimensional reconstruction resulted from a series of tilt projections of the region marked by the square vertices in (A). A partial cell volume 97 nm thick was used to produce this image at 5-nm resolution. The dense network of actin filaments is shown in red, ribosomes in green, and membrane in blue. A portion of the plasma membrane is seen in the top left corner and circular membrane vesicles are also seen in the lower part (center and to the left). The membrane vesicle in the lower middle part (corresponding to the white square region in (A)) appears to be decorated by ribosomes, thereby corresponding to a section of the rough endoplasmic reticulum. SOURCE: O. Medalia, I. Weber, A.S. Frangakis, D. Nicastro, G. Gerisch, and W. Baumeister, “Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography,” Science 298:1209-1213 (2002). magnetic resonance imaging (MRI) machines in clinical imaging. CT and MRI machines have allowed physicians to peer noninvasively into the human body, permitting precise three-dimensional visualization of organs and tissue and allowing, for example, rapid diagnoses for stroke and accident patients. The majority of CTs and MRIs currently in use are able to visualize objects about 1 cubic millimeter in size, a useful scale for clinical diagnosis. By the same token, cryo-ET and single-particle cryo-EM should allow researchers to visualize cells, their organelles, and the crucial biomolecular assemblies at a near molecular resolution, the appropriate spatial dimension to uncover their functional behavior.
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Inspired by Biology: From Molecules to Materials to Machines HYPERRESOLUTION OPTICAL MICROSCOPY Light microscopy, especially fluorescence microscopy of specifically tagged macromolecular components, has become the mainstay technique of cell biology. Its advantages are relative ease of implementation, tremendous adaptability and facility for labeling specific proteins and nucleic acids, and real-time and three-dimensional imaging. A big disadvantage with which optical microscopy normally contends is its limited spatial resolution relative to molecular dimensions. The spatial resolution for discriminating two objects in a conventional optical microscope is limited to about 250 nm owing to diffraction of the light by the imaging objective. Of course, many cellular components are much smaller than that. Several new techniques have been described that overcome this diffraction barrier in optical microscopy. The first general method is termed “structured light.” It is possible to form a spot of light or a grid with greater resolution by various nonlinear techniques. One such approach, called stimulated emission depletion (STED) microscopy, uses pairs of precisely timed and shaped laser pulses to generate an excitation area—point spread function (PSF)—much narrower than the standard 250 nm wide diffraction-limited spot. Figure 4.3 shows a schematic diagram of the optical arrangement and the mechanism for narrowing the PSF. For the fluorescent probe Atto532, a blue laser (470 nm) excites fluorophores with the usual diffraction-limited spatial distribution. A few nanoseconds later, an orange (670 nm) laser pulse triggers stimulated emission to quench the excited state of most of the fluorophores. This quenching is a reversible process, and the individual fluorophores can be excited and deexcited hundreds of times. The quenching beam is made donut-shaped by phase modulation, allowing probes only at the center of the original spot to remain excited. The reason the remaining PSF is so narrow is that de-excitation saturates abruptly at zero excitation (negative excitation does not occur) and a very intense STED pulse switches all of the probes except those within a few nanometers of the center. In Figure 4.3, the resolution achieved is 66 nm. There is no intrinsic limit to this narrowing, and 20 nm resolution has been demonstrated. Gains in axial resolution are also achieved by using two converging objective lenses on opposite sides of the specimen, and irreversible photobleaching is minimized by allowing triplet states to relax between excitation-quench pulses. Images at high resolution are obtained by raster-scanning the sharp PSF excitation spot over the sample and sequentially collecting the fluorescence emission from each spot to reconstruct the distribution of fluorescent probes in the specimen. In one experiment, synaptotagmin-labeled synaptic vesicles were resolved much more clearly in STED images (Figure 4.3c) than by standard confocal microscopy (Figure 4.3b). Fluorophores can be switched on and off by photophysical means other than
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Inspired by Biology: From Molecules to Materials to Machines FIGURE 4.3 Schematic diagram showing (a) operation of stimulated emission depletion (STED) microscopy, (b) images of fluorescent-labeled synaptic vesicles using standard confocal microscopy, and (c) same vesicles imaged with STED microscopy. Scale bars are 500 nm for (b) and (c). SOURCE: K.I. Willig, S.O. Rizzoli, V. Westphal, R. Jahn, and S.W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440:935 (2006). stimulated emission. Certain fluorescent proteins, organic dyes, and pairs of closely spaced cyanine dyes are photoswitchable between fluorescent and nonfluorescent metastable states using two different wavelengths. Again, switching these probes off is saturable, allowing the equivalent of STED narrowing of the PSF but at much lower laser intensities. The disadvantage of fluorochrome switching is that the image collection times are much longer.
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Inspired by Biology: From Molecules to Materials to Machines Individual fluorescent molecules can be localized to within a few nanometers by collection of sufficient photons and fitting an appropriate kernel function to find the center of their PSF. Repeatedly switching a few single fluorophores on and off so that they are spatially separated allows determination of the position of each one at nanometer precision. After many cycles of excitation and reversible quenching, the overall spatial distribution emerges. Various versions of this scheme have been termed photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). This group of special fluorescence microscopic techniques and further developments are highly likely to accelerate understanding of spatial distributions, dynamics, and signal transduction in a broad range of molecular and cell biological problems. X-RAY METHODS The discovery of X-ray diffraction from crystals by von Laue and Bragg nearly 100 years ago marked the beginning of developments for visualizing the three-dimensional atomic structures inside crystals. Indeed, X-ray crystallography has since made a tremendous impact in materials sciences, physical sciences, and biology. It has now reached a point where, as long as appropriate high-quality crystals are obtained, it can determine any structure. However, many biological samples such as whole cells, organelles, viruses, and many important protein molecules are difficult or impossible to crystallize and are hence not accessible to crystallography. Currently, there are two successful approaches to high-resolution, full-field X-ray imaging of noncrystalline samples: one that uses a high-resolution lens and another that does not use a lens but requires coherent illumination. Both of these imaging techniques have demonstrated rapidly improving resolution: 15-30 nm. While the first approach requires a high-resolution X-ray lens similar to that of a standard optical microscope, it does not require a source with high degree of coherence. In fact, using a laboratory X-ray source, sub-50-nm-resolution, three-dimensional imaging has been achieved. The second approach does not require an X-ray lens but requires a source with a high brilliance, such as a third-generation synchrotron source or the upcoming fourth-generation free-electron X-ray laser source. Newly developed X-ray imaging techniques are expected to have a major impact on biomolecular materials research by facilitating fundamentally new ways of characterizing events at the nanometer scale and also as a function of time. In addition to bridging the resolution gap between optical and electron microscopy, they offer many unique capabilities resulting from the high penetration power (at short wavelengths) of X-rays for nondestructive and time-lapse imaging. Using nanoparticles as markers, X-ray three-dimensional cryotomography with nano-
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Inspired by Biology: From Molecules to Materials to Machines meter resolution will allow the study of many important biomolecular processes. Nondestructive three-dimensional tomography will play an important role in the development of future-generation nanostructured biomolecular materials with the desired chemical, mechanical, and functional properties. X-ray Tomography A schematic illustration of a lens-based X-ray full-field tomographic imaging microscope is shown in Figure 4.4 (left). It consists of an X-ray source, a high-efficiency condenser lens focusing X-rays onto the sample, a high-precision rotation stage, an objective zone plate lens, and a high resolution charge-coupled device detector. Spatial resolution better than 15 nm has been demonstrated with 8 keV synchrotron X-rays. Using a laboratory X-ray source, a full field X-ray microscope with 50 nm resolution has recently been developed. High-resolution X-ray tomography opens up new avenues to nondestructively explore the internal structure of optically opaque solids with nanometer-scale resolution, previously not possible with other analytical techniques. As a proof of concept, Figure 4.4 (middle) shows a rendered three-dimensional image of an advanced copper-interconnect-based integrated circuit (IC) chip with 120-nm feature size. FIGURE 4.4 (Left) Schematic of a zone-plate-based full-field three-dimensional X-ray microscope operating in phase contrast mode. The principle of operation is very similar to that of a visible light microscope, where the visible light source is replaced by the commercially available X-ray source and the glass lenses are replaced with equivalent X-ray imaging optics. The Zernike phase ring alters optical path length for some scattered X-rays to obtain contrast by constructive/destructive interference with nonscattered rays. (Middle) An example of three-dimensional X-ray tomography with 120-nm feature size: volume rendering of a modern IC chip with sub-50-nm spatial resolution obtained with a laboratory-based X-ray microscope. (Right) Malaria-infected red blood cells imaged with a laboratory hard X-ray microscope (8 keV) in phase contrast mode. Dark protrusions from the cell boundaries are indicative of infection by malaria parasite. SOURCE: Wenbing Yun, Xradia, Inc.
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Inspired by Biology: From Molecules to Materials to Machines To fully utilize the high penetration power and to image weakly absorbing objects, such as biological cells, the phase contrast technique has been applied to full-field imaging X-ray microscopy. For structures containing mostly low atomic number elements, such as biological specimens, phase variations provide much more contrast than absorption, especially for X-ray energies greater than a few keV. Figure 4.4 (right) shows an image of malaria-infected red blood cells obtained from a laboratory X-ray microscope operating at 8 keV in the phase contrast mode. In the next decade and beyond, the spectral tunability and high brilliance of third-generation synchrotron X-ray sources will allow three-dimensional X-ray microscopy techniques to be employed to answer many key questions in biomolecular materials and processes: What are the elements? How are they arranged? What is their chemical nature? and What is the nature of the defects and how do they alter function? X-ray Diffraction A second X-ray based approach making rapid progress is the use of coherent X-ray diffraction to provide three-dimensional images of noncrystalline systems without requiring a focusing lens. When a coherent wave of X-rays illuminates a noncrystalline specimen, the far-field diffraction intensities are continuous and weak. This continuous diffraction pattern can be sampled at a frequency finer than the inverse of the specimen size (that is, oversampled), which corresponds to surrounding the electron density of the specimen with a no-density region: the higher the sampling frequency, the larger the no-density region. It has been shown that when the no-density region is larger than the electron density region, the phase information is uniquely encoded in the diffraction pattern and can be recovered directly by an iterative process that takes advantage of the knowledge that the electron density outside the object is zero and within the object is positive. The first successful experimental demonstration of coherent diffraction imaging was carried out in 1999. Since then, it has been successfully applied by several groups for imaging a variety of samples ranging from nanocrystals and biomaterials to double-walled carbon nanotubes. In a recent proof-of-principle experiment, coherent X-ray diffraction imaging was used to study E. coli, a eubacterium of typical size 0.5 × 2 μm, transformed with a recombinant yellow fluorescent protein (YFP) that could be marked by potassium permanganate precipitates. The detected densities bear a strong resemblance to the pattern of fluorescence seen from comparable bacteria examined with the confocal microscope (Figure 4.5). Since no X-ray lenses are needed in this technique, the resolution of coherent X-ray diffraction imaging is only limited by radiation damage to the samples. X-ray experiments have indicated that radiation damage can be greatly reduced by
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Inspired by Biology: From Molecules to Materials to Machines FIGURE 4.5 Coherent X-ray imaging of E. coli bacteria. Left: An image directly reconstructed from a coherent X-ray diffraction pattern. The dense regions inside the bacteria indicate the distribution of His-tagged YFP labeled with KMnO4 precipitates. The semitransparent regions are devoid of YFP. Right: Individual bacteria are seen using transmitted light (A, D) and fluorescence (B, E), where the YFP (green) is seen throughout most of the bacteria except for one small region in each bacterium that is free of fluorescence (arrows). C and F show the fluorescent image superimposed on the transmitted light image. SOURCE: J. Miao, K.O. Hodgson, T. Ishikawa, C.A. Larabell, M.A. LeGros, and Y. Nishino, “Imaging whole Escherichia coli bacteria by using single-particle X-ray diffraction,” Proceedings of the National Academy of Sciences USA 100:110 (2003). freezing the samples to liquid nitrogen temperatures and sub-10-nm resolution should be achievable. X-ray free electron lasers also offer promising prospects with their ultrashort and extremely intense pulses. In this case, radiation damage could be circumvented by recording the diffraction pattern from single macromolecules before they are destroyed. By using many identical copies of the molecules, a three-dimensional diffraction pattern could be assembled, which could then be directly converted to an image by using phases recovered by oversampling. Small-Angle X-ray Scattering Synchrotron techniques of small-angle X-ray scattering (SAXS) are important structural probes of disordered or partially ordered nanostructured complex substances, including biomolecular materials. Owing to its relative ease of use and availability at several synchrotron X-ray sources worldwide, SAXS is a primary method for in situ studies of phase behavior, intermacromolecular interactions, structures, and the kinetics of formation of bioassemblies on multiple length scales, from nanometers to micrometers. Among the many systems on which SAXS has had a significant impact is the elucidation of the basic structural units of the β-amyloid fibers implicated in Alzheimer’s disease. In this condition, the deposited misfolded β-strands spontaneously assemble into stacked β-sheets as a result of hydrophobic interactions
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Inspired by Biology: From Molecules to Materials to Machines techniques, and applications developed by scientists remain simplistic by nature’s standards. Although significant progress has been made in assembling unique architectures of narrow dispersity, achieving nature’s idealistic standard of synthetic control with an unlimited monomer set has remained elusive. Indeed, tremendous opportunities exist in marrying computational modeling and theory, new synthetic and biological engineering methods, and precise characterization techniques in this area. Coupling diverse yet complementary areas will certainly facilitate and allow the design of novel smart and dynamic materials with extraordinary function, far from equilibrium. Synthetic Methods for Materials Synthesis Extensive efforts have been devoted to modifying existing materials to perform various functions. Synthetically evolving materials for a desired application offers the most simplistic and rapid route for functional and novel material creation. This concept has been commonly applied in the field of sensors. Synthetic receptors are often formed by molecular imprinting of the most commonly utilized material, polyethylene. Ethylene and functionalized vinyl monomers are polymerized around a template and this analyte molecule can then be removed, which endows such systems with molecular recognition properties. Existing materials have also been commonly utilized and modified for drug and nucleic acid delivery. Polyethyleneimine (PEI) has been studied since the 1960s to aid biomolecule separation but is now widely studied for nucleic acid delivery. Many examples of modifying the PEI backbone exist, for example, via PEGylating (to reduce toxicity and promote serum stability) and conjugating carbohydrates for tissue targeting. Although readily available and scalable, off-the-shelf materials often suffer from polydispersity. Thus unlimited opportunities exist in developing novel synthetic methods and materials that encompass existing and newly synthesized monomers assembled by conventional and novel routes. The advent of dendrimer synthesis has been a significant advance toward synthetic structural control. Macromolecules such as polyamidoamine (PAMAM) dendrimers can be synthesized via divergent or convergent routes (Figure 4.13), yielding completely monodisperse structures with well-defined architectural features that have been compared to artificial proteins. PAMAM is monodisperse, readily scalable, and commercially available. Thus much work has also focused on synthetically evolving this structure for a number of applications, ranging from drug delivery and synthetic vaccine development to disease diagnosis. Many elegant and diverse architectures are continuing to be designed and synthesized. For this reason, opportunities exist in this subfield to develop elegant structural architectures endowed with creative chemical functionality specifically and/or asymmetrically placed on monodisperse dendritic macromolecules.
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Inspired by Biology: From Molecules to Materials to Machines FIGURE 4.13 Methods of dendrimer synthesis via divergent (top) or convergent (bottom) means. SOURCE: Andrew Shipway. Available at http://www.ninger.com/dendrimer/. Well-defined materials, control in dispersity and architecture, and understanding the role of sequence on macromolecular conformation and function are the paradigms for materials synthesis. A main emphasis is to develop catalysts that yield unique architectures with highly defined sequences and controlled molecular weight (low dispersity). To broadly apply these catalysts toward controlled materials synthesis, the systems must be compatible with a variety of functional groups, which can be a significant challenge. The discovery of Grubbs’ catalyst and the subsequent generations of these unique ruthenium catalysts have facilitated the design and synthetic control over innumerable polymeric systems, ranging from adhesives to multivalent glycopolymers that are being used to understand cell surface receptor spacing and signaling interactions. In addition, the “click reaction,” copper-catalyzed Huisgen azide-alkyne cycloaddition, is a high-yielding reaction that can be performed in aqueous conditions and has facilitated the synthesis of a rich library of functional macromolecules. Incredible opportunities exist in developing unique catalytic systems that promote coupling in a user-friendly and efficient manner and can be carried out in the presence of oxygen and water to create highly functional materials.
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Inspired by Biology: From Molecules to Materials to Machines Materials Synthesis Using Natural Machinery Although numerous metal-based catalysts offer some control over stereochemistry, regiochemistry, and dispersity, often the catalyst can contaminate the final material, which may cause unwanted toxicity or interfere with proper material function. In addition, these catalysts do not play a role in the structure, conformation, or supramolecular assembly of synthetic material architecture in solution, all of which have been shown to be essential for proper function and reproducibility. To this end, researchers have been inspired by the synthesis and assembly control allowed by the intrinsic biological machinery. Adapting and engineering cellular systems to manufacture, assemble, and scale up completely synthetic materials in a controlled and monodisperse fashion affords an exemplary model in synthetic materials chemistry. Researchers have shown that a number of enzymes, utilized by biological systems for degrading biomacromolecules, can catalyze polymerization reactions with nonnatural monomers. Functional materials can be formed easily and rapidly in a regio- and stereoselective manner with narrow polydispersity. Chemical control of this sort is very difficult or impossible on the macroscale using traditional chemical methods. For example, lipases, enzymes normally utilized to degrade ester bonds in lipids, have been shown to catalyze the formation of polyesters with low dispersity. Widespread opportunities exist in this field to understand and utilize existing enzymes and/or biological engineering of enzymes to facilitate specific coupling reactions and assembly of novel materials in aqueous and/or organic conditions. Evolving enzymes and biological assembly machinery such as chaperones to catalyze reactions and assemble materials in a reproducible manner would be powerful. By hijacking and engineering the promiscuous cellular machinery, researchers have also shown that novel materials can be created by incorporating nonnatural amino acids into protein structures containing functional groups not normally found in biological systems. This allows the specific placement of reactive sites in a polypeptide such as alkynes and azides that can be utilized for specific and selective bioconjugation with, for example, the click reaction. The uses of cellular systems to synthesize these materials (as opposed to a protein synthesizer) also allows for the proper assembly and folding of polypeptides into precise protein conformations, which is essential for protein function. Extensive opportunities are available in engineering prokaryotic and eukaryotic cells not only to synthesize but also to assemble supramolecular structures. Such unique potential and promise represent the future of materials chemistry: control over synthetic sequence, dispersity, and conformation.
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Inspired by Biology: From Molecules to Materials to Machines Materials Synthesis Using a Natural Toolbox It has been shown that noncovalent interactions play as essential a role in function as do covalent bonds and are the ultimate model and inspiration for control and performance. By understanding the workings of the natural toolbox, researchers can model biological reactions, understand biological principles, and alter the chemistry to build unlimited functional materials. Utilizing amino acids, peptides, nucleosides, nucleotides, lipids, carbohydrates, and synthetically modified versions of these molecules, many bioinspired assemblies can be created by conventional and unique chemical routes. One approach is to utilize natural monomers because of their incredible biodiverse workings and mechanisms. Carbohydrates and polysaccharides alone store energy and promote and/or discourage cellular and biomolecule interactions, recognition, adhesion, and signaling. Simple and complex saccharide-based systems are therefore being exploited in the development of novel materials, ranging from sensors for shiga toxin to increasing specificity for targeting the delivery of various drugs. Moreover, their hydrophilic structures are highly biocompatible and can be consumed and degraded in biological systems. For this reason, carbohydrates are being utilized to build synthetic vehicles to increase intracellular delivery efficiency and lower toxicity of the drug and nucleic acid delivery process (Figure 4.14). Numerous opportunities are available to build biomimetic structures utilizing natural tools. For example, if synthetic viruses can be built to deliver nucleic acids into cells in a specific, efficient, and nontoxic manner, a paradigm shift from small molecule to macromolecular therapeutics could occur and revolutionize modern medicine. In addition, multivalent materials have been shown to serve as inhibitors and effectors for various biochemical pathways and have promise as novel drugs and research tools. Very subtle structural changes in such biomolecular systems are FIGURE 4.14 (a) The general structure of novel carbohydrate-based polymers (the length, n, can be varied between 56 and 100) that are efficient intracellular nucleic acid delivery vehicles. The synthetic structures, polymerized via click chemistry, contain a disaccharide for lowering toxicity and heterocycles, amides, and amines that facilitate self-assembly via electrostatic interactions with DNA into (b) viral mimetic nanoparticles. SOURCE: S. Srinivasachari, Y. Liu, G. Zhang, L. Prevette, and T.M. Reineke, “Trehalose click polymers inhibit nanoparticle aggregation and promote pDNA delivery in serum,“ Journal of the American Chemical Society 128:8176 (2006). Copyright 2006 American Chemical Society.
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Inspired by Biology: From Molecules to Materials to Machines known to affect the performance and efficacy of biochemical pathways; a major challenge in this area is to elucidate the chemical structure-biological property relationships to develop advanced and bioresponsive systems. To this end, significant opportunities exist to understand advanced biomaterial structure-activity relationships (SAR) in a manner similar to traditional small-molecule medicinal chemistry to optimize biomaterial behavior. Nucleosides and amino acids also provide researchers with a rich toolbox of monomers and macromers that can be linked by covalent or noncovalent means onto synthetic systems, providing advanced performance. Antibodies, essential for biological immunity, have been utilized extensively for sensor and bioassay development owing to their specific and selective recognition properties. Peptide sequences, such as the transactivator of transcription sequence derived from HIV, have endowed nanoparticles, drugs, and nucleic acid vectors with enhanced cellular penetration. Moreover, synthetic polymers formed with amino acid monomers (created either by conventional synthesis or by phage display) have been exploited to improve cellular uptake and delivery of drugs and promote cellular infiltration into biopolymer scaffolds for tissue engineering. Novel ligation chemistries with nucleosides and amino acids have also created completely new materials such as peptoids and peptide nucleic acids, which are not subject to enzymatic degradation in biological systems and may yield diverse applications from artificial transcription factors to antisense agents. The challenges with exploring and exploiting such materials lie in the fact that researchers are limited by the lack of biological knowledge and the unavailability of functional peptides and other biomolecules to enhance biomaterial performance. Thus many opportunities exist in understanding, creating, and modifying the tools and coupling chemistries to create unique protein and nucleic-acid-like materials and in exploration of these structures for sensor, therapeutic, and diagnostic development. Macromolecular Assembly Routes Intrinsically coupled with synthesis is the ability to assemble macromolecules into novel structures that control function. This ability to create accurate, specific, and reproducible three-dimensional structures is the holy grail of materials synthesis. To this end, understanding the routes and mechanisms of macromolecule assembly via noncovalent interactions is as important as developing new covalent coupling routes that link molecules together. Unique opportunities in materials synthesis are unfolding that exploit both covalent and noncovalent interactions such as hydrophobic, hydrophilic, and electrostatic interactions and metal coordination and H-bonding. For example, novel monomers can be polymerized via H-bonding and metal coordination (Figure 4.15a), and the backbone of a polymer can be functionalized via noncovalent interactions after polymerization (Figure 4.15b).
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Inspired by Biology: From Molecules to Materials to Machines FIGURE 4.15 Routes to create supramolecular polymers via noncovalent interactions. (A) Main-chain supramolecular polymers and (B) side-chain supramolecular polymers. SOURCE: M. Weck, “Side-chain functionalized supramolecular polymers,” Polymer International 56:453 (2007). Many opportunities arise to form innovative materials by this plug-and-play design, whereby a library of structures can easily and rapidly be formed. Structures created via noncovalent synthetic routes also offer potential to create self-healing systems because the molecular structure can be self-corrected by atomic rearrangement due to the specificity and directionality of hydrogen or coordination bonds. Similarly, many possibilities also exist in template-directed polymerization strategies; for example, monomers could be assembled via H-bonding along a preexisting polymer backbone and then be polymerized. This strategy may allow the assembly of complex material structures in a manner similar to the transcription of DNA to RNA. Other unique supramolecular materials can be formed by a combination of hydrophobic and hydrophilic interactions. Novel supramolecular polymers have been created by peptide amphiphiles (Figure 4.16). Such polymers are a product
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Inspired by Biology: From Molecules to Materials to Machines FIGURE 4.16 Peptide amphiphiles: The hydrophobic lipid tales are on the inside of the fibers and the peptides are displayed on the fiber surface. Each fiber is approximately 6 to 7.5 nm in diameter and 1 μm in length. Growth factors have been incorporated, which endows these fibrous structures with tissue regeneration properties. SOURCE: K. Rajangam, H.A. Behanna, M.J. Hui, X. Han, J.F. Hulvat, J.W. Lomasney, and S.I. Stupp, “Heparin binding nanostructures to promote growth of blood vessels,” Nano Letters 6:2086-2090 (2006). Copyright 2006 American Chemical Society. of the self-assembly of peptide-lipid hybrid molecules that have been shown to form micellelike fibers. Also, block copolymers have been shown to assemble into core-shell nanoparticles, polymer-based micelles with well-defined size and shape. As shown in Figure 4.17, chemical reactions can also be performed on the micelle shell (e.g., covalent crosslinking), creating a stable shell. The core may also be removed, yielding hollow nanocapsules that can house a variety of guest species such as drugs and diagnostic agents. These nanocapsules can also offer controlled release of these substances by incorporating degradable linkers in the capsule shell. Many other novel methods of monodiperse nanoparticle formation are beginning to unfold using novel molding and templating techniques. Tremendous opportunities exist in examining noncovalent polymerization and assembly methods
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Inspired by Biology: From Molecules to Materials to Machines FIGURE 4.17 Polymer micelles can be formed by noncovalent interactions. Top: The shell can be chemically crosslinked to stabilize the nanoparticles. Bottom: Atomic force microscopy displays the nanoparticle shape and size. SOURCE: Karen L. Wooley, Washington University, St. Louis, Mo. that yield biomolecular structures. Although challenges remain in understanding and controlling the creation, morphology, and reproducibility of such structures, exceptional properties will result once researchers understand and can manipulate these processes. Lastly, the ability to pattern molecules on substrates offers another means to control the synthetic assembly of macromolecules in three-dimensional space.
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Inspired by Biology: From Molecules to Materials to Machines Molecular patterning and lithography are a powerful means of assembling structures via both covalent and noncovalent methods on substrates. Lithography (dip pen, photo, electron beam, and so forth) has proven to be very effective at the nanoscale to design and develop a number of devices dependent on the control, placement, and conformation of macromolecules. For example, proper and accurate patterning, placement, and conformation of nucleic acids on substrates have offered powerful detection methods for applications ranging from gene sequencing to disease diagnosis. Opportunities in three-dimensional patterning of biomolecules such as growth factors in biocompatible gels and scaffolding will certainly play a role in creating novel tissue regeneration systems to promote cell signaling, angiogenesis, and tissue formation. OPPORTUNITIES AND CHALLENGES In this chapter, the committee discussed many emerging areas in the modeling and analysis of biomolecular materials and processes. New experimental tools are facilitating cutting-edge experiments that when closely coupled to theoretical and computational analysis, are providing new research opportunities in biomolecular materials and processes. Some of the challenges to further advancement are listed here along with some possible opportunities. Challenge: Achieving angstrom resolution in electron and X-ray imaging Opportunity: Unprecedented elucidation, at the molecular level, of the structural and operational principles of many important cellular processes Challenge: Studying the structure, dynamics, and kinetics of assemblies of biomolecular systems using X-ray scattering techniques Opportunity: Elucidating the dynamical processes involved in RNA genomes packing into viral capsids and probing the collective behavior of motors moving on their natural tracks Challenge: Probing reactions using neutron scattering at timescales of microseconds or longer Opportunity: The ability to probe dynamical behavior as well as structural correlations in biomolecular materials and processes Challenge: Developing techniques for seeing correlations in space and time (for example, neutron correlation spectroscopy and correlated neutron imaging) rather than in q (momentum) and frequency (energy) space, as is done in neutron inelastic scattering Opportunity: Allow one to look at motions on a particular length scale, such as the size of some domain of a protein, and watch the time dependence of the motion in different environments
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Inspired by Biology: From Molecules to Materials to Machines Challenge: Improving the temporal and spatial resolution of single-molecule techniques and integrating them into studies of larger macromolecular complexes that approach the complexity of actual cellular machines Opportunity: Designing artificial biomolecular machines from insights into folding and self-assembly of complex biomolecules Challenge: Predicting the native conformations of macromolecules and the mechanisms and rates of conformational transitions Opportunity: Designing bioinspired macromolecules that can perform specific functions or serve as building blocks for functional supramolecular structures Challenge: Developing rigorous multiscale algorithms that bridge the molecular and mesoscopic scales Opportunity: The ability to understand and predict how formation and function of supramolecular structures depend upon the molecular building blocks Challenge: Developing efficient, stochastic, spatially resolved simulation methods that can study dynamical phenomena characterized by cooperation and feedback Opportunity: The development of stimuli-responsive materials, like cells, that can perform precise functions Challenge: Developing methods to study the thermodynamics of small systems and understanding how noise can be exploited or avoided in collective dynamical phenomena to effect a desired function Opportunity: The design of nanoscale biomaterials through greater understanding of their thermodynamics and stochastic fluctuations Challenge: Developing a rigorous theoretical understanding of how to describe systems far from equilibrium Opportunity: Greater understanding of all the topics outlined in this report since many biological systems and functional biomaterials operate far from equilibrium Challenge: Manipulating the organization of hierarchical assemblies (the secondary, tertiary, and quaternary structures) of biomolecular materials Opportunity: The ability to create macromolecular, or even cellular, synthetic biomolecular materials from molecular building blocks Challenge: Having the ability to fuel directed assembly by novel reactions, templating agents, or hijacking the cellular machinery, such as chaperones (proteins that can assemble complex biomolecules into discrete structures) Opportunity: Unprecedented control over the synthesis of new and complex biomolecular materials
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Inspired by Biology: From Molecules to Materials to Machines SUGGESTED READING Als-Nielsen, J., and D. McMorrow, Elements of Modern X-Ray Physics, West Sussex, England: John Wiley and Sons, 2001. Bacon, G.E., Neutron Diffraction, Glasgow, New York: Oxford University Press, 1975. Grunewald, K., O. Medalia, A. Gross, A.C. Steven, and W. Baumeister, “Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: Implications of crowding,” Biophysical Chemistry 100(1-3):577-591 (2003). Hawker, C.J., and K.L. Wooley, “The convergence of synthetic organic and polymer chemistries,” Science 309:1200 (2005). Langer, R., and D.A. Tirrell, “Designing materials for biology and medicine,” Nature 428:487 (2004). NRC (National Research Council), Controlling the Quantum World: The Science of Atoms, Molecules, and Photons, Washington, D.C.: The National Academies Press, 2007. NRC, The Role of Theory in Advancing 21st Century Biology: Catalyzing Transformative Research, Washington, D.C.: The National Academies Press, 2008. NSF, Cyberinfrastructure Vision for 21st Century Discovery, Arlington, Va.: NSF, 2007. Available online at http://www.nsf.gov/od/oci/CI_Vision_March07.pdf. Last accessed March 27, 2008. NSF, Simulation-Based Engineering Science: Revolutionizing Engineering Science through Simulation, Report of the National Science Foundation Blue Ribbon Panel on Simulation-Based Engineering Science, Arlington, Va.: NSF, 2006. Available online at http://www.nsf.gov/pubs/reports/sbes_final_report.pdf. Last accessed March 27, 2008. Neuman, K.C., and S.M. Block, “Optical trapping,” Review of Scientific Instruments 75(9):2787-2809 (2004). Ozin, G.A., and A.C. Arsenault, Nanochemistry, A Chemical Approach to Nanomaterials, Cambridge, England: Royal Society of Chemistry, 2005. Tomalia, D.A., and J.M.J. Frechet, “Discovery of dendrimers and dendritic polymers: A brief historical perspective,” Journal of Polymer Science Part A: Polymer Chemistry 40:2719 (2002). Zlatanova. J., and K. van Holde, “Single-molecule biology: What is it and how does it work?” Molecular Cell 24(3):317-329 (2006).