Travels to the Nanoworld.
Miniature Machinery in Nature and Technology
1. Welcome to the nanoworld
Imagine a motor measuring a few hundredths of a thousandth of a millimeter, running on and on and on. Or a data storage device squeezing seven megabytes (five "high density" floppy disks) into a thousandth of a millimeter. Or a catalyst converting the inert nitrogen gas from the air into ammonia at room temperature and atmospheric pressure.
Such extraordinary achievements are sometimes dreamt up when scientists discuss new technologies operating in the nanometer area of the length scale, which are therefore collectively referred to as "nanotechnology". Scientists use the prefix "nano" to refer to a billionth (10-9) of a metric unit. One nanometer (1 nm), therefore, is a billionth of a meter, or a millionth of a millimeter (Fig. 1).
We are talking about complicated and highly efficient machines, which are only allowed a few millionths of a millimeter in size. Unbelievable? Not at all, for evolution has solved these problems more than a billion years ago. The motor mentioned above exists already -- it is a system mainly consisting of the proteins actin and myosin, and is known to power our muscles. The data store, also known as a chromosome (i.e. a very long stretch of DNA wound up in a complicated way) determines our genetic identity.
And the catalyst, an enzyme named nitrogenase, is a specialty of the nodule bacteria, which live in symbiosis with certain plants and provide them with freshly made nitrogen fertilizer produced from air and water.
And these are just three examples out of an enormous number of tricky technical problems which living cells can apparently handle with little effort. The secret behind this success story, the underlying principle which has been proven the best (if not the only) way to efficiency on a small scale by three billion years of evolution, is the modular design principle. Nature's nanotechnology relies on long, chain-like molecules, fabricated from a small set of building blocks. The whole management of genetic data is based on an alphabet of just four letters. Most functions of a living cell are performed by proteins consisting of only 20 different amino acids. However, this simplicity is rather deceptive, as the combination of these building blocks to a long chain can happen in an astronomical number of different ways. If we wanted to "invent" a rather small protein with 100 amino acid residues, we would have the choice between 20100 (i.e. 10130, or a number with 131 digits) possibilities.
Let us have a closer look at these "natural nanomachines", to see whether they could serve as models for scientist and engineers developing new technologies. We will start with the smallest parts and work our way up the length scale into the world of the cell.
Molecules: The building blocks of life
Atoms are generally regarded as the fundamental building blocks of matter. Although physicists can split or fuse them under extreme conditions, they will stay intact in all biologically relevant environments and in all technological applications short of a nuclear power station. (They may win or loose a few electrons in chemical reactions, but that doesn't change their mass very significantly.) The physical properties of atoms are crucial for the way in which they assemble to form molecules. Molecules, their formation, reactions, transformations are the domain of chemistry. But if the early stars of our universe had not fused an awful lot of hydrogen atoms to provide a whole range of different (heavier) atoms, there wouldn't be much chemistry to talk about.
Molecules can consist of as little as two, or of as many as thousands of atoms. The latter may be called macromolecules (i.e. "big" molecules) although they are still far too tiny to be visible through a light microscope. (Using visible light as a probe -- as we do when we are looking at things -- determines a physical limit to the resolution of small objects. As the wavelength of visible light ranges from 400 to 800 nm, molecular objects smaller than 400 nm cannot be seen in the strict sense of the word. Structural information can be obtained by using radiation with shorter wavelengths, such as electrons or X-rays.) Macromolecules are normally put together through repeated reactions of small molecules, a process called polymerization. When chemists first produced polymers, they made long chains of just one type of building blocks, called homopolymers. Many well-known plastic materials fall into this category, such as polyethylene (PE), polyvinylchloride (PVC), etc. In contrast, biological macromolecules are made of a meaningful sequence of different building blocks selected from a limited set. Therefore, they are also referred to as heteropolymers. Heteropolymers can store information and carry out functions, the combination of which makes them the perfect material for life. Without molecules, there wouldn't be any life.
But just how small are atoms and molecules? Atoms can't really be measured, because they are wrapped in clouds of electrons, which don't have sharp boundaries. But if you take half of the distance between two atoms of the same kind in a molecule as a measure of their radius, they are all less than one nanometer. By this definition, the diameter of a hydrogen atom is 0.06 nm, while the sulphur atom, which is 32 times heavier, measures 0.2 nm across. Small molecules may be up to several nanometers long. Macromolecules can extend up to micrometers, or be wound up to compact shapes with diameters in the 10 to 100 nm range (Fig. 1).
And it is on this length scale that the macromolecules of the living cell store information, process it and convert it into function. Desoxyribonucleic acid (DNA), possibly the most prominent molecule of our time, is in charge of the information storage, while proteins fulfill the mechanical or chemical functions. Ribonucleic acid (RNA) can do both and is therefore regarded as a promising candidate for the role of the ancestral molecule, which made evolution possible before the complex DNA-RNA-protein machinerie came up.
These molecules normally act individually as independant machines in the nanometer-sized network of the cell's business. Some examples of well-defined molecular function units will be discussed in part II. In contrast, if we handle molecules, we normally have huge numbers of them. An amount of protein that is visible for the naked eye and can be weighed out on a laboratory balance, can contain millions of billions of molecules. For instance, a milligram of the enzyme uricase, which is commonly used to assay the concentration of uric acid in the blood, contains six million billion molecules -- and when a physician is using the enzyme for a diagnostic assay, they are all doing the same thing. It is as if we were only able to see a tree when it comes in the company of the whole South American rain forest.
In order to construct machines on the nanometer scale, we will have to build macromolecules which are nearly as efficient as their biological counterparts, and we will have to learn to give each individual molecule a task, and to check it performs well. First advances in that direction will be described in part III of this book.
However, sticking atoms together to make macromolecules does not necessarily produce nanomachines. Because the latter owe their strength -- somewhat paradoxically -- to weak interactions.
Interactions: The weakest are the best
Organic chemistry was born when the German chemist Friedrich Wöhler (1800-1882) demonstrated that the molecules of life are normal chemical species in a sense that they can be synthesized from inanimate substances -- in the first instance, it was urea, which he obtained from ammonium cyanate in 1828. Since then, organic chemists have been making and breaking bonds between carbon atoms and half a dozen other species such as hydrogen, oxygen, nitrogen, sulphur, phosphorous, in order to create new or interesting molecules, or just to re-create natural products. However, with their classical methodology of making, breaking and rearranging firm chemical bonds beween atoms (known as "covalent bonds") they will never be able to create anything remotely similar to a cell. Although biological macromolecules are built from covalently linked atoms, this type of binding is much too rigid and inflexible for their three-dimensional functional architecture and for interactions with other molecules in the cell. Breaking a stable covalent bond requires either a catalyst, a large excess of one of the reacting species, or -- in the laboratory -- high temperatures and non-aqueous solvents. (One of the few covalent bonds easily formed and broken under physiological conditions, the disulphide bridge, is indeed used in the cell to stabilize three- dimensional structures.)
In the cell, three-dimensional folds and assemblies of several molecules are held together by the so-called weak interactions (Fig. 2). These include:
Hydrogen bonds, for instance, provide the force which keeps the two strands of the DNA double helix together. In the three-dimensional structures of proteins, they help with the formation of structural elements such as the alpha-helix and the beta-pleated sheet. Salt bridges often help an enzyme recognize its substrate. Van-der-Waals interactions are so short-ranged that they only become effective when two molecules or parts of molecules have complementary shapes and click into a jigsaw like association. The hydrophobic interaction keeps the lipid double- layer together which forms the membrane surrounding every cell and some special compartments within cells. It also plays an important role in the structure formation of proteins (II.2.).
All these bonds can easily be dissolved and rebuilt by subtle changes in conditions. In many cases, their quality of being as easy to open as a velcro tightening is a major requirement for the function of the systems they help to build and stabilize. The DNA double helix, for instance, has to be opened locally so that it can be "read" by the enzymes which either copy it to make more DNA or transcribe it to make RNA. And when the oxygen-storage protein of the muscle, myosin, carries an oxygen molecule, the latter is deeply buried within the structure of the protein. In order to take it up or set it free, the protein has to rearrange its structure in such a way that a tunnel is opened between the oxygen binding site and the rest of the world. But weak interactions are not only needed for these relatively rapid local rearrangements. In addition, they enable the association of macromolecules to form highly complicated systems without any help from molecules which are not part of the final structure. This phenomenon is known as self-organization.
Self-organization: Together we will make it
The factory in which the gut-colonizing bacterium Escherichia coli produces its proteins, the (bacterial) ribosome, consists of two subunits, containing a grand total of 52 protein molecules and three long strands of RNA. Although more than a dozen research groups have been trying for more than two decades to determine the exact structure and function of this machinery, this has not yet been achieved.
Researchers have divided the particle into its molecular components, separated all the 55 types of macromolecules, and studied them individually. More surprisingly, they found that if they recombined the aqueous solutions of those components coming from the smaller subunit, they obtained fully functional small subunits. The recipe for the larger subunit is only a little bit more complicated, involving two subsets of components, one of which should only be added after the other one has had time to assemble, and a shift in the buffer conditions. Having assembled the two subunits, one can proceed by mixing these and will end up with complete ribosomes which will synthesize proteins as if nothing ever happened. The formation of this extremely complex structure has happened just like that, by four mixing steps, without any scaffolding or input of additional information about the target structure.
This example may be spectacular, but it is by no means unique. It points to an important principle in the nanotechnology of life. All machine parts are built in a way so that they can associate to functional machinery on their own. They don't need an engineer, blueprint or scaffolding. They carry their destination encoded in their structures. In a similar way, scientists can reassemble complete viruses, such as tobacco mosaic virus (TMV, Fig. 3), or complex cellular structures such as microtubuli, the tube-like threads of the cell's skeleton. However, you should not try this with your computer or VCR. It would be a rather expensive way of demonstrating how 20th century engineering falls short of the standards set by nature.
A remarkable example of how researchers have learnt their lesson from nature and used self-organization to create an artificial ion channel will be discussed in chapter III.1. However, this is a rather singular case. Although the reconstitution of natural systems which tend to self-assemble in a similar way as the ribosome has been performed in the reaction tube decades ago (TMV 1972, large ribosomal subunit 1974, small subunit 1968), this phenomenon has only rarely been used to construct artificial molecular systems. The new branch of chemistry which mainly deals with weak interactions and self-organization -- kown as supramolecular chemistry -- is still in its infancy (III.1.).
Having watched the cellular nanomachines as they put themselves together, you may wonder what these tiny marvels do when they are finished.
Catalysis: Chemical reactions fast and accurate
Proteins can, for instance, serve as structural elements in the cell's architecture (e.g. tubulin) or as carriers for small molecules or ions (e.g. hemoglobin) but most of them have the task of speeding up (catalysing) chemical reactions. In extreme cases, they can make reactions which would otherwise require millions of years run to completion within seconds. Proteins with a catalytic function are called enzymes. The dogma that only proteins can play this role was overturned in the 1980s, when researchers discovered catalysts made of RNA only and called them "ribozymes".
Why does a cell need enzymes? The obvious answer is that it doesn't have the time to wait for slow reactions to occur. But there is another equally important reason: it needs enzymes to direct the production processes in its chemical factory. Catalysts are -- by definition -- not allowed to govern the direction of a reaction. They only accelerate the arrival of the equilibrium distribution between states, which is defined by the conditions such as temperature, pressure, and so on, by lowering the energy barrier between the initial and the final state (Fig. 4). However, this apparently modest influence can move quite a lot. For instance, if one chemical could theoretically get involved in alternative reaction paths, they could catalyse only one of them. This way, a highly specific catalyst -- and enzymes tend to be the most specific catalysts we know -- could completely change the range of products obtained from a given reaction mix.
Furthermore, enzymes can couple one reaction to another. This way, reactions which are energetically unfavourable, e.g. the synthesis of macromolecules, can be driven forward by energy-providing reactions, such as the cleavage of certain small molecules. (To be more exact, we should use the criterion of what chemists call the free energy G, which accounts for both the energy and the entropy (disorder) balance of a reaction.)
Many enzymes outperform the corresponding technical catalysts by orders of magnitude. For instance, there is no technical catalyst producing ammonia from the elements (hydrogen and nitrogen) at ambiant temperature and pressure, as the nitrogenase of nodule bacteria does (II.1.).
Some enzymes are used in households, for stain removal, as additives in washing powders or for curdling milk to make curd cheese. Enzymes which can degrade proteins (proteases) are used in cosmetics, and perms can be laid with the help of the enzyme urease.
Some enzymes have created their own specific applications in the laboratory, in procedures which scientists could not even have dreamt of before a specific enzyme was discovered. Among the best known highflyers are the restriction endonucleases developed by bacteria to fight viruses. Now they are indispensable for molecular biologists, who use them to fragment nucleic acids into well defined pieces. Even more spectacularly, the availability of extremely heat-stable DNA- polymerase from thermophilic bacteria paved the way for the polymerase chain reaction (PCR) of Jurassic Park fame, which enables molecular biologists to make millions of copies starting from just one piece of DNA. While a small sample containing only a "countable" number of DNA molecules was utterly useless in pre-PCR times, the so-called amplification procedure enables researchers to do anything with it -- at least anything short of cloning dinosaurs.
And some enzymes are already used in industrial production, mainly in simple reactions, such as the degradation of starch to make sugar. More than 20 million tons of sugar are produced this way every year, requiring 15000 tons of the enzyme amyloglucosidase. In Brasil, the fermentation of carbohydrates to produce technical ethanol has been boosted as part of a national programme to reduce the country's dependence on petrol imports. Furthermore, enzymatic processes are becoming more and more important in the production of pharmaceuticals as well as in food processing.
Although nature has millions of different enzymes and we are far from using this potential to a significant degree, many technical applications would benefit, if we could make similarly specific catalysts to measure, especially to speed up reactions which do not occur in biology. One might also hope that these synthetic enzymes could have a longer shelf-life than their natural counterparts. Various routes to this goal will be discussed in III.1.
Directing the chemical reactions of metabolism by selctive catalysis is a really clever feat, but in order to avoid getting things messed up, the cell also has to allocate spaces for each process.
Compartimentation: Keeping your cells tidy
The first step towards the confinement of this network of chemical reactions which we call life was taken when cells started being cells. They surrounded their precious little selves by a double layer membrane (which can in many cases be further shielded and reinforced by a cell wall, and further layers) so that their own chemical processes would not so easily be disturbed by the outside world.
But we also find walls and barriers within cells. We (people, cats, plants, yeasts ...) belong to the group of lifeforms enjoying a cell nucleus, collectively called the eukaryotes. While this nucleus contains most of our DNA, there are more compartments in the eukaryotic cell, which all have a specific set of functional tasks and often a fairly tongue-twisting name, such as endoplasmic reticulum, mitochondrion, etc. (Fig. 5). All that matters for our current purpose is that the cell appears to have secluded areas for specific functions, resembling the way in which houses are divided into living room, dining room, kitchen, bedrooms etc.
Obviously, this organization requires even more different sorts of nanomachines. Walls between compartments must be built, or rather build themselves, as we would suspect after having discussed self-organization above. So we won't need cranes or scaffolds. But once the walls are there, we will need means of transport between the rooms. A door or catflap won't do, as we want to control the traffic between the rooms. A simple regulated valve might do, if we just want molecules to get from one room with plenty of their kind to an empty one. However, quite often the cell needs to transport molecules against this trend of equal distribution. In this case it could use the principle of coupling the transport with an energy-consuming process, as discussed above.
Within the rooms of the house as well as in the single-roomed bacterial cell, some scientists have expected to find a chaotic random floating of all molecules. However, it begins to emerge that even the soluble enzymes have some kind of spatial organisation as well. Nanomachines are sometimes arranged in an assembly line, where the product of one step can be directly passed on to become the raw material of the next. For instance, molecular chaperones known to supervise the folding of freshly synthesized proteins have been found in close contact with the ribosomes which make the proteins (II.2.).
Only a few years ago (1994), scientists have managed to arrange biological macromolecules or similarly complex systems with nanometer precision, at least in two dimensions. Using the method presented in chapter III.2., nano- biotechnologists will be able to construct a biotechnological assembly line, along which the substrates can be handed on from one enzyme to the next without any loss of time or substance. Although, with the discussion of whole cells, we are leaving the nanoscale and heading towards the visible world, we shall now have a quick look at the macroworld of life, before returning to the molecular scale.
Evolution: Molecules to organisms
From the Big Bang through to the rise of green plants and vertebrates, matter has increasingly become organized in bigger and more complex structures. Subatomic particles became atoms, atoms became small molecules (which could form big things like planets, but not complex things like living cells), small molecules became macromolecules, which gave rise to cells, which evolved to form multicellular organisms, which grouped together to form herds, flocks or learned societies ...
This admittedly rather crude account of the history of our universe covers fifteen billion years on the time scale and fourteen orders of magnitude on the length scale. The theory of evolution by the interplay of mutation and selection provides convincing connections for most of this way, at least from the first macromolecule that catalysed its own duplication -- possibly a variant of today's RNA -- through to the current population of the earth with millions of species.
Some researchers even believe that the evolutionary principles have started shaping our history even further back in time and down in scale. Atomic scale defects in the otherwise regular lattices of clay minerals may have been the first kind of hereditary information. This hypothesis implies that a pre-evolution has taken place in the realm of atoms and inorganic solid states, which then may have provided a scaffold for the first organic information molecules. Even today we observe stunning capabilities of proteins and cells which can direct the precipitation of inorganic minerals in crystalline as well as in amorphic phases, leading to such diverse structures as bones, teeth, egg shells, mollusc shells, or pathogenic urate crystals (II.1.).
The question of how cells form complex organisms is beyond the nanoscopic focus of this book. However, it should be noted that communication between cells, which is essential for every multicellular organism, relies on complex molecular systems, many of which may be useful models for information scientists and computer developers.
The "one to all" function is often performed by hormones and the corresponding receptors. Self-organization is, again, involved when receptor complexes integrate into membranes. Molecular recognition with the help of weak interactions is needed for the specific binding between hormone and receptor, which then triggers a reaction cascade.
For the site-directed delivery of information, our body has its own telephone network, also known as the nervous system. In addition to the phenomena discussed above, electric voltages and currents play a major role here. At the best-described site of the nervous system, the retina of the eye, light is one further information carrier to be considered. Signal conversion beween light, electricity, and chemical energy on the length scale of cell receptors certainly is one of the targets to be set for future technological developments.
Technology: Back to molecules
In a sense, scientists are now walking the way back on which humankind evolved. Fifteen billion years took us from femtometers to meters, from subatomic particles to human beings, who are now trying to go back to the small worlds.
The first tools which early people made and applied, were on the scale of their natural tools, their hands and arms. They may have used sticks if their arms were too short to reach a fruit. Or stone blades if their finger nails were too blunt to dissect a prey.
Later on, early cultures used pulleys, levers, wheels etc. to erect amazing buildings on the gigantomanic scale of the pyramids or Stonehenge. And unknowingly, they used microorganisms to produce beer and bread. But they did not try to observe or manipulate the invisibly small parts of the world. The atoms proposed by the Greek philosopher Democritus of Abdera (* ca. 460 BC)remained a philosophical postulate for more than two millenia.
The microworld only opened up when the light microscope, which was invented in the Netherlands in 1590, became a fashion during the 17th century. The Dutch shopkeeper Antoni van Leeuwenhoek (1632-1723) was the first to develop a microscope good enough to discover microbes (1675).(Van Leeuwenhoek made his discoveries as a total outsider to the science establishment of his time. He did not even know any Latin, which was the official language for scientific publications. Eventually, he became a fellow of the Royal Society.)
Even in the 19th century, watchmakers were the only people to fabricate small structures. They were operating down to the 0.1 mm scale, using a magnifying glass. Chemistry, which evolved to become an exact science during the early 19th century, and a leading industry afterwards, had a strong tendency to go for big things, rather than for small ones. Although 19th century chemists could not work out what molecules were in a physical sense, they found comfort in the observation that these chemical entities behaved in a predictable way, if you had billions of billions of them in your reaction tube. To be on the safe side, they defined their standard quantity of matter, the mole in a way that it contains ca. 6*1023 molecules.
Only when electronic components became important in the second half of our century, and miniaturisation led to more useful and faster equipment, fabrication on the micrometer scale became a mass industry.
Windows into the nanoworld have been opened since the middle of our century by techniques such as electron microscopy, X-ray crystallography, neutron scattering, and nuclear magnetic resonance. Chemists have learned during the past 200 years to handle molecules, describe their structures, and create novel molecular structures. However, they have always dealt with macroscopic amounts of the substances, containing billions of billions of molecules. And there have always been limits to the size and complexity of the molecular systems which could be analysed. Furthermore, the science of the huge molecules, called macromolecular chemistry, has always been a stepchild of chemistry. It could neither aspire to become a classical subject, as inorganic, organic and physical chemistry are, nor to become an independent discipline like biochemistry.
Making tools for the fabrication on the nanometer scale is something we are only just beginning to learn. Only now the subject areas of biochemistry, chemistry, physics and biology, which deal with natural nanoscale systems or try to produce artificial ones, approach one another. Only now chemists start using the power of weak interactions and the principle of self-organization to make synthetic molecules similarly efficient as biological systems. Only now have methods in materials sciences been miniaturised to a degree that nanometer scale structures can be etched out of a semiconductor material and electronic elements as well as mechanical machine parts can be produced on this scale.
When technology enters a new dimension, this can potentially change the world. Much as the discovery of microbes following the development of microscopes, or the triumph of computers after the invention of microchips, the technologies which will result from the conquest of the nanoworld may revolt not only the world of science but the daily life as well. Whether or not nanotechnology is likely to become the next industrial revolution will be discussed in part IV. Current prophecies will be critically assessed. Predicted applications of nanomachines range from medicine to space travel, from data processing to the protection and healing of the environment. We shall once again realize that the nanoworld, although invisible, shapes our visible world.
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