Table of Contents
INTRODUCTION
Bionanotechnology, being a synonym for nanobiotechnology, is a rapidly growing field that encompasses contributions from various disciplines, ranging from engineering and computational sciences to physics, chemistry and biology. Hence, there is an increasing need for reviews and textbooks that provide an introduction to biomolecular sciences and their impact on nanotechnology. Living cells are made up of complexes, which carry out many of the functions essential for their existence, differentiation, and reproduction. In many cases the malfunction of these proteins can be a source of disease; for example, myosin mutations, particularly in the head and neck region of the molecule, can result in inherited diseases such as familial hypertrophic cardiomyopathy. An understanding of the mechanisms of these proteins may provide a guide for therapy. Many of these complexes can be described as “molecular machines” or “molecular motors” or “molecular devices,” depending on their sizes, complexity and tasks.
Indeed, this designation captures many of the aspects characterizing these biological complexes: modularity, complexity, cyclic function, and, in most cases, the consumption of energy. In molecular machines or motors, a rotary or linear movement is used for motility, nucleic acid processing, folding or unfolding, or as transducers of light or chemical energy. Examples of such molecular machines are the replisome, the transcriptional machinery, the spliceosome, the ribosome, but also smaller machines, residing in the plasma and organellar membranes, are known as ion and protein transporters, or as the bacterial flagellar motor.Modern Molecular MachineryAs a consequence of the evolution of life from a single primordial cell, all known living things on earth share a common molecular plan. All living things are made of four basic molecular building blocks: protein, nucleic acid, polysaccharide and lipid. Other small molecules are specially synthesized for specific functions, but the everyday work of the cell is performed by the four basics.
The earliest cells chose these materials to the exclusion of others, and subsequent generations of cells, right up to our own, have been forced to work with them. Two different approaches are taken to synthesize these molecules, resulting in characteristic forms and functions. Proteins and nucleic acids are built in modular form by stringing subunits together based on genetic information. Proteins and nucleic acids may be built in any size and with subunits in any order. This gives remarkable flexibility to the form and function of these molecules. In contrast, lipids and polysaccharides are built by dedicated machines. Each new type of lipid molecule requires the creation of an entirely new suite of synthetic machines. Likewise, a new suite of machines must be created to build each new type of polysaccharide linkage. The result is that lipids and polysaccharides appear in fewer forms than proteins and are used in much more limited, albeit essential, roles. Our distant relatives developed a standard for biological information, choosing a particular 20 amino acids to be used in proteins, encoded by five types of nucleotides found in the nucleic acids DNA and RNA. Today, every protein is made of these 20 amino acids (at least initially). In their defense, these primordial cells chose an excellent set of building blocks, including flexible and rigid components, charged, uncharged, acidic, basic and neutral amino acids, large and small amino acids, and several with attractive chemically reactive properties (Figure 3).
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The amino acids may be used to create proteins with a wide range of properties. These include very flexible proteins with changeable shapes and very rigid crosslinked proteins designed to retain their shape under harsh conditions. Other proteins are highly basic or highly acidic, designed to perform their jobs under extreme acidic or alkaline conditions. Some are covered with carbonrich groups that repel water and seek out membranes for binding; others have polar surfaces and perform their duties in the watery cytoplasm. Modular synthesis allows proteins to be built in many shapes and sizes. As a consequence, most of the processes of modern cells are performed by proteins. Evolutionary legacy, however, places several limits on the design of proteins. As noted above, proteins are limited to the 20 components encoded in the DNA genome.
Evolution also limits the size of proteins, limits them to aqueous environments and requires that they automatically assemble themselves within the crowded confines of the cell. In spite of these limitations, the breadth of protein form and function in modern cells is remarkable. The size of a protein is limited by the error rate of the protein-synthesis machinery, which in theory could produce a protein of any length. Missense errors, which misread the genetic information and substitute an incorrect amino acid at one position, occur at an average frequency of about 1 in 2,000. For a protein composed of 500 amino acids, one out of four proteins will typically have an error, but nearly every protein of 2,000 amino acids will have one. More important, however, are processivity errors, which cause protein synthesis to abort prematurely. These errors have been estimated to occur at a rate of about 1 in 3,000, so long proteins of several thousand amino acids are only rarely constructed in full. The average size of a typical protein chain, 300 to 500 amino acids, is the compromise adopted by most cells. Error rates keep the chain length low, so larger proteins must be built as complexes of multiple protein chains.Symmetry of ProteinsThe process of evolutionary selection has yielded an unusual result: Evolution of proteins favors perfect symmetry.
The majority of soluble and membrane-bound proteins found in cells are symmetrical complexes formed by several subunits. Most proteins are oligomeric, composed of multiple copies of one or more types of subunits. Nearly all of these oligomeric proteins are also beautifully symmetrical, with identical subunits packed in identical environments. A complex interplay of conflicting functional needs has driven evolution to this surprisingly aesthetic conclusion. The major evolutionary force is the need for large proteins. Large proteins are preferred over smaller proteins and peptides for several reasons. Some functional roles simply require a molecule that is physically big. Large protein complexes form structural elements that span entire cells; they form rings that encircle DNA and rulers that measure lengths of DNA; they create pores of many sizes through cell membranes; they form large spherical containers for storage and delivery and small cylindrical containers that create exactly the proper environment for protein folding. Large proteins are also well suited for cooperative functions, such as allostery and multivalent binding, which require a molecule with several identical active sites. Multivalent binding increases the binding strength of a molecule to a target by reduction of entropy. Once one site on the protein has bound, the other sites are held in close proximity to the target, increasing their probability of binding.
Many of the molecules of the immune system have a distinctive shape, composed of many flexible arms, in order to take advantage of this cooperativity. Large proteins also have attractive physicochemical characteristics. They are more stable against denaturation, having a more stabilized internal structure than small proteins.Biomolecular Flexibility and DynamicsEngineers in our macroscopic world typically build rigid structures that stoically resist the forces of nature. Nature, however, has taken a different approach, developing machines that flex over the course of their action. Is a totally rigid nanostructure needed or even desired? Apparently not. In fact, biological molecules take advantage of flexibility for many aspects of their function. Many of these functions would be severely compromised, or not even possible, given a rigid molecule.
Subtle motions can have surprisingly large effects on reaction rates or assembly. Biological molecules are perfectly placed to take advantage of these subtle motions. The step-by-step optimization provided by evolution allows a moderately active protein to be improved, through small changes modifying structure and flexibility, to yield a machine ideally tailored to fulfill its function. This process is easy for evolution but far more difficult for biotechnological design. We design our machines in one step, instead of through many small random optimization steps, and we expect to get it right with a minimum of tweaking and redesign. Thus, to anticipate all of the subtle effects of motion, our design techniques must be accurate enough to predict conformation and flexibility of molecules at scales far smaller than the radius of an atom.
All biological molecules are flexible to some extent and are battered into different conformations by the constant pressure of surrounding water and the kinetic energy of their own atoms. At physiological temperatures, biological molecules constantly flex. Most of the interactions holding a protein together are conserved—covalent bonds remain connected, hydrogen bonds and salt bridges link portions of the chain—but entire elements of secondary structure flex, bending slightly or separating momentarily from the globule. These motions are often termed “breathing.” Breathing is essential in the function of myoglobin, a deep red protein that stores oxygen in muscle cells. Oxygen is bound to myoglobin in a pocket that is completely buried within the protein. Looking at the static structures provided by x-ray crystallography, there are no channels leading into or out of the pocket. For the oxygen to enter and exit, the molecule must breathe, transiently forming channels that allow passage. Many proteins use a carefully designed change of shape to regulate their action. These allosteric (“other shape”) proteins are composed of several subunits, each of which performs identical functions. In the simplest model of their action, each subunit may adopt two conformations, one functionally active, the other less active. Regulation is performed by propagation of the shape change from one subunit to its neighbors.
For instance, phosphofructokinase, a key enzyme in sugar metabolism, uses allosteric regulation to modify its action. Phosphofructokinase is composed of four identical subunits (a tetramer), each containing a reactive site for the sugar molecules. The tetramer also contains binding sites for the energy molecule adenosine triphosphate (ATP) in the cleft between subunits. When ATP binds to this second site, it forces the entire enzyme complex into a different shape, which is less active than the original form. In the cell, this regulation is used as a negative-feedback loop. ATP is one of the final products of the sugar-breaking process that the enzyme performs. When ATP is plentiful, it binds to the regulatory site in phosphofructokinase, shutting down its own synthesis. The enzyme that performs the opposite reaction, is also allosterically regulated. Many protein chains rely on “induced fit” to mediate their function. The chain may remain in a partially unfolded conformation that only completely folds when it binds to its target. Induced fit may be used to create doorways that allow ligands to enter protein cavities that are shielded from the surrounding environment. HIV-1 protease is an example. The active site is a cylindrical tunnel, with the cleavage machinery at its center.
Somehow, a polypeptide must be threaded through this tunnel in order for the cleavage reaction to occur. This problem is solved through the use of two flexible flaps that cover the top of the tunnel. When free in solution, these flaps are disordered, opening a path to the active site. When the protease wraps around its target, the flaps close, forming a stable structure that positions the polypeptide accurately for cleavage. Flexible linkages are common in the molecular world. Protein chains may be made more flexible through addition of many molecules of the amino acid glycine, which are less hindered in bond rotation because of the lack of a side chain, or through addition of many charged residues, which favor exposure to solvent over forming a compact globule. The rigid kink formed by proline, surprisingly, is also commonly found in flexible regions, because it does not fit comfortably within compactly folded structures. The immune system contains many examples of flexible linkages that enhance multivalent binding. ProspectsBiological molecules are examples of solved problems in nanotechnology— lessons from nature that may be used to inform our own design of nanoscale machines. The entire discipline of biotechnology has emerged to harvest this rich field of biological wealth. We routinely edit and rewrite the information in DNA to build custom proteins tailored for a given need.
Today, for instance, bacteria are engineered to produce hormones, genes for disease resistance are added to agricultural plants, and cells are cultured into artificial tissues. Principles of protein structure and function also yield insights for nanotechnological design and fabrication. The diversity of protein structure and function shows the power of modular, information-driven synthesis, as well as the limitations imposed by modular design once a dedicated modular plan is chosen. Proteins demonstrate that extended, complementary interfaces are essential prerequisites for molecular self-assembly. The prevalence of protein complexes proves that error-prone synthesis may be accommodated through the use of subunits and symmetry to build large objects accurately and economically. And contrary to our macroscopic experience, motion and flexibility may be assets, not liabilities. The principles observed in the mobile, organic shapes of biological molecules may be applied to the controlled rectilinear forms of diamondoid lattices, fullerines or whatever nanoscale primitives are ultimately successful. We must not be too impatient, however. Nature has had some three or four billion years to perfect her machinery; so far, we have had only a few decades.ResearchThe construction of more complex molecular machines is an active area of theoretical and experimental research.
A number of molecules, such as molecular propellers, have been designed, although experimental studies of these molecules are inhibited by the lack of methods to construct these molecules.[citation needed] In this context, theoretical modeling can be extremely useful[citation needed] to understand the self-assembly/disassembly processes of rotaxanes, important for the construction of light-powered molecular machines.[54] This molecular-level knowledge may foster the realization of ever more complex, versatile, and effective molecular machines for the areas of nanotechnology, including molecular assemblers.Although currently not feasible, some potential applications of molecular machines are transport at the molecular level, manipulation of nanostructures and chemical systems, high density solid-state informational processing and molecular prosthetics.[55] Many fundamental challenges need to be overcome before molecular machines can be used practically such as autonomous operation, complexity of machines, stability in the synthesis of the machines and the working conditions.[5]In 2018, an international team of researchers, led by researchers from the University of Osaka, Japan, created a molecular machine in which elements of a mechanical ratchet were used. The main advantage of this machine is that it provides movement in only one direction. In addition, some features of the structure of the molecular machine provide the best balance between the produced motion and chemical reactivity of the molecules that make up it, that is a problem in itself.
BIBLIOGRAHY
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