Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
2 Understanding Biomolecular Processes: Toward Principles That Govern Biomaterial Design The application of new technologies such as DNA chips and fluorescent label- ing of molecules has led to remarkable progress in the ability to collect detailed data about biological processes. Advances in genetics and proteomics are produc- ing huge amounts of data. New knowledge of gene regulation and cell signaling is resulting in an ever more detailed understanding of these complex phenom- ena. With this increasing amount of data comes increased understanding of the mechanisms that underlie many of these processes. For example, the understand- ing of the role of restriction enzymes has led to completely new applications in gene manipulation. These enzymes selectively cut strands of DNA to protect cells against viral invasion, yet they do not cut the cellâs own DNA. As another example, the discovery of small interfering ribonucleic acids (siRNAs) will have enormous impact on studies of gene regulation and cell signaling. The list of such discoveries is constantly growing. This improved understanding of the principles that underlie biological dynam- ics and function also creates new opportunities in biomolecular materials. It is now possible to design materials that not only mimic the properties of biological materials but also mimic the function and underlying principles of biological systems. This represents a significant step forward for biomolecular materials. Current research could lead to qualitatively new materials and to qualitatively new functions and methods for use of these materials. Such possibilities become feasible when new knowledge of biological processes is combined with the ability to fabricate new materials. While good progress has been made so far, the potential impact of new func- 10
Understanding Biomolecular Pro cesses 11 tional materials through continued understanding and exploitation of biological processes is enormous. In this chapter, specific examples of research on biomo- lecular processes are discussed. The focus of the chapter is on learning the prin- ciples of biomolecular processes, which could then be used to design biomolecular materials. Imagine that one could . . . ⢠Create sensors having the exquisite sensitivity and accuracy of the immune system, able to detect minute quantities of molecules with a very high precision. ⢠Create new biomolecular materials with highly adaptable and controllable properties based on the mechanical design principles of cells, where bio- molecular motors can actively control the stiffness of the networks that give the cell its rigidity. ⢠Assemble new materials with the incredibly detailed precision made pos- sible by interactions that result from the sequence of oligonucleotides. ⢠Engineer advanced materials that mimic evolution and adapt their proper- ties to address new environmental pressures or to self-heal disruptions. ⢠Develop advanced materials that self-replicate, storing structure and func- tion information in the materials themselves, just as is done in the genomes of all living species. These revolutionary scientific goals represent the future impact of the new materials that will be possible through increased understanding and utilization of biomolecular processes. Following discussion of certain areas of research, specific challenges and opportunities are discussed at the end of this chapter. Multiple cooperative interactions Nature has evolved impressive means to sense and respond to a wide variety of stimuli. For example, in response to many external stimuli, individual cells can modulate their mechanical properties, shape, growth rate, motility, secretory functions, and the biochemical and charge characteristics of their surfaces. The extraordinary precision with which they sense environmental cues and the exquisite control with which they modulate their properties to affect specific functions is not matched by any synthetic material. The ability to even crudely mimic this ability of cells is expected to find application in a myriad of technologies. The key process that facilitates this precision, sensitivity, and selectivity, and the exquisite control in response, is recognition based on the many weak but highly cooperative interactions that are buttressed by positive and negative feedback mod- ules spanning a spectrum of time and length scales. Scientific understanding of the
12 Inspired by Biology principles underlying such hierarchically arranged cooperative processes involving feedback is still rudimentary. With an understanding of these principles, scientists will be able to create materials with unprecedented functional capabilities. The process of unraveling the principles will be expedited by bringing theoreti- cal and computational approaches together with experimental approaches such as genetic, biochemical, and imaging experiments. Expertise in the statistical mechan- ics of complex systems from the study of soft materials will also be a great asset. In addition, the effects of stochastic fluctuations must be included and properly treated. Ideas from network theory may also help researchers to understand coop- erative processes that take place on very different length and time scales. Research at the crossroads of statistical mechanics, materials science and engineering, and molecular and cell biology should pay dividends for both fundamental biological research and the understanding of these essential highly cooperative processes and interactions. Some examples of research focused on understanding the specificity of detection and the precision of response in biological systems follow in the next subsections. Cells Many living cells carry out specific functions when they sense certain external stimuli. One example of cells that function this way is the T lymphocytes, also known as T cells. They have evolved to deal with pathogens that are no longer in the blood or on mucosal surfaces but have penetrated other cells. Because they combat pathogens that have not been previously encountered, they are critical components of the adaptive immune system in higher organisms such as verte- brate animals. They can rapidly and sensitively detect the presence of biological and chemical hazards. Moreover, they can detect minute quantities of hazardous molecules without frequent false positive responses. Thus mimicking T cells would be one way of overcoming an important challenge facing society todayânamely, the rapid and sensitive detection of biological and chemical hazards in the envi- ronment, including unknown pathogens that could be engineered, perhaps from existing agents. Specialized cells, called antigen-presenting cells (APCs), display molecular signatures of the pathogen on their surface (Figure 2.1). Antigen-derived pro- teins are cut up into small peptide fragments by enzymes in APCs. These peptide fragments can then bind to major histocompatibility proteins, and this complex of peptide (p) and major histocompatibility (MHC) is the molecular flag of the pathogen displayed on the surface of APCs. Peptides derived from proteins of the host organism can also bind to MHC molecules, and these self pMHC molecules are also displayed on APC surfaces. T cells can detect as few as 10 antigen-derived pMHC molecules in a sea of tens of thousands of self pMHC molecules.
Understanding Biomolecular Pro cesses 13 FIGURE 2.1â T cell (shown in blue) interacting with an antigen-presenting cell (in green). The latter display molecular signatures of unknown pathogens. T cells can detect fewer than 10 such molecules based on cooperative interactions between membrane-bound and cyto- plasmic molecules. SOURCE: Michael L. Dustin, Kimmel Center for Biology and Medicine of the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, Program in Molecular Pathogenesis. An understanding of how T cells operate is still emerging. Recent results from studies that bring together the physical and biological sciences suggest that a deep understanding of how T cells use cooperative interactions and feedback regula- tion of signaling cascades for sensitive detection could develop in the coming years. Related studies of other cellular components of the immune system (e.g., natural killer (NK) cells and macrophages) that serve as sentinels have also been illuminating. Further work along these lines will result in an understanding of the biomolecular processes that could then be harnessed to design synthetic materials that can mimic the specificity of the cells that comprise the immune system.
14 Inspired by Biology Understanding how cells of the immune system function is greatly aided by advanced experimental technologies that provide vivid images of the spatiotempo- ral evolution of key cellular components. The activation of a T cell (and, indeed, other types of cells) is an emergent property in that it is the consequence of coop- erative dynamic events that involve a myriad of membrane-associated and cyto- plasmic components. An understanding of the mechanistic principles is essential for the future creation of cell-mimetic materials that will incorporate only those components necessary to affect a particular stimuli-dependent response. More generally, the exquisite sensitivity of cells to their environment and their complex yet detailed response to stimuli can be harnessed in many other ways to create new materials. For example, cells can themselves be used as detectors in many different sensors, from specific assays to test for disease or individualized response to drugs, to highly selective and sensitive detectors of pathogens. This use requires an interface between the cell, its control and response circuitry, and the more tradi- tional electronic circuitry of modern instrumentation. This interfacing of systems represents an important challenge in biomaterials research. Another potential use of cells themselves is in personalized medicine. For example, the response of cells from an individual could be tested against different drug combinations to opti- mize the choice for the individual. All these uses require research into the behavior of cells and into the new biomolecular materials required to create the interface between the cell and ancillary electronics or other readout mechanisms. Cell-mimetic Materials Future research opportunities also exist in the creation of bioinspired systems that mimic the behavior and properties of T cells. These will probably be based on synthetic vesicles that contain the key biochemical elements of the signaling machinery and secretory apparatus identified by studies of the biomolecular pro- cesses inherent in T cells. One specific class of candidate materials that may suit this purpose are polymersomes, capsules formed from bilayers of complex amphiphiles (for example, short peptide amphiphiles or co-assembled cationic-anionic amphi- philes). Polymersomes are stable structures into which molecular functionality can be incorporated. A major challenge is to determine how to make them interact with the environment in a more flexible manner as cells of the immune system do. For example, can they be made to open and close âporesâ in response to signals? The challenge here is the development of structures that serve as the support surface and encapsulate the cell while remaining robust and flexible. It is also important to understand the organization of components on living cell membranes and synthetic vesicles. The challenge here is to develop systems that can both sense the stimuli and simultaneously respond to them in some pro- active way. This effort will benefit from a combination of spectroscopy, materials
Understanding Biomolecular Pro cesses 15 FIGURE 2.2â Hydrogel microwells used to create microarrays of single live lymphocytes. Micrograph at right shows array of live B cells. SOURCE: H. Kim, R.E. Cohen, P.T. ÂHammond, and D.J. Irvine, âLive lymphocyte arrays for biosensing,â Advanced Functional Materi- als 16:1313 (2006). Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission. science, and biology. In addition, platforms where materials such as polymersomes can sample the environment will also be essential for developing sensing devices that mimic the immune system. High-throughput experimental techniques may prove particularly valuable. One example is the patterned surfaces that can mimic lymphoid tissue in living systems (see Figure 2.2). Processes far from equilibrium One of the distinguishing features of virtually all biological systems is that the description of materials and kinetics using equilibrium statistical mechanics often no longer applies. This is mainly because the molecular motors and other molecules that are present convert chemical energy, usually in the form of adenosine triphos- phate (ATP), into mechanical energy, increasing the level of mechanical activity in the cell. It also results in fluctuations within the cell that can appear remark- ably similar to Brownian motion but that are not driven solely by thermal effects. Interestingly, when Brown first observed the motion that now bears his name, he attributed this motion to âvitalâ processes due to living objects, and it was only after he observed the same effect in clay and other inanimate objects that he real- ized that the motion is in fact ubiquitous. It was, in fact, Einstein who ultimately confirmed the purely thermal origin of what is now known as Brownian motion.
16 Inspired by Biology However, certainly within the cell, and probably in many other places, the result of constant but random motor activity is fluctuations that have many features in common with thermally induced Brownian motion, much like those first imagined by Brown during his seminal studies. The nonequilibrium fluctuations that result from motor activity have a sig- nificant impact on the behavior of all the dynamics within the cell. They can affect the mechanical properties of the structures within the cell, and they can also affect the signaling of the cell and its response to stimuli. Thus a better understanding of these effects is essential. More generally, application of all the tools of statistical mechanics to the dynamics of biological processes is hampered by the fact that so many things are way out of equilibrium, so that it will be essential to develop new theoretical and conceptual tools that can directly address such nonequilibrium processes. Clearly the nonequilibrium transport of molecules is critical for cell function and often accomplished by molecular motors. These are discussed further in Chap- ter 3. Another critical transport process is that of ions. The precise measurement, molecular description, and elaborate analysis of ion transport through ~1-Ã -wide channels combine to give what is probably the best example of rigorous physical thinking on a biological material. The determination of the structure of a chemical- and voltage-dependent potassium channel, together with the electrical observation of single channels, allows researchers to speak quantitatively of the mechanisms that control the channelâs opening and closing, to allow potassium ions to move out of a firing nerve cell. At the next level, the nanometer level, there has been sys- tematic work not only on sizing channels to study the physics of transport through well-defined structures but also on determining how these channels might prove to be conduits for specifically designed antibiotics or nutrients to enter cells. Detailed understanding of transport processes will facilitate new applications. For example, can the understanding of ion transport be generalized to nonbiological systems, where control of charge can be a mechanism to control the structure or function of biomolecular materials? While motor activity within the cell drives nonequilibrium fluctuations, the cell is, nevertheless, always near room temperature, and many biochemical pro- cesses can be described by traditional equilibrium statistical mechanics. This is particularly true of the enzymatic reactions that are so critical to the function of the cell. The interplay between the equilibrium and nonequilibrium phenomena will provide much insight into the nature of many essential cell functions, and this represents an important area for further investigation. Moreover, as these processes become better understood, new ways to harness the use of cells and the tissues that are constructed from them will surely emerge.
Understanding Biomolecular Pro cesses 17 Design Principles for Mechanics The cell is a remarkable construction that combines both specific function and flexibility of performance with an array of mechanical properties. It is highly controllable and highly adaptable, and its properties can change significantly in response to external stimuli. The cell can change its shape and become motile; it can both sense and respond to forces in its environment. At the same time, the cell is sufficiently rigid to maintain its own shape. Understanding the design principles that control the mechanics of the cell and other living systems would facilitate the development of biomolecular materials that share these physical properties. The elasticity of a cell comes from several different load-bearing structures: The cytoskeleton is an elastic network throughout the cell, made primarily of actin filaments and many cross-linking proteins. Microtubules are the stiffest rodlike element in the cell and provide both structural support and physical pathways for transport of material within the cell. Intermediate filaments form a structural component that provides elasticity to the cell and bears tension. The stiffness of the networks that make up the cell is highly controllable. The networks are under internal tension, which is balanced both by adhesion of the surface of the cell to the surrounding medium and by the compressional load-bearing capability of the microtubules. Internal tension is provided by activity of biomolecular motors within the cell. The elasticity is highly sensitive to the degree of internal prestress, providing a sensitive control mechanism through regulation of motor activity. Cells exert forces in many ways: Molecular motors within the cell can provide forces of several piconewtons each and, operating in concert, can exert much larger forces. The elasticity of the networks that make up the cell can also provide a force when they are strained. The cell is also constantly remodeling its shape, and the polymerization of the network components during the course of this remodeling also provides a force. The cell adheres to its surroundings and can exert a tension on the matrix, coupled through focal adhesions, the points where the cell is adhered to the external environment. Motor activity within the cell is coupled to the matrix through these focal adhesions to exert the external force. Cells also respond to the elasticity of their surroundings; for example, the differentiation of stem cells is strongly influenced by the stiffness of their surroundings. Molecular motors, in conjunction with the cytoskeleton, control cell shape, division, targeted intracellular transport, and many other cellular motions. These motors are briefly described in Chapter 3. Muscle contraction is an extreme example of cell motility that allows higher organisms to maintain posture, move, walk, and swim. At the microscopic level, the contractile organelle is termed the sarcomere. Micrometer-sized filaments of polymerized actin and myosin interdigitate and slide to produce force and shortening. The molecular mechanism of the sliding follows the general operational properties of protein motors described in Chapter 3.
18 Inspired by Biology Hundreds of sarcomeres arranged in series and in parallel within the muscle cell increase the force and motion to the macroscopic levels required to achieve locomotion. Muscle contraction is highly adaptable to the conditions of work, and individual muscle cells vary considerably in their speed, metabolic requirements, and resistance to fatigue. The transduction of metabolic energy into work is more than 50 percent efficient. Man-made actuators for animal and robotic locomotion have generally been based on different principles, such as electrical or magnetic forces. The energy density of these actuators is usually lower. Understanding the principles of muscle contraction and, even more particularly, how they self-assem- ble into the highly regular sarcomeric structure may lead to bioinspired actuators, which could be used in man-made devices as prosthetics or as actuators or drives in other devices. Another important mechanical function within cells is the transport of materi- als. As the cell grows or remodels, material must be transported from one location to another. Given the size of a cell, one would expect reasonably rapid diffusion. In fact, the interior of a cell is a very crowded environment, slowing any diffusive transport. As a result, the cell typically relies on molecular motors to actively trans- port materials. Mimicking such active transport could qualitatively change the way transport is accomplished in biomolecular materials. This would make it possible to specifically target what and where things are transported, using active processes that overcome the limitations inherent in random diffusion. The number of certain molecules in a cell can be very low, especially those involved in gene expressionâfor example, DNA, specific messenger RNAs (mRNAs), and transcriptional and translational regulators. The randomness of both the interactions and the conformational transitions of these molecules leads to fluctuations in the content of a given protein in cells that are otherwise identi- cal. Theoretical and experimental studies have shown that such fluctuations are inevitable at the low copy numbers of regulators and the rates of cellular processes. So-called âgene noiseâ and its dynamic characteristics have been detected from variations of mRNA and protein expression between cells. These fluctuations limit the precision of expression in cellular regulatory networks, but they may also be advantageous for development, adaptation, and evolution by facilitating the sampling of nearby alternative states that may improve cell function. Signaling processes such as those that may be important to mimic in creating cell-mimetic materials can also involve molecules present in small copy numbers. The influence of stochastic fluctuations on such signaling processes has received less attention. It is possible that signaling processes have developed ways to quench the deleteri- ous effects of fluctuations while exploiting them to generate phenomena such as discrete decisions. Biomimetic and nanoscale materials may also exhibit statistical fluctuations when very small copy numbers are involved; these need to be taken
Understanding Biomolecular Pro cesses 19 into account in the design of the material, as they may be advantageous for adapta- tion to changing conditions. The adaptability of the mechanical properties of the cell serves as a model for the properties that can be achieved by biomolecular networks. However, to fully exploit the remarkable properties of such materials, it is essential to determine the underlying design principles that determine their properties. What gives small amounts of these materials such very high strength? What is the underlying cause of the highly nonlinear behavior? What is the role of the specific cross-linker? How does the cross-linker determine the network architecture? What impact does the architecture have on the mechanical properties of the network? How is the nonlinearity controlled? If these basic materials design principles are understood, they should provide the requisite guidance for fabrication of new materials with similar properties. For example, a consequence of malaria infection is that the mechanical properties of the red blood cells are altered, making it difficult for them to squeeze through blood vessels. If one could change the mechanical properties of cells, one might be able to address significant problems like malaria. Can materials be designed that can change their mechanical properties in response to environmental cues? Can an intrinsically hard material be designed that becomes soft when it senses certain stimuli? Such a capability might allow a material to enter compartments that oth- erwise exclude it and then carry out some function. Can the control circuitry of the cell, which depends on the highly regulated activity of biomolecular motors, be adapted to other materials? This would allow the stiffness of a material to be changed by several orders of magnitude by tuning motor activity. Knowledge of the fundamental design principles of the cell will facilitate the building of bioinspired structures, formed either directly from biomolecular materials or, alternatively, from strictly synthetic nonbiological analogs. Such bio- inspired materials can be designed to mimic the behavior and dynamics of the cell and to recreate its remarkably adaptive and highly controllable mechanical proper- ties. Any material fabricated with these design principles would, ideally, be scalable: The same principles could apply, for example, to the construction of macroscopic networks, which could have similar strength-to-weight ratios and which would have similar controllable mechanics. These materials would represent a truly new class of material. self-ASSEMBLY, directed ASSEMBLY, and spatiotemporal Assembly Assembly of complex structures is ubiquitous in nature, from the one-of-a- kind patterns of individual snowflakes to the membrane that surrounds every cell. Assembly is a common paradigm in materials, biological and nonbiological
20 Inspired by Biology alike, but the mechanisms driving the assembly processes are profoundly different. Evolution has perfected assembly mechanisms that lead to remarkably complex structures in biology; examples include microtubules, cell membranes, viruses, and a myriad of other structures that far surpass traditional materials in both design and functionality. Assembly processes can be divided into several categories. Self-assembly refers to the spontaneous organization of preexisting components under the influence of forces acting among the components. Self-assembly is generally considered to be a reversible process, tunable by varying a thermodynamic parameter such as tem- perature or density, and one that can be controlled through judicious design of the components. Typically, self-assembled structures form based on thermodynamic principles in which free energy is minimized subject to constraints. In polymers, liquid crystals, metal alloys, and other nonbiological materials, van der Waals and electrostatic interactions between atoms and molecules conspire to produce bulk materials whose elementary building blocks self-organize into stable equilibrium patterns. This same mechanism is also observed in colloidal and nanoparticle sys- tems, where additional solvent-mediated interactions contribute to self-assembly. Self-assembly is not always sufficient to ensure the organization of molecular or other building blocks into highly organized structures. Sometimes the system can get stuck in kinetic traps. To overcome these limitations, guided or directed assembly exploits the application of external fields, such as electric, magnetic, or shear, to help align the assembling particles, affording another means for structural organization. Alternatively, templated self-assembly involves the use of scaffolds or templates to provide a pattern on which building blocks order; such templates can be used to promote the formation of a desired structure in preference to competing structures with similar free energies. Nature exploits all of these categories of assembly processes. However, nature also uses a much more sophisticated assembly process that combines many spatial and temporal scales. For example, precise specific interactions among proteins lead to the self-assembly of highly organized complex structures, often with no known analogue in nonbiological systems. These structures may be dynamic, stable far from equilibrium, and may reorganize based on dynamically changing interac- tions among the constituents, leading to complex, emergent behavior. Processes carried out in living cells, for example, depend on the spatial organization of many different chemical components. These assembly processes are often hierarchical: Self-assembly of elementary building blocks results in secondary building units, which assemble into tertiary building units, and so on. This hierarchical assembly can lead to powerful functionality. However, a fundamental understanding of these spatiotemporally correlated assembly processes remains elusive. Such understand- ing is essential if we are to exploit the mechanisms that control many biological
Understanding Biomolecular Pro cesses 21 systems. It is also essential to be able to mimic these assembly processes to create new materials that possess new functionality. One important example of directed assembly that has already been widely investigated is the use of DNA as a building block to program the self-assembly of larger structures. For materials, this approach holds the promise of fabricating more complex structures than are currently attainable. Both DNA and RNA are molecular materials for programmable assembly in which the selective affinity of base pairs on strands of these molecules is exploited for fabricating nanostructures with designed geometries. Through judicious design of the base pair sequence, oligonucleotide strands can be fabricated to bind reversibly with complementary strands. This unique property of DNA allows this âmolecule of lifeâ to be used as an architectural element, both as a building block itself and as a linker of building blocks; this has resulted in new areas of research, such as DNA computing, struc- tural DNA nanotechnology, and programmable self-assembly (Figure 2.3). Like RNA, DNA assembles into structures other than the double helix, includ- ing hairpins and multiway junctions. Cohesive or âstickyâ ends on these motifs allow for their use as architectural elements for larger nanostructures, including cubes (as in Figure 2.3), truncated octahedral rings, knots, bricks, and three- dimensional crystalline arrays. These DNA structures and nanoarrays can further serve as templates for nonbiological materials and as scaffolds for nanoelectronic components and nanomechanical devices; examples include a bipedal walker and a translation device. The field of structural DNA nanotechnology is an emerging area of biomolecular materials research at the intersection of the physical and biological sciences. Another potential application is in computation; DNA nanostructures have been used as molecular building blocks for self-assembled tilings, which can, in turn, be used for molecular computation. The first two-dimensional example of this was an âexclusive orâ (XOR) logic function. Hierarchical Self-assembly A classic example of precise hierarchical self-assembly is the virus. All viruses are made up of protein coats (called capsids) that protect the viral genome. Cap- sids are self-assembled from groups of proteins called capsomers, which interact noncovalently to create the capsid structure; in some viruses, chaperones help to direct their assembly. In about half of all viruses, the capsid is roughly spherical and takes the shape of a perfect, 20-sided polyhedron or icosahedron composed of integer multiples of 60 proteins. Influenza, herpes simplex (HSV-1), human rhinovirus (which causes the common cold), and hepatitis B are all examples of icosahedral viruses. Other capsid shapes include prolate spheroids or ovoids (aber- rant flock house virus (FHV) and alfalfa mosaic virus (AMV)), cones (HIV), and rods (tobacco mosaic virus and H5N1, or avian flu, virus).
22 Inspired by Biology FIGURE 2.3â DNA cube with six different cyclic strands. Their backbones are shown in red, green, yellow, magenta, cyan and dark blue. Each nucleotide is represented by a single colored dot for the backbone and a single white dot for the base. Note that the helix axes of the molecule have the connectivity of a cube. However, the strands are linked to each other twice on every edge, making this molecule a hexacatenane. The red strand is linked twice to the green, cyan, magenta, and dark blue strands and only indirectly to the yellow strand. Each edge of the cube is a piece of double helical DNA, containing two turns of the double helix. SOURCE: Nadrian C. Seeman, New York University. The controlling factors that govern the self-assembly of proteins into these highly ordered structures precisely, rapidly, and repeatedly to propagate an Âinfection in living organisms are not yet understood. Many theoretical models exist, most involving the complex interplay of specific and directional noncovalent interactions among protein building blocks. Control of these interactions and the subsequent assembly process would allow the design of antiviral drugs to interrupt virus replication or the fabrication of empty viral capsids containing diseaseÂ-Âfighting
Understanding Biomolecular Pro cesses 23 drugs. Moreover, an understanding of the basic design and assembly principles of these structures would permit the use of complex, hierarchical self-assembly processes to construct many other completely different structures, either from proteins or from inorganic nanoparticles designed to assemble as proteins. New âpatchyâ particles with diameters between 1 and 1,000 nm are now being syn- thesized with unprecedented anisotropy as building blocks with directional and specific interactions for assembly into complex structures. Furthermore, viruses themselves, or bioinspired structures imitating viruses, could be used as building blocks for the construction of new materials on a larger scale. For example, highly precise and symmetric capsid and capsidlike structures can serve as templates for novel new materials such as oriented quantum-dot nanowires. The use of viruses or other biomolecular materials such as proteins, including engineered proteins, as structured building blocks to create larger structures represents an important opportunity in hierarchical self-assembly. Another example of hierarchical self-assembly is implicated in amyloid dis- eases, in which peptides assemble into beta-sheet tapes, which assemble into ribbons (double tapes), fibrils (twisted stacks of ribbons), and ultimately fibers (entwined fibrils). Such fibrils are thought to play a critical role in diseases such as Alzheimerâs and Pickâs. Building block chirality is thought to be very important in the hierarchical assembly of many such biological structures, but the process underlying the order of various subprocesses in this type of assembly is poorly understood. By contrast, assembly in hard materials such as ceramics and metals is not typically hierarchical, and in soft materials and complex fluids such as surfac- tants and block copolymers the hierarchy is often limited to the self-organization of secondary aggregates into ordered liquid crystalline lattices. An understanding of how nature exerts spatiotemporal control over the assembly of groups of building blocks to create precise structure at successive scales would be of great value. For example, it could allow development of drugs to constructively disrupt the fibril formation responsible for Alzheimerâs. In addition, it could open the way to the exploitation of similar principles to create hierarchically arranged structures with nonbiological materials. Complex Spatiotemporal Assembly In many biological systems, different spatial patterns form over time. One such example is provided by the formation of the immunological synapse. As described earlier in this chapter, the orchestrators of the adaptive immune response are a class of cells called T cells. When they interact with antigen-presenting cells (APC) and recognize the molecular markers of pathogens, different types of receptors and ligands organize themselves into specific spatial patterns that evolve with time (Figure 2.4).
24 Inspired by Biology FIGURE 2.4â Formation of the immunological synapse: T cells in contact with a supported planar bilayer. (A) Images of contact formation as seen with interference reflectance micros- copy and (B) as seen with fluorescing Oregon green. SOURCE: A. Grakoui, S.K. Bromley, C. Sumen, M.M. Davis, A.S. Shaw, P.M. Allen, M.L. Dustin, âThe immunological synapse: A molecular machine controlling T cell activation,â Science 285:221 (1999). Panels A and B show how a T cell interacting with a supported bilayer mimics the APC that contains ICAM-1 and pMHC. These images are taken looking up. Panel A shows the time evolution of the shape of the T cell during synapse forma- tion. The darker the color, the closer the apposition between the T cell membrane and the supported bilayer. Panel B is an overlay of peptide-MHC molecules (green) and adhesion molecules (red) concentrations in the intercellular junction. Movies that make these observations of the spatiotemporal evolution of protein patterns and cell shape vivid can be seen online. Similar spatiotemporal patterns were first observed at the junctions between a T cell and an APC. These spatial patterns are thought to form by a guided self-assembly processes. The intrinsic tendency of receptor-ligand pairs of different sizes to separate due to coupling between membrane elastic forces and topographic size differences is amplified by cytoskeletal motion triggered by T cell signaling. Although there is still no consensus on the exact function of these spatiotemporal patterns, the patterns are thought to mediate specific functions. A fundamental understanding of such guided self-assembly of spatiotemporal patterns of molecules could be exploited in the design of biomolecular materials that perform different functions over time. Even more complex examples of a spatiotemporal, hierarchical assembly process are motility organelles such as the bacterial flagellum and membranous organelles such as the Golgi apparatus and the endoplasmic reticulum. These are âMovies of these observations are available at www.sciencemag.org/feature/data/1040037.shl. Last accessed March 30, 2008.
Understanding Biomolecular Pro cesses 25 examples of highly ordered, precise constructs, whose structure arises from self- assembly and is required for functionality. Bacterial flagella, for example, contain an intricate, 20-nm-diameter helical filament self-assembled from flagellin pro- teins. Depending on the filamentâs stress state, the proteins within can pack in either a left-handed or a right-handed configuration. This transition in configu- ration reorients the swimming direction of the cell. It arises from subnanometer conformational changes in the protein subunits that constitute the flagellum. A quantitative understanding of how applied stresses and torques lead to an overall polymorphic transition from one chirality to another has remained elusive. Such an understanding could provide the basis for nonbiological sensors, actuators, and other nanomechanical devices. Assembly promises to remain an overarching theme of research at the intersec- tion of biology and materials in the foreseeable future. Hierarchical organization, external fields, and biomimetic motifs for guiding self-assembly, and dynamic self-assembly in dissipative systems, are likely to grow as key themes. Assembly of dynamic structures that are responsive to external stimuli, such as the bacterial flagellum, holds great promise for nanotechnology. The exploitation of anisotropic, noncovalent interactions at nano and colloidal scales is an important biological approach that could be applied to the assembly of nonbiological materials. Break- throughs in understanding biological assembly processes, and in mimicking it to create new materials and devices, will revolutionize materials fabrication and development. Self-replicating, self-healing, and evolving materials A tenet of all biology is that organisms have evolved to their current state and continue to evolve as they are subjected to environmental pressure. This has allowed finding effective solutions to problems. They are not necessarily the best solutions, but they are solutions that work. As a result, biological species are constantly chang- ing, adapting, and evolving. Can similar design principles be applied to materials? A key feature of evolu- tion is the genetic encoding of information, which provides an essential means for modification and allows the modifications to become permanent. A living organism responds to environmental pressure by changing its behavior or struc- ture. While this response may ultimately be specified in the genetic code, there is also strong evidence that many changes do not involve direct modification of the genetic code; instead, they involve modification of the complex response of the entire system, exploiting the multiple cooperative interactions and feedback regu- lation discussed earlier in this chapter. While evolution is highly complex and still poorly understood, the potential for mimicking it is enormous. This could allow the development of materials that actively adapt to their surrounding environment.
26 Inspired by Biology It would also make possible the development of materials that modify their behav- ior to perform new functions in response to changing conditions. These materials could be self-healing, repairing themselves upon being damaged. Self-replicating Materials An important feature of living systems is that all the information for the indi- vidual member of the species is encoded in the genome. Indeed, each cell has all the information required for reproduction encoded within its genome. This is the key to both producing and reproducing life. This is also the essential challenge: to create a biomaterial that mimics this information content. The challenge might be met by using the same material and sequence information as is used in nature. Considerable effort is being focused on the use of DNA to assist directed self-assembly of colloidal particles. This methodology exploits the precision and specificity with which DNA can bind. Thus, for example, the formation of colloidal crystals and crystalline alloys by binding with DNA oligonucleotides has already been demonstrated, and similar structures can certainly now be fabricated. Even if the basic principles are completely understood, and even if they can be incorporated in a material, significant challenges would remain to manufacture the material. Again it is likely that the optimal way to do this, at least initially, is to follow the lead of biology and to use biological materials directly. While this will limit the number and type of materials that can be produced, it will allow known principles to be exploited directly. As researchers learn more about the principles, it is conceivable that other materials will be made following those principles. These could lead to the fabrication of a whole new class of materials. Self-replicating materials are now being made by another means, called syn- thetic biology. Organisms such as E. coli are being programmed to perform specific activities, usually mimicking specific computer gates such as functions of Boolean logic, making the bacteria work in ways that are analogous to simple computers based on binary logic. This represents an opportunity to harness some of the meth- odology of nature for other uses. Ultimately, the functions will reflect functions found in biology rather than in the physical sciences. This will call for knowing much more about the specific functions in biological systems. While synthetic biol- ogy can reproduce functions familiar to more traditional computation, possibilities well beyond these could be explored. For example, might not some of the complex calculations performed by living organisms be designed into synthetic biology sys- tems, and might not such a capability be harnessed to perform computations?
Understanding Biomolecular Pro cesses 27 Self-healing Materials One distinguishing feature of living organisms is their ability to heal. This ability could take the form of preventing an invasion of foreign substances, as exemplified by the immune system discussed above. Alternatively, it could entail regrowing regions that die or regenerating regions that are injured. Because all the structural and functional information is encoded in the genome, each por- tion of the living system can, in principle, be regenerated. Stem cells could be one resource for constructing self-healing systems. Understanding the mechanisms by which stem cells differentiate into specific cells might allow similar adaptability in biomimetic systems. There are numerous other examples where a deeper understanding of interac- tions at the molecular or cellular level could lead to other self-healing systems. For example, the adsorption of biomolecules at interfaces could serve as a geometric constraint that allows new function and interactions to occur, and these could be harnessed to create alternative forms of self-healing materials. Many biologi- cally important interactions, such as those between membrane proteins, occur at interfaces, and they remain poorly understood. As our understanding of such interactions improves, they can be exploited to create new materials. The key to understanding many of them is a better comprehension of the effects of the inho- mogeneity at the interface of the dielectric constant of the material. Materials That Evolve An essential feature of biology is the adaptability of living systems to their envi- ronment and to changing environmental pressures. Understanding the way living systems adapt will help us to design biomolecular materials with the same traits. Living organisms also adapt over time, through the process of evolution. Adapta- tion occurs at the molecular level, through the evolution of new proteins; at the cellular level, as cells evolve in their function and responses; and at the level of the organism as species evolve. Understanding the process of evolution, particularly at the molecular level, may be able to show a new way to the development of more sophisticated biomaterials, as described in the examples below. One endeavor where the principles of evolution are already being exploited is âdirected evolution,â a way to create new enzymes. This approach adapts the Darwinian process of evolution to create new molecules by subjecting the origi- nal enzyme to externally applied pressure and then screening and selecting the improved species. It can be an effective method for creating new enzymes while simultaneously providing new insights into the nature of evolution itself. The concept is also being applied to develop new microbes and cells with improved performance. For example, a directed evolution approach is being used with yeast
28 Inspired by Biology cells to create cell lines that can produce ethanol more efficiently. Such an example suggests that some evolutionary traits could be exploited to create biomolecular materials. Improved understanding of existing evolutionary pathways would allow the more rational design of new pathways, but the biggest challenge would be to make the biomaterial evolve itself by subjecting it to external pressures. Representations of the genomes for all living species and knowledge of the full expression of each gene, the functions of the proteins each gene produces, and, most important, the complex signaling and coupling between the expression levels of these proteins and their complex control circuits are still in their earliest stages. Current techniques to exploit this genetic information to create new materials use only the most rudimentary parts of this knowledge. As the amount of detail that is known increases, and as a more general understanding begins to emerge, increas- ingly sophisticated methods will be developed to more fully exploit the complex systems of control and signaling that are inherent in living systems. Opportunities and Challenges In this chapter, the committee has identified many areas where research is elucidating the underlying processes of biology. This knowledge is the basis for developing new technologies and making new biomolecular materials and pro- cesses. The committee discussed the essential nonlinearity in biological processes, illustrating this in particular through the behavior of cells, and considered its implications for new materials, with the creation of materials with cell-mimetic capabilities a prime example. The committee discussed the conversion of chemical energy to mechanical energy by molecular motors, with the consequence that most biological materials cannot be described by equilibrium processes. The committee considered the design principles for mechanical properties of the cell. It also dis- cussed the myriad forms of self-assembly in nature and the implications for new biomaterials and processes if they could be mimicked. The committee considered as well two remarkable properties of living organismsânamely, their ability to evolve in response to new conditions and their ability to regenerate or heal them- selves in response to damage. These properties will open up opportunities for new biomolecular materials. Some of the challenges to scientific understanding where new understanding is beginning to emerge are listed below. They are followed by mention of some opportunities that might arise if researchers are able to address the challenges. ⢠A unique feature of many response and signaling systems in biology is their use of cooperation to create a response to stimuli. This allows them to achieve a very high degree of precision and sensitivity, while reducing spurious false positives.
Understanding Biomolecular Pro cesses 29 âOpportunity: Biosensors that combine high sensitivity and high precision âOpportunity: Biomimetic structures that mimic the specificity of T cells in identifying and selecting pathogens ⢠The large number of motors that convert chemical energy into mechanical energy ensures that the properties of living species cannot be described with an equilibrium description but must instead be described in the framework of nonequilibrium systems. âOpportunity: New description of biological function for materials development ⢠The essential design principles that describe the mechanical properties of a cell are not fully understood. Some cell components are under tension, while others balance this and are under compression. They are under a steady pre-stress, which apparently drives them into a nonlinear elastic state. The relationship between these properties and the mechanical behav- ior of the cell remains undetermined or only poorly determined as does the biological rationale for this behavior. However, the design principles for the elasticity of the cell must be understood if its behavior is to be mimicked. âOpportunity: Actively controlled biomolecular materials using molecu- lar motors âOpportunity: Highly adaptable and controllable biomolecular materials ⢠Biological systems possess very high specificity in their ability to recog- nize molecules and to respond and control themselves when this recogni- tion occurs. This ability to very precisely recognize specific targets can be exploited to create new functional materials. âOpportunity: Exploit specificity of DNA interactions to fabricate bio- molecular materials âOpportunity: Use viruses as building blocks for the assembly of more complex materials âOpportunity: Mimic viral function in synthetic materials ⢠Biological systems are the ultimate example of the construction of highly complex structures and systems from simple and common building blocks. This is accomplished by very fine control of the spatiotemporal assembly. Mimicking this behavior would allow changing the paradigm of manufac- turing by following the model of biological systems. âOpportunity: New manufacturing capability that relies on self-assembly ⢠Much of the diversity in all living species comes from their ability to evolve, changing both their structure and their function in response to external pressure. Understanding the details of how this is accomplished will allow the development of synthetic bioinspired materials that are able to evolve themselves when external pressures are applied.
30 Inspired by Biology âOpportunity: Design new enzymes or microbes with improved functionality âOpportunity: Improved efficiency for biofuel production âOpportunity: Materials that are self-healing to recover from disruption ⢠All living species carry the full information about their structure and function, as well as information about the nature of the complete sys- tem. This information is carried through sequence-specific structure, and understanding the details of this information storage and propagation will facilitate the design of bioinspired systems that incorporate this ability. âOpportunity: Materials that can self-replicate âOpportunity: Materials that can adapt by changing the stored information âOpportunity: Modification of materials properties with analogues of RNAi Suggested Reading Arnold, F.H. âDesign by directed evolution,â Accounts of Chemical Research 31:125 (1998). Janmey, P.A., and D.A. Weitz, âDealing with mechanics: Mechanisms of force transduction in cells,â Trends in Biochemical Science 29:364 (2004). Mizuno, D., C. Tardin, C.F. Schmidt, and F.C. MacKintosh, âNonequilibrium mechanics of active cytoskeletal networks,â Science 315:70 (2007). Nicolis, G., and I. Prigogine, Self-Organization in Nonequilibrium Systems, New York, N.Y.: John Wiley & Sons, 1977. Thorpe, M.F., and A.E. Carlsson, The Role of Theory in Biological Physics and Materials: A Report to the National Science Foundation. Available online at http://biophysics.asu.edu/workshop/report/pdf/bio_mat.pdf. Last accessed on March 24, 2008. Whitesides, G.M., and B.A. Grzybowski, âSelf-assembly at all scales,â Science 295:2418-2421 (2002).
