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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 (pro- teins, carbohydrates, lipids, and nucleic acids) that give rise to a variety of supra- molecular 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 understand- ing 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 76
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 77 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 noncrys- talline supramolecular complexes requires continual advancement in the develop- ment 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 âsingle-molecule biophysics.â 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 observa- tions 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, inter- pret experimental results, and examine alternative design motifs for biomaterials. Some of the potential advances to be achieved from next-generation experi- mental tools and computational methods inspire research efforts. Imagine that one could . . . â¢ View cells in atomic to molecular detail and at a time resolution of milliÂ seconds, appropriate to observe the events during neuronal synaptic trans- mission or chromosome replication. â¢ Measure the forces and motions of nanobiomachines directly and on the millisecond to microsecond timescale. â¢ Determine the sequence of an individual DNA molecule, providing the ultimate sensitivity for forensic purposes or diagnosing inherited disease. â¢ Understand how biological machines conquer the chaotic and crowded conditions of the cell. â¢ Use principles of biological recognition to design new molecular interac- tions ab initio. â¢ Predict how molecules with a specific sequence of monomers will adopt a specific conformation and self-assemble into precise supramolecular structures.
78 Inspired by Biology â¢ Use cell-mimetic cooperativity to design biosensors that can detect minute amounts of hazardous substances without noise-induced false positives. â¢ 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 reconstruc- tion 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 pro- jected 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 inte- rior 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 Ã 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 non- covalent 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
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 79 FIGURE 4.1â The structure of an assembled infectious P22 bacteriophage virion as revealed by three-dimensional cryo-EM at 17 Ã 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, âThe structure of an infectious P22 virion shows the signal for headful DNA packaging,â 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
80 Inspired by Biology B FIGURE 4.2â (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, âMacromolecular architecture in eukaryotic cells visualized by cryoelectron tomography,â 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, per- mitting 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.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 81 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 micro- scope 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. ÂSeveral new techniques have been described that overcome this diffraction barrier in opti- cal microscopy. The first general method is termed âstructured light.â 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 areaâpoint spread function (PSF)âmuch narrower than the standard 250 nm wide Âdiffraction- 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 exci- tation spot over the sample and sequentially collecting the fluorescence emission from each spot to reconstruct the distribution of fluorescent probes in the speci- men. In one experiment, synaptotagmin-labeled synaptic vesicles were resolved much more clearly in STED images (Figure 4.3c) than by standard confocal micros- copy (Figure 4.3b). Fluorophores can be switched on and off by photophysical means other than
82 Inspired by Biology FIGURE 4.3â 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, âSTED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,â 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.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 83 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-
84 Inspired by Biology 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 imag- ing 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 micro- scope with 50 nm resolution has recently been developed. High-resolution X-ray tÂomography opens up new avenues to nondestructively explore the internal struc- ture 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-Âinterconnect- based integrated circuit (IC) chip with 120-nm feature size. FIGURE 4.4â (Left) Schematic of a zone-plate-based full-field three-dimensional X-ray micro- scope 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 exam- ple 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.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 85 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 bioÂ molecular 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 Ã 2 Âµm, transformed with a recombinant yellow fluorescent protein (YFP) that could be marked by Âpotassium 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
86 Inspired by Biology FIGURE 4.5â 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, âImaging whole Esch- erichia coli bacteria by using single-particle X-ray diffraction,â 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 impor- tant 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 Î²-amyloid fibers implicated in Alzheimerâs disease. In this condition, the deposited misfolded Î²-strands spon- taneously assemble into stacked Î²-sheets as a result of hydrophobic interactions
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 87 and twist around a fiber axis. It is expected that SAXS studies will elucidate the structure and formation kinetics of many biomolecular material systems, including the misfolded proteins that cause amyloidosis diseases such age-related macular degeneration, type II non-insulin-dependent diabetes, and bovine spongiform encephalopathy (âmad cowâ disease). The combined application of SAXS and cryo-EM, a growing focus of research in the next decade, complements electron and X-ray tomography as powerful probes of noncrystalline biological structures. The X-ray methods quantitatively measure statistically averaged structures in reciprocal space, whereas cryo-EM provides a direct space model (although at lower resolution) of its subjects. Several recent studies of higher-order assembly of cytoskeletal microtubules and filamen- tous actin have in fact demonstrated the importance of the combined techniques in elucidating such partially ordered hierarchical structures. Neutron Scattering Neutron scattering and diffraction provide detailed information on the struc- ture and dynamics of biomaterials and systems across time and length scales that range from pico- to nanosecond of time resolution and from 1 to 10,000 Ã of spatial resolution. Neutrons are scattered from atomic nuclei (as opposed to X-ray scat- tering by electrons) and are thus exquisitely sensitive to hydrogen atom position, content, and dynamics in biological materials. This feature allows the structure and composition of complex or composite biomaterials to be determined and dis- tinguished according to the bulk hydrogen content of the individual components. Moreover, neutrons scatter differently from hydrogen and its deuterium isotope, so that in mixed systems, individual components can be selectively labeled with deuterium in order to highlight them. The power of the technique is thus most fully realized when combined with synthetic capabilities that allow the design and production of specific, random, or uniformly deuterium-labeled macromolecules to permit selected components of structure, dynamics, and interactions of macroÂ molecular structures to be analyzed in situ in multicomponent systems in solu- tion, at surfaces, or in single crystals. Neutron scattering is thus a powerful tool for characterization and analysis of complex structure-function and interfacial relationships between membrane, polymer, and macromolecular systems at the intersection of biology and materials science. Because neutrons interact with nuclei rather than electrons, neutron-scattering lengths show little variation across the periodic table. Many of the heavier compo- nents of mixed or complex materials that are opaque to X-rays and that dominate X-ray scattering are then virtually transparent to neutrons. This property facilitates the lighter, hydrogenated components (polymers, peptides, proteins, lipids, nucleic acids, or solvents) of complex systems, composites, or phases to be highlighted
88 Inspired by Biology and analyzed in situ. At the atomic and molecular levels, neutron diffraction can pinpoint hydrogen atom positions in such materials. This feature can provide fundamental insight into catalytic processes in enzymes or of the proton Âshuttling/ relay pathways involved in biological processes (Figure 4.6, left). In complex bio- p Â olymers such as cellulose, an important potential source of renewable Âethanol, neutron diffraction experiments have mapped the detailed hydrogen bonding interactions that mediate its macroscopic material properties (Figure 4.6, right). The enhanced visibility of hydrogen atoms in water, substrates, and proteins allows direct determination of protonation state and thus helps to provide a more complete picture of atomic and electronic structures in macromolecules. This is beneficial for determining enzyme mechanisms, for studies of ligand binding interactions and, since complete D2O water molecules are prominent in neutron density maps, for detailed analysis of the structure and dynamics of water in hydra- tion layers at the protein-solvent interface. Neutron diffraction can determine the pattern and extent of H/D isotope substitution in proteins, the solvent accessibility of individual amino acids, the mobility and flexibility of interesting domains, and the H/D exchange dynamics themselves. Incoherent scattering from a sample containing H and D is strongly dominated by the motions of the H nuclei, which in neutron-scattering experiments mainly reflect the motions of the macromolecular side chains to which they are bound. Inelastic neutron-scattering experiments on dedicated time-of-flight and filter- FIGURE 4.6â Left: Location of hydrogen or deuterium atom at the active site of Âendothiapepsin in a 2.1 Ã resolution Laue diffractometer (LADI) structure. SOURCE: Dean Myles, Oak Ridge National Laboratory. Right: Hydrogen bonding interactions in cellulose from neutron fiber diffraction. SOURCE: Y. Nishiyama, P. Langan, and H. Chanzy, âCrystal structure and h Â ydrogen-bonding system in cellulose I from synchrotron X-ray and neutron fiber diffrac- tion,â Journal of the American Chemical Society 124:9074-9082 (2002). Copyright 2002 American Chemical Society.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 89 analyzer instruments provide high-quality data on the inelastic structure factor and vibrational densities of states in the energy domain from a few meV to a few hun- dred meV, which serve as constraints on dynamic models of atomic bonding and even their structures. The ability to systematically highlight, isolate, and probe the dynamics of specific H-labeled residues in situ within their natural environments is valuable for the study of biological and model biophysical/Âbiotechnological applications. At the mesoscale, neutron scattering is sensitive to the bulk hydrogen atom content and composition of materials and can be used to characterize and deter- mine the structure of mixed and complex systems and phases. For example, the bulk neutron scattering characteristics of proteins, nucleic acids, lipids, and car- bohydrates all differ significantly from one another (Figure 4.7). This natural dif- ference in contrast can be exploited to locate individual components in functional biological assemblies such as histones or ribosomes. Small-angle neutron scattering (SANS) experiments allow the influence of protein or chemical cofactors and ligands on both structure and dynamics to be monitored in solution and at near-physiological conditions. The packing interac- tions of natural or bioinspired materials with synthetic substrates are detectable at the interface with synthetic nanostructures and scaffolds. Applications at the inter- section of biology and materials science include characterization of functionalized nanomaterials such as DNA-, protein-, or peptide-coated carbon nanotubes or polymeric assemblies. When such interactions occur at planar surfaces, interfaces, or layered phases, neutron reflectivity experiments can be exploited. These experi- ments can be extremely powerful in characterization and help to design new classes of biodevices that incorporate active biological or bioinspired agents in monitoring devices. The neutron contrast can allow marker, signaling, or receptor proteins, peptides, or nucleic acids to be discriminated from host substrates or supports that are composed of polymer or lipid matricesâinformation that can be difficult if not impossible to obtain from other techniques. In hybrid or functionalized materials and composites, neutron scattering allows the in situ structure and dynamics of D-labeled polymers and proteins incorporated into devices to be analyzed directly. For example, solar fuel-producing molecular photovoltaic structures are a promis- ing future concept. The availability of new neutron-scattering facilities will contribute greatly to these efforts. For example, the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory will provide the worldâs most intense beam of cold neutrons for biomaterials research. The SNS will allow probing at length scales of nearly 10,000 Ã and timescales of 400 ns. The order of magnitude gains in performance at such new facilities will make possible the analysis of whole new classes of macroÂ molecular materials and processes and can be used to guide the design, synthesis, and assembly of natural and synthetic components into functional units.
90 Inspired by Biology Contrast Variation 9.0 8.0 D-DNA 7.0 scattering length density ( 10-10 cm-2) D-protein 6.0 -3 Matching Point cm.Ã 5.0 14 DNA 4.0 scattering length density 10- RNA 3.0 Protein 2.0 water 1.0 Phosphatyl Choline 0.0 0 10 20 30 40 50 60 70 80 90 100 D2 O (%) %D O FIGURE 4.7â Contrast variation. Plot showing the neutron-scattering length density as a function of H2O/D2O solvent content for some common biomaterials. SOURCE: Dean Myles, 4-7.eps Oak Ridge National Laboratory. Single-Molecule Probes A major recent advance for investigations of the macromolecules that make up biomolecular materials is the development of instrumentation and protocols to investigate physical behavior of individual molecules. So-called single-molecule biophysics reveals the mechanics, biochemistry, structural biology, and dynamics of biomolecular processes, complementing classical methods for understanding them. Some important aspects of biomolecular function are usually obscured in studies of ensembles of molecules due to the averaging of heterogeneities and dynamic variations among their individual functional units. This ambiguity is removed by detecting the reaction trajectory directly for each molecule under study. Signals from single molecules are noisy partly because the specimen is so tiny but also because the individual molecules exhibit large variations in their orientations and conformations due to thermal fluctuations and bombardment by solvent
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 91 molecules. This noisiness is a real expression of the random, stochastic nature of molecular interactions, so that the facile detection of such processes is fundamental to understanding biomolecular mechanisms, especially nanoscale phenomena. Dramatic progress in understanding biological macromolecules, materials, and cellular function has derived from genetics, structural biology, and biochemical experiments on ensembles, solutions, and suspensions of proteins and nucleic acids and in live cells. High-resolution structures derived from X-ray crystallography and cryo-EM are revealing snapshots of particular states in the reaction path- ways. These conformations must be fit into the context of the functional mecha- nism by measurements of the path, kinetics, and equilibrium of the enzymatic r Â eaction sequence. Classical steady-state biochemistry, rapid reaction kinetics, and p Â hysiology provide much of this information, but important characteristics of the individual molecules that are crucial for their behavior are very difficult to detect in ensemble experiments. Individual chemical reactions, including the elementary steps of biological enzymatic pathways, are stochastic, which means that their stepwise progress is probabilistic rather than regular and clocklike. Thus virtually every dynamic characteristic fluctuates greatly. Researchers do not often detect the randomness of molecular events because observations usually average the behavior of billions of molecules. But all nanodevices, including natural biomolecules and human-made nanomachines, function in chaotic conditions due to these thermal and quantum fluctuations. Taking account of this aspect of physics at the small scale is a major challenge for designing biomolecular materials. Some of the important characteristics of single biological macromolecules that are averaged out in ensemble observations are listed below. Each of them is directly accessed by single-molecule methods. â¢ Conformational fluctuations. The stochastic nature of individual reactions and randomness implies that each molecule follows a different temporal trajectory and possibly even a different reaction path. Surmounting an activation barrier for a reaction to proceed is a ubiquitous example. Some biomolecules harness thermal motions to gain brief access to the edges of their conformational distributions, thereby achieving a functional advantage. â¢ Reversals. Any isomerization or binding reaction that is not too far from its equilibrium undergoes many reverse reaction steps along with its forward progress (for example, most reactions in biological processes). â¢ Pauses. DNA-processing enzymes that expose and replicate the genome and the ribosome that translates the genetic code into amino acid sequences exhibit brief, sequence-dependent pauses with important regulatory consequences, such as termination or splicing. â¢ Inhomogeneities. Complex macromoleucles have individual chemical differ- ences, such as (1) protonation, (2) redox state, and (3) post-translational chemical
92 Inspired by Biology modifications such as phosphorylation and methylation. Thus each molecule may display physiological variations from its nominally identical partners. These dynamic and structural variations are usually invisible in ensemble measurements. In some specialized systems, single-molecule biophysics reveals special signals that are intrinsically difficult to obtain in bulk assays: â¢ Rotational motions and wobble. These are very common in enzyme mecha- nisms, but in a suspension of macromolecules the orientations of the Âindividual mol- ecules are random, and rapid tumbling tends to make the suspension isotropic. â¢ Stepping distance. The primary output of molecular motors and DNA- p Â rocessing enzymes is progress along cytoskeletal or nucleic acid tracks. The indi- vidual motions are difficult to synchronize in an ensemble, so only the average progress is detected. â¢ Mechanical forces and elasticity. Molecular motors, DNA-processing enzymes, cytoskeletal filaments, and other subassemblies are the mechanical actuators and structural supports for the tissue. The mechanical characteristics of the cells and macroscopic tissue are a sophisticated composite of the individual molecular devices. â¢ Conductivity. Ion channels and transporters are ubiquitous membrane com- ponents that control and modulate the composition of cellular and tissue compart- ments. In addition, the electrical performance of these intrinsic membrane proteins produces sensory, neural, and cardiac signaling and many other receptor-effector responses. One of the earliest applications of single-molecule approaches used patch electrodes to measure the conductance and dynamics of single-ion channels. This development led to the 1991 Nobel prize in physiology or medicine. â¢ Binding and folding energy landscape. Spontaneous or assisted folding of proteins and RNA enzymes (ribozymes) are essential steps in their assembly into native, functioning macromolecules. Chemical interactions between most macro- molecules and their smaller liganding partners produce the overall system response. The average behavior is the composite of the individual assembly and interaction events, which are seldom detectable except by studying one molecule at a time. Single-Molecule Instrumentation Some of the most prominent single-molecule techniques that have been suc- cessful for elucidating the dynamic and energetic aspects of biological macro- molecules are the âoptical trap,â also called the âlaser tweezers,â single-molecule fluorescence microscopy, scanning probe microscopy, such as the atomic force microscope, and single-ion channel electrophysiological recording of membrane currents. The first two of the methods are described here as examples.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 93 Optical Trap Tightly focusing an optical beam by a high-numerical-aperture microscope objective forms an intense spot of light that attracts small objects having a higher refractive index than their surroundings. For instance, a 0.5- to 1-Î¼m polystyrene bead with a refractive index n of 1.57 (water has an n of 1.33) acts like a tiny lens that deflects or scatters the rays of a tightly focused infrared laser beam. The trans- fer of momentum from the bead to the infrared photons when they are scattered implies a reaction force between the photons and the bead. This force causes the bead to move toward the position of highest intensity, the center of the focused spot of light. The force becomes zero when the particle is balanced in the center of the spot. Thus the tightly focused beam is termed an optical trap (or, alternatively, laser tweezers). When biological molecules are attached to the bead, the forces and stepping or folding motions that pull the bead away from the center of the trap can be measured at the functionally relevant scales of piconewtons and nanometers. Optical traps have been used to study molecular motors, DNA-processing enzymes (Figure 4.8), the folding and unfolding of proteins, and ligand-receptor interactions. The forces and length steps of molecular motors and the enzymes that replicate and transcribe DNA, untwist it, and translate the DNA code are fundamental characteristics, necessary to understand the relationship among their enzyme activities, the energetics of their internal conformational changes, and their function in movement and progress along their biological tracks. Figure 4.8 shows the geometry and mechanical recordings from an experiment on the enzyme RNA polymerase (RNAP), the cellular macromolecular machine that transcribes DNA sequences into messenger RNA. The recordings (panels B and C) show the distance between two beads tethered together by the RNAP and a DNA molecule (panelÂ A). This distance decreases as the enzyme translates along the DNA while synthesizing an mRNA molecule. The translocation sometimes pauses and back- tracks, probably to correct errors (panel C). Detailed kinetics of these processes and the actual trajectory of the reaction waited until the technology progressed to resolve the individual events. Total Internal Reflection Fluorescence Microscopy Detecting the fluorescence from single organic dyes (for example, rhodamine), fluorescent peptides (for example, green fluorescent protein), or semiconduc- tor nanocrystals (for example, quantum dots) has become a relatively routine laboratory procedure. Specific attachment of one of these probes to a biological molecule allows the tracking of its position and the measurement of its orienta- tion and rotational motions, internal distances that change between conformations and interactions with neighboring molecules and binding partners. In order to
94 Inspired by Biology A B C FIGURE 4.8â Single-molecule in vitro assay using dual optical traps to investigate RNA poly- merase, the enzyme complex that transcribes DNA to synthesize messenger RNA. (A) Two tightly focused infrared optical beams are depicted as pink waists. The blue spheres rep- resent small 0.5-Âµm and 0.7-Âµm polystyrene beads. During synthesis of the mRNA, RNAP (green) moves forward on the DNA (blue) as it elongates the nascent RNA (red). The smaller bead (right) is bound to a single molecule of RNAP by biotin-avidin (yellow cross), while the larger bead (left) is bound to the downstream end of the DNA by an antibody-antigen complex (yellow polygon). (B and C) Recordings of the distance between the two beads at a constant stretching force of 8 pN. At each position along the DNA template, RNAP may slide backward along the template (bracket in B), causing transcription to pause temporarily and recover. These events occur randomly or at particular DNA sequences. SOURCE: J.W. Shaevitz, E.A. Abbondanzieri, R. Landick, and S.M. Block, âBacktracking by single RNA polymerase molecules observed at near-base-pair resolution,â Nature 426:684 (2003). detect a single fluorescent molecule, it is electronically excited by a laser beam, and then the longer-wavelength photons that are subsequently emitted (fluores- cence emission) are captured by a photon-counting detector or a sensitive video c Â amera. The detector must be capable of registering 500Â-10,000 photons per second above Â spurious instrumental dark counts. This performance is achievable with commercial Â cameras, photomultipliers, and silicon avalanche photodiodes. The samples must be very clean to reduce background contamination down to the
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 95 level that fluorescence from contaminants is well below the emission of the target fluorophore. Even with these characteristics, the sample volume that is interrogated by the instrument usually has to be limited to the region containing the fluorophores of interest to sufficiently lower fluorescence from contaminants. The excitation volume is often reduced by using the tightly focused exciting beam in a confocal microscope or the evanescent electromagnetic field that is present near a total internally reflective interface, giving total internal reflection. Figure 4.9a shows this geometry for a single-molecule total internal reflection fluorescence (TIRF) microscope. The evanescent wave extends only 100-200 nm into the aqueous sample compartment. Thus only fluorescent molecules on or very near the surface are excited for fluorescence. The orientation of the fluorescent probe is determined by time-multiplexing the exciting input polarizations. Distances are measured by fluorescence resonance energy transfer (FRET) which is sensitive to the spacing between two fluorophores on labeled protein domains in the 30- to 70-nm range. The location of an individual molecule can obtained at 1- to 2-nm precision by fitting the distribution of Âemitted light to a Gaussian-shaped function, a method termed fluorescence imaging at one nanometer accuracy (FIONA). These signals have been used to determine the mechanism for stepping in several of the molecular motors, structural changes in RNA switches, and many other mechanistically important characteristics. In the example shown in Figure 4.9b, a molecular motor, myosin V, is walking along its cytoskeletal filament, actin. The alternating large and small translocation steps of a fluorophore located on one of the myosin âheadsâ observed in this kind of recording provide strong evidence for a hand-over-hand mechanism, as shown in the cartoon (Figure 4.9c). Theory and Computation Synergies between theory, computation, and experiment are well established in materials science and are beginning to emerge in the biological sciences. These past and recent successes, however, point to some pressing needs. Examples of challenges that need to be confronted and overcome in order for theory and com- putation to play an important role in the discovery and design process include the development and refinement of fundamental theoretical frameworks for describing properties far from equilibrium and in small (nanoscale) systems; the development of efficient, multiscale simulation methods that can describe the full complexity of biological and bioinspired structures and biomaterials and can be applied to the spatiotemporal evolution of many-component systems; ways to analyze informa- tion content in biomolecules; and providing the infrastructure (hardware, software, and communication) to support these activities.
96 Inspired by Biology C x A 7 4 nm 37 n m - 2 x B 2 3Â±3 nm 5 2Â±4 nm 1 9. 2 4 5 .5 nm 2 7. 2 nm 4 8. 8 nm 7 4 .3 nm 74 n m 1 8. 7 nm 5 8. 6 nm 1 5 .4 nm 37 n m + 2x 4 8 .3 nm 6 6 .6 nm 8 2. 1 nm 7 2 .5 nm 2 3. 7 nm 6 7 .2 nm 56 . 6 nm 37 37 25 . 8 nm 2 6. 0 nm A nm B nm A 6 8 .2 nm 51 . 5 nm 23 . 5 nm 6 8. 4 nm 5 2 .0 nm 2 6 .5 nm 7 3 .5 nm 6 8. 9 nm FIGURE 4.9â Single-molecule fluorescence imaging assay to monitor position at nanometer precision (FIONA). The experimental geometry is indicated schematically in (A). A laser beam is reflected from the coverslip-water interface, producing a nonpropagating, Âspatially 4-9 decaying electromagnetic oscillation, termed an evanescent wave. This fluorescence excita- tion field extends into the sample compartment only 100-200 nm, thereby reducing back- ground intensity relative to conventional epifluorescence or confocal excitation. (B) Position of a rhodamine fluorescent probe attached to a myosin V molecular motor while the motor translocates along a filament of the cytoskeletal protein, actin. The probe tilts and moves stepwise, either 74 nm at a time (blue circles) or with alternating 52-nm and 23-nm steps (dark, half-filled symbols). (C) Cartoon showing the origin of the alternating large and small steps when myosin V walks hand-over-hand along actin. The yellow and grey actin-binding motor domains swap places on each step along the filament. If the probe is located nearer the motor domain, it moves 74 nm every other step. SOURCES: Yale E. Goldman, University of Pennsylvania; and A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin, âMyosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localiza- tion,â Science 300:2061 (2003). Advances in these areas and the unabated increase in processor speeds could make it possible for theory and computation to play a major role in the design of new superstrong materials with memory and recognition; cell-mimetic materials that exploit feedback and stochastic fluctuations for carrying out stimuli-Âresponsive functions; sensors that detect hazardous molecules with unprecedented sensitivity and specificity; self-replicating materials; and systems yet to be imagined. Some specific topics worthy of consideration are outlined below.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 97 Modeling and Computer Simulation This section is focused primarily on classical computer simulations, but it is important to emphasize that results from such simulations usually need to be augmented by analytical approximations, quantum mechanical calculations, and phenomenological models in order to glean a proper understanding of mechanistic and dynamic principles. Molecular Simulations Molecules are the building blocks of all biomolecular processes and biomateri- als, and so the simulation of molecular structure and molecule-specific properties is important. Typical molecular simulations are based on molecular dynamics, Monte Carlo methods, or Brownian dynamics methods. There are many examples of prob- lems pertinent to biomolecular processes and biomaterials that could be addressed using these methods. Consider foldamers (Figure 4.10), polymers with monomer FIGURE 4.10â Examples of three beta-peptide foldamers, each of which forms stable helices in water. Left: 14-helix. Middle: 12-helix. Right: alpha-helix. SOURCE: Samuel H. Gellman, University of Wisconsin at Madison.
98 Inspired by Biology sequences that are designed so that they collapse into unique native conforma- tions that can perform proteinlike functions such as catalysis, ion transport, and energy transduction. An outstanding question is how to determine the monomer sequence that will fold into a given target native structure. This is a computational challenge that is similar to the grand challenge of predicting protein structure from knowledge of the sequence of the peptide backbones. There are several important bottlenecks for computations that could answer such questions. Many of the bottlenecks are the same as those identified for com- putational materials science, computational nanoscience, the chemical sciences, and energy research. For example, there is a need for more efficient methods for sampling large conformational spaces and for finding rare (but important) d Â ynamical trajectories that traverse complex potential energy surfaces. More accu- rate Âatomistic force fields that include quantum effects and can treat heterogeneous materials, better models for water and polarizability, improvements in implicit rep- resentation of solvation, and faster algorithms for computing electronic transport using first-principles methods are also required. The development of multiscale simulations that can accurately describe bio- molecular assemblies beginning from their fundamental protein building blocks represents a new challenge in terms of both their underlying theoretical basis and their computational implementation. As one illustrative example, Figure 4.11 depicts the multiple scales that exist for the actin filament (the major component of the cellular cytoskeleton). These scales begin at the atomistic level with the basic actin monomer building block depicted at the right in Figure 4.11. As with most such biomolecular assemblies, the atomistic-scale is then coupled to one or more intermediate (meso-)scales, and this in turn is coupled to the near-continuum scale (in this case the cytoskeleton network). This multiscale coupling means that the FIGURE 4.11â Example of a multiscale simulation approach to the study of an actin filament. The smallest scale is shown at right, and the model depicted becomes increasingly coarse- grained from right to left. SOURCE: Gregory A. Voth, University of Utah.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 99 behavior at the level of the atomistic-scale (molecular) domains is ultimately cru- cial in determining the collective properties at larger scales. Such problems abound in biology and biomaterials. Some critical challenges in addressing such problems are developing better ways of constructing simplified mesoscale potentials starting from atomistic representations to make the computations tractable; improving multiscale methods for global optimization of structures; and developing scale- bridging algorithms incorporating Monte Carlo methods, molecular dynamics, or other simulation methods. Modeling and Simulation of Cellular Processes The molecular and mesoscopic models described above should interface with theoretical and computational studies focused on understanding how cells func- tion and how these principles may be mimicked. In particular, information on bioÂmolecular interactions emerging from such calculations and experiments are inputs to studies of dynamic phenomena in cells. Cellular functions, and that of future cell-mimetic materials, are the result of cooperative dynamic phenomena that involve a myriad of membrane-proximal and cytosolic components (see FigureÂ 4.12). Because experimental tools to interrogate such processes are becoming avail- able, analogous computational studies are also emerging. Such calculations can be invaluable complements to the experiments in the quest for a mechanistic understanding of the underlying principles. Two classes of computational Âstudies are being pursued. The most common is based on mean-field descriptions of the pertinent phenomena in terms of ordinary differential equations. Such an approach cannot account for the spatial organization of components or the effects of Âstochastic fluctuations, features that are often proving to be important in biologi- cal systems. It is expected that spatial organization of components and stochastic fluctuations will also play an important role in the design of the various biomimetic systems that were envisaged in preceding sections of this report. Currently, spatially resolved stochastic simulations of dynamical phenomena in cells are carried out using recently developed variants of the Gillespie algorithm (or continuous-time Monte Carlo methods). Some important advances were recently realized by bring- ing together such computational studies and experiments. However, these Âmethods are extremely computation-intensive and difficult to apply as the complexity of the phenomena being studied increases. The understanding of biomolecular pro- cesses and concomitant design of biomolecular materials will be greatly aided by faster and more efficient algorithms for the simulation of spatially resolved, stochastic dynamic phenomena in cells. Another critical need is algorithms that can carry out parameter sensitivity and bifurcation analyses using such simula- tion tools (when the closed-form differential equations are not known). This is
100 Inspired by Biology ï¢ ï§ ï¡ ï FIGURE 4.12â The complexity in a signaling system. SOURCE: Joyce B. Easter, Virginia Wesleyan College. because in spite of advances in experimental technologies and molecular simula- tion methods, many molecular and mesoscopic parameters will be unknown, and it is imperative to establish the parameter range over which a mechanistic principle or design criterion is robust. It is possible that developing such methods will also impact computational investigation of phenomena that occur on larger scales (for example, collections of cells or macroscopic biomaterials with built-in nanoscale functionality). Studies of biological systems, from gene regulatory systems to the response of cells to therapeutics, are benefiting from the computational approach of Bayesian networksâprobabilistic graphical models that represent a set of variables and their causal influences. Combining this approach with models at molecular scales that provide input probabilities to the Bayesian network models is an important area for future growth. Field-theoretic simulations have been developed to study complex pattern for- mation in soft matter such as polymers and other complex fluids. These simulations
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 101 typically start from a free-energy function derived from statistical mechanics that describes the thermodynamic properties of the material. These models are powerful because molecular details are included at a coarse-grained level for the prediction of structures on long length and time scales. Driven systems such as biological systems that are not described by a free-energy function pose challenges for this approach, and additional processes must be considered that include energy input and output. Such approaches sometimes map on reaction-diffusion models and are used to study the emergence of spatiotemporal patterns in complex chemical systems. Combining these two related simulation methods to describe the large- scale assembly of biological components is an important opportunity for future research. As described later, a proper theoretical understanding of nonequilibrium phenomena remains elusive. Access to High-Performance Computing Environments Supercomputer centers play an important role in computational research on bimolecular materials and processes. However, many computations for bioÂ materials design require faster swaps of information than can be provided by grid computing or require resources that are more dedicated than are available at current superÂcomputer centers. Thus there is often a need for large local compu- tational resources. Various computing environments are being put to use to meet these requirements. Clusters of processors are now ubiquitous, and cost-effective ways of maintaining and operating them are emerging in various institutions (for example, centers for mid-range computing and co-location facilities). The field could be advanced if funding agencies and research institutions were to jointly develop mechanisms for providing and renewing local computational resources so that computational labs can remain at the forefront of the ever-evolving state of the art in computing environments. Another emerging and much publicized paradigm is distributed computing (for example, folding@home and SETI), which aims to harness computers all over the world to carry out specific computations. Its efficacy needs to be evaluated in the future as more data on performance become available. The next several years will see the development of petascale computers able to perform computations at an unprecedented 1015 floating-point operations per second (petaflops). These fast machines will take advantage of increases in pro- cessor speed as well as sophisticated multichip architectures for the simulation of biological processes 1,000 times faster than is currently possible. This means that simulation will be able, in principle, to model molecular processes over a millisecond time scales with femtosecond resolution. However, to be able to use these ultrafast supercomputers will require the (unprecedented) development of software algorithms capable of efficiently utilizing these new architectures, and
102 Inspired by Biology the development of such software has yet to begin in a substantive way. Efforts in this direction must begin at once, so as to leverage the enormous investment now being made in petaflop computers. Informatics and Data Mining Storing, accessing, mining, and sharing large amounts (terabytes to petabytes) of information generated by simulations and experiments pose a continuing chal- lenge for research in all fields of science and engineering. There are many examples of successful databanks in the biological sciences but far fewer in materials science, although many online resources do exist. Tools developed over the next decade, such as those based on Fedora, wikis, and the like, will revolutionize the sharing of data among researchers around the world as databanks increase in Âfunctionality and ease of access and use. The growing use of metadata tags in data files and all electronic resources should be encouraged, since metadata allow the creation of relationships among related data, which in turn allows for intelligent searching of related objects. Standards for interoperability, security, and data integrity will be required. The integration of federated databases with modeling and simulation codes will provide new opportunities for scientific discovery and prediction. Public Domain Codes While commercial software has an important place in scientific research, it is not always well suited to the needs of individual researchers and can limit the types of studies performed owing to its black box nature. At the same time, academic research codes developed by university research groups are not easily shared and are often used only within the group, leading to a duplication of effort. Thus the devel- opment and maintenance of public domain, open source codes are to be strongly encouraged. Interoperability and portability of codes within and across communi- ties will necessitate the widespread adoption of standards and responsibilities. The Need for Theoretical Advances Infrastructure for computational research is essential because results emerging from such research complement experimental work. Computational studies allow elaborating the consequences of mechanistic hypotheses and the calculation of material properties for specific systems. While synergy between computation and experimentation can provide some insights that go beyond the specific system studied, the development of overarching principles that govern the behavior of classes of systems requires theoretical studies as well. An understanding of general
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 103 principles is crucial for enabling the a priori design of materials with desired prop- erties. Therefore, theoretical studies should be strongly encouraged. Theoretical studies can be divided into two broad classes. The first comprises research that employs known fundamental principles to develop a deep under- standing of classes of phenomena. This type of research is often initiated by the experimental observation of a puzzling phenomenon but can turn into the formu- lation of new ideas and predictions that lead to further experiments. Research of several kinds will be invaluable. The understanding of collective dynamical phe- nomena in cells, especially how fluctuations are used and avoided, underpins many biological phenomena and should guide the design of biomimetic systems. Many interesting materials that are being studied and imagined today involve nanoscale systems. Understanding of the thermodynamics of such small systems is still not as mature as that of bulk materials. Fundamental studies in this regard will be a welcome addition to the knowledge base required for the design of biomolecular processes and materials that function on the nanoscale. Systematic and rigorous ways to bridge scales in computational studies will be important for the develop- ment of computational algorithms that could be used for design of biomaterials. A second class of theoretical studies comprises efforts to establish fundamental new ideas that are currently not available. Such problems need not ever be driven by a specific class of phenomena but may impact understanding of a vast array of scientific and technological questions. As has been noted several times in this report, many functional materials work under conditions far from equilibrium. Developing an understanding of the theoretical principles that govern the behav- ior of systems at equilibrium or close to equilibrium constitutes one of the major theoretical scientific advances of the twentieth century. For example, the Renor- malization Group Theory provides researchers with the concepts necessary to think about the behavior of systems near a critical point and classifies different systems into universal classes. Theoretical approaches to study the dynamics of systems close to equilibrium are also well established. Such general theoretical frameworks are not available today for systems far from equilibrium. This is largely true even for nonequilibrium steady states, although some advances in treating reaction- d Â iffusion systems in this regard appear to be promising. Two challenges that hinder the development of such theoretical principles are the inability to a priori identify slow degrees of freedom and the lack of the equivalent of fluctuation-dissipation relations far from equilibrium. The latter difficulty implies that although noise correlations can be very important in determining properties, they can only be inferred phenomenologically or guessed. Fundamental theoretical advances that address these (and related) problems will be a very important component of the arsenal of tools for understanding biomolecular processes and for developing bio- materials with precise functional properties.
104 Inspired by Biology Synthesis of Biomolecular Materials Our knowledge of the reactions, transformations, and mechanisms that govern the chemical behavior and synthesis of small molecule systems is rich and diverse. Researchers have sufficient mastery over small molecule organic synthesis with stereo- and regiospecific control. Researchers have also gained the ability to fully characterize complex small molecular structures with a number of spectroscopic techniques. Chemists have successfully isolated, characterized, and fully duplicated the synthesis of many natural products that have played an extremely important role in ameliorating human health and quality of life. However, at the macroÂ molecular, supramolecular, and nanoparticle scale a large gap exists; researchers have not gained nearly as exquisite a level of synthetic control. Translation of chemical concepts from the small molecule scale to the macromolecule scale is a difficult and often impossible task owing to the increased complexity of intricate material-based systems. Fully characterizing the sequence and conformation of such elegant and multifaceted architectures is also currently problematic, yet is of the utmost importance to accurately equating structure to function. At the most fundamental and essential level, materials researchers must acquire the ability to creatively synthesize, modify, and manipulate novel macromolecules and nano- systems with atomic-level control to broadly and accurately design and apply nanoscale materials to achieve revolutionary scientific advances. Indeed, macromolecule synthesis and assembly have been mastered in nature. Biomolecular function arises from the sequence, structure, and conformation by both covalent bond formation and noncovalent interactions within and among macromolecules. Sequential atomic arrangement, hierarchical assembly (discussed in Chapter 2), three-dimensional conformation, and allosteric interactions play an incredible role in the performance and activity of biomolecules. For example, DNA and RNA are supramolecular polymers built from only four monomers. Yet, in addition to the monomer sequence, three-dimensional conformation is important for coding the synthesis and assembly of diverse functional proteins. Proteins, too, are created by the differential arrangement of only 22 amino acids linked together through identical amide bonds. Nature uses this simplistic monomer set to create an infinite and complex library of proteins with functions ranging from cellular signaling and transport to complex molecule synthesis, assembly, and degradation. These extraordinary features are completely dependent on molecular-level com- positions and intramolecular and intermolecular interactions, which together in large numbers and over multiple dimensions determine the macromolecular and supramolecular conformation on the nanometer scale. In contrast to the simplistic set of biological monomers that make up such complex biofunctional architectures, a large number and diversity of monomers are available to scientists to create synthetic systems. Yet, the materials, synthetic
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 105 techniques, and applications developed by scientists remain simplistic by natureâs standards. Although significant progress has been made in assembling unique architectures of narrow dispersity, achieving natureâs idealistic standard of synthetic control with an unlimited monomer set has remained elusive. Indeed, Âtremendous 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 mate- rial, polyethylene. Ethylene and functionalized vinyl monomers are Âpolymerized 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. PolyÂ ethyleneimine (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 read- ily 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 fea- tures 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.
106 Inspired by Biology FIGURE 4.13â 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 understand- ing 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 Âmaterials synthesis, the systems must be compatible with a variety of functional groups, which can be a significant challenge. The discovery of Grubbsâ 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 sur- face receptor spacing and signaling interactions. In addition, the âclick reaction,â 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.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 107 Materials Synthesis Using Natural Machinery Although numerous metal-based catalysts offer some control over stereoÂ chemistry, 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, conforma- tion, 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 uti- lized to degrade ester bonds in lipids, have been shown to catalyze the formation of Â 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 conforma- tions, 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.
108 Inspired by Biology 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 conven- tional 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 4.14â (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, âTrehalose click polymers inhibit nanoparticle aggregation and promote pDNA delivery in serum,â Journal of the American Chemical Society 128:8176 (2006). Copyright 2006 American Chemical Society.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 109 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, sig- nificant 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 degrada- tion in biological systems and may yield diverse applications from artificial tran- scription 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 understand- ing, 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 syn- thesis. To this end, understanding the routes and mechanisms of macroÂmolecule assembly via noncovalent interactions is as important as developing new covalent coupling routes that link molecules together. Unique opportunities in Â 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-Âbonding and metal coordination (Figure 4.15a), and the backbone of a polymer can be functionalized via noncovalent interactions after polymerization (FigureÂ 4.15b).
110 Inspired by Biology FIGURE 4.15â Routes to create supramolecular polymers via noncovalent interactions. (A) Main-chain supramolecular polymers and (B) side-chain supramolecular polymers. SOURCE: M. Weck, âSide-chain functionalized supramolecular polymers,â Polymer Inter- national 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-Âbonding 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
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 111 FIGURE 4.16â 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 7.5Â nm in diameter and 1 Âµm 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, âHeparin binding nanostructures to promote growth of blood vessels,â 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 oppor- tunities exist in examining noncovalent polymerization and assembly methods
112 Inspired by Biology FIGURE 4.17â Polymer micelles can be formed by noncovalent interactions. Top: The shell can be chemically crosslinked to stabilize the nanoparticles. Bottom: Atomic force m Â icroscopy displays the nanoparticle shape and size. SOURCE: Karen L. Wooley, Washing- 4-17 new ton 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.
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 113 Molecular patterning and lithography are a powerful means of assembling struc- tures 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 accu- rate 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 biomol- ecules 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. â¢ Challenge: Achieving angstrom resolution in electron and X-ray imaging âOpportunity: Unprecedented elucidation, at the molecular level, of the structural and operational principles of many important cellular processes â¢ Challenge: Studying the structure, dynamics, and kinetics of assemblies of biomolecular systems using X-ray scattering techniques âOpportunity: Elucidating the dynamical processes involved in RNA genomes packing into viral capsids and probing the collective behavior of motors moving on their natural tracks â¢ Challenge: Probing reactions using neutron scattering at timescales of microseconds or longer âOpportunity: The ability to probe dynamical behavior as well as struc- tural correlations in biomolecular materials and processes â¢ 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 âOpportunity: 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 depen- dence of the motion in different environments
114 Inspired by Biology â¢ Challenge: Improving the temporal and spatial resolution of single-mole- cule techniques and integrating them into studies of larger macromolecular complexes that approach the complexity of actual cellular machines âOpportunity: Designing artificial biomolecular machines from insights into folding and self-assembly of complex biomolecules â¢ Challenge: Predicting the native conformations of macromolecules and the mechanisms and rates of conformational transitions âOpportunity: Designing bioinspired macromolecules that can per- form specific functions or serve as building blocks for functional supraÂ molecular structures â¢ Challenge: Developing rigorous multiscale algorithms that bridge the molecular and mesoscopic scales âOpportunity: The ability to understand and predict how formation and function of supramolecular structures depend upon the molecular building blocks â¢ Challenge: Developing efficient, stochastic, spatially resolved simulation methods that can study dynamical phenomena characterized by coopera- tion and feedback âOpportunity: The development of stimuli-responsive materials, like cells, that can perform precise functions â¢ Challenge: Developing methods to study the thermodynamics of small systems and understanding how noise can be exploited or avoided in col- lective dynamical phenomena to effect a desired function âOpportunity: The design of nanoscale biomaterials through greater understanding of their thermodynamics and stochastic fluctuations â¢ Challenge: Developing a rigorous theoretical understanding of how to describe systems far from equilibrium âOpportunity: Greater understanding of all the topics outlined in this report since many biological systems and functional biomaterials oper- ate far from equilibrium â¢ Challenge: Manipulating the organization of hierarchical assemblies (the secondary, tertiary, and quaternary structures) of biomolecular materials âOpportunity: The ability to create macromolecular, or even cellular, synthetic biomolecular materials from molecular building blocks â¢ 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) âOpportunity: Unprecedented control over the synthesis of new and complex biomolecular materials
Probes and Tools for B i o m o l e c u l a r M at e r i a l s R e s e a rc h 115 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, âProspects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: Implications of crowding,â Biophysical Chemistry 100(1-3):577-591 (2003). Hawker, C.J., and K.L. Wooley, âThe convergence of synthetic organic and polymer chemistries,â Science 309:1200 (2005). Langer, R., and D.A. Tirrell, âDesigning materials for biology and medicine,â 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, âOptical trapping,â 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, âDiscovery of dendrimers and dendritic polymers: A brief historical perspec- tive,â Journal of Polymer Science Part A: Polymer Chemistry 40:2719 (2002). Zlatanova. J., and K. van Holde, âSingle-molecule biology: What is it and how does it work?â Molecular Cell 24(3):317-329 (2006).