Besides the fundamental competition between the top-down and bottom-up approaches in nanotechnology, there are some basic aspects for organizing structures and functions at the molecular level. The recent challenges to the development of nanotechnology are marked by a group of general requirements: selection of suited building units, overcoming the restrictions of planar technology, shrinking of nanofabrication facilities, sustainable production and management of life cycles, organization of autonomy and communication at the nano-level, and the optimization of power consumption and energy management. Looking at the natural principles in the construction, synthesis, and function of proteins helps in understanding the principal differences between the currently applied technologies and the characteristics of biomolecular mechanisms in cells. This view allows formulating seven basic rules to meet the general requirements for future developments in molecular nanotechnology.
The discussion on the strategies for future nanotechnology are often focused on antagonistic points of view such as the “top-down” and “bottom-up” concepts, “lithographic fabrication” and “self-organization”, or “solid-state devices” and “soft matter”. The model character of the organization of living systems at the nanometer level is mostly reduced to one side of these pairs: bottom-up, self-organization, and soft matter. Furthermore, the recent industrial nanotechnology of chip technology seems to prove that these aspects are much less relevant for practical use than the traditions of top-down, lithography, and solid-state technology. However, the continuous reduction of critical dimensions, the constant validity of Moore’s law, and the recent approach to lithographic structures in molecular dimensions demand for a new discussion on the technical future and the role of new concepts in nanotechnology [1–3].
Nanotechnology is one of the cutting-edge research areas of the last two decades. Thus, nanotechnology is currently applied in important fields of industry and in everyday life, and it is also promoted through accelerating research [4, 5]. However, from a more skeptical point of view, the recent state of the art of nanotechnology is marked, as a long-term tradition in nanoparticle and nanomaterial development, by a gradual improvement of lithographic techniques and by a more or less chaotic stepwise experimentation and modeling in many special fields. Important expectations and the visions of nanotechnology pioneers are hardly fulfilled by the technological progress of the last decades. This concerns, for example, the shrinking of machines to nanodimensions [6–12], the substitution of planar technology with three-dimensional architectures [13–15], the development of autonomous-acting nanorobots [16–18], or a technical approach for “artificial life” [19–23]. In particular, the comparison with the construction principles and functions of nanosystems in living nature teaches us how far our technical products are away from the level of the machine-like micro- and nanostructures generated by biomolecular mechanisms [24, 25]. Proteins demonstrate a huge spectrum of molecular structures and functions at the nano level, among them fibers and tubes, linear and rotational nanomotors, pumps and valves, and synthesis and recycling machines. Thus, we can state that we are not yet able to reach the quality of “natural nanotechnology” at the moment [26, 27]. Therefore, we should ask, “What can we learn from nature concerning the development of new strategies for the advancement of nanotechnology?” [28–31]. This would help us not only to accelerate the progress and improve the efficiency of nanotechnology research, but also to develop nanotechnological systems and mechanisms with high compatibility to cells and organisms [32, 33] and with the potential of integration in healthy ecosystems.
2 Technical requirements and recent limitations of molecular nanotechnology
The required compatibility with natural systems and the ecosphere must not affect the technical functions of future nanotechnological products. The question is not how we can efficiently develop new technical solutions at the nanoscale and separate them from the natural environment, but how we should define the strategies for system architectures, technologies, and functions by integrating the ecological aspects and technical requirements. From this fact and from the above-discussed general aspects, seven challenges arise for the future development of nanotechnology (Figure 1):
The recent nanotechnology uses only a very small part of the possible combinations of building blocks and functional elements . A new strategy is needed for the rational synthesis and assembly of building blocks in order to gain access to all interesting directions in the theoretical space of combinatorial coupling of atoms, molecules, and nanoparticles [35–37]. However, the number of actually used building units should be restricted by the criteria of rational production, processing, and recycling. The key issue is to find the criteria for the selection of canonical types that fulfill the requirements for synthesis, assembly, application, and ecological compatibility at the same time.
Planar technology is the fundamental basis of recent lithography and, therefore, the basis for nearly all micro- and nanodevices used in computers, machines, and communication systems [38, 39]. Moore’s law dictates the introduction of a new approach behind the reduction of linear extensions during the next one or two decades. It is unavoidable to transfer nanotechnology and large-scale integration into the third dimension. How can we find a way into the third dimension? Is planar technology a suitable basis for this step? Might it be necessary to think about general concepts of “dimensionality management”? What about a step back in dimensionality to find a promising new way?
Car-producing and microchip-producing machines are always working through the same mechanisms as in computers: they follow a linear sequence of steps of orders. Moreover, many chemical plants work with the same sequential processes. However, these production plants have become larger and larger, and more and more expensive during the last decades. How can we shrink the size of our production facilities to nanodimensions? How would it be possible to project the “principle of continuous-flow production” to the nanoscale?
The recent technical systems are tough and mechanically robust in comparison to living beings. This enables the systems to function reliably, and therefore we can trust them to work under different conditions. However, to realize those functions, we need the usual recent technological materials and large machines for their production. This excludes the use of self-organization mechanisms and the achievement of high eco-compatibility. How do we construct nanotechnological systems for use with self-organization mechanisms, and how do they become highly integrable into a natural recycling system and a sustainable eco-management?
Recently, we have come to expect that all devices have a lifetime of several years or even decades. They have to be robust enough to perform during their whole lifetime, and then they have to be disposed thereafter. This technical expectation contradicts the laws of chemical kinetics and the organization of life. Chemical processes have their characteristic time constants. The dynamics of cells and organisms are marked by cycles, periods, and generation times. Ecological interactions are based on the adaptation to yearly cycles and to the intrinsic time scales of the involved organisms and populations. What is the reason for the integration of different time scales? Do we have to develop technical systems with different life cycles? How can we do that?
Nanotechnological systems should not only be mobile devices in a traditional sense. “Mobile” simply means that these devices can be moved. However, complex functions and interactions demand “automotive nanosystems”. This demand is not focused on the idea of a completely autonomous technical system but on functional system components with a high degree of freedom even in individual motion in space. The vision of nanotechnology means not a simple shrinking of the sizes of robots, but it involves individual-acting nanocomponents under a reliable control and with efficient collaboration with other subsystems. This includes a balanced ratio between autonomous actions and discipline, and between self-sustainment and system integration. It demands, to a certain extent, individual intelligence and efficient communication between single nanosystems. How can they be realized? What do we have to do to enable the exchange and processing of information beyond digital electronics? How should problem-adapted information channels be realized, and how can they be used for defining a limited mobility and controlled autonomy for future nanosystems?
The recent technical world is split into two fundamental groups of devices. From the point of view of thermodynamics, we have to distinguish between passive tools like a hammer or a dam and active machines like a computer or a car. The first kinds are more or less stable and work only in the presence of an outside action, such as through the hand of a worker or the flowing water of a river. The second ones are driven by a potential, by consumption of energy, and, in most cases, by an electrical power supply. The power of technical culture is attributable to the application of both groups. However, how can they be brought together at the nanoscale? How can an equilibrium support the functions of actively working nanosystems? Which driving forces and which channels for energy harvesting can be used? What is available to minimize power consumption and heat release? What do future nanosystems have to do for energy dissipation and for entropy export?
These seven problems will be considered in the following sections with respect to the state of the art in the understanding of natural nanosystems, particularly proteins, which are considered an impressive model for technical concepts in nanosystem research and technology.
3 Limitation of types: variability and control of combinatorial explosion by chemical modularity
Proteins are the main biomolecular component in all cells. They are responsible for many different functions, from structure and mechanical transport over very diverse recognition and sensing functions, to catalysis and replication . All cellular processes are based on the properties and functions of proteins. This wide spectrum of reliable functions can only be covered by molecules with very different geometrical structures and a large variety of chemical affinities.
The most astonishing fact is that these incredible universal challenges are based on one class of molecules only and on a strict modular concept with only 20 basic modules – the proteinogenic amino acids. These basic units are obviously selected by using a small set of important criteria. They are small and simple in chemical structure, and they are made of a constant part that is responsible for coupling and a variable part that is responsible for specific chemical functions. Moreover, these variable parts can be characterized by a few pairs of antagonistic properties (Figure 2), as follows:
lipophilicity and hydrophilicity, and, correspondingly, protic and non-protic character;
small space requirement and larger space requirement;
stiff or flexible structure;
lower or higher electronic polarizability;
negative or positive charge in case of polar residuals.
This also makes proteins a very attractive class of compounds for molecular nanotechnology [41, 42]. From a chemical point of view, many more classes of molecules than proteins and nucleic acids  could be taken into consideration for the construction of functional organic macromolecules. In principle, many more than 20 amino acids can be used for building peptides. However, in rough approximation, nature does not need more than these 20 types to meet all its requirements, from the simplest bacterium to complex plants and animals.
The facts that there is a restriction on this single class of molecules and that only 20 building units are sufficient for nearly all the required functions support the assumption that nature has chosen a very good – and probably the best possible – way of biochemical evolution. It can be concluded that a bottom-up approach for nanotechnology has to define basic construction systems for both molecular and nanoparticle or hybrid assembly [44–47]. The number of classes, types, and key modules for the development of one or a few construction systems should consequently be restricted. When such a system is established, then it could be of interest to use or create a complementary set of other molecules from a larger spectrum of chemical classes, as is found in nature.
4 Limitation of spatial dimensions
The world and all real objects including all living beings are constructed and organized in three dimensions. The possibilities of arrangement of objects increase with the number of dimensions. Thus, the arrangement of objects in two dimensions is more complex than that in one dimension, and the arrangement in three dimensions is more complex than that in two dimensions. That is why a reduction in the number of dimensions means an important reduction in complexity. A linear order (one dimension) can be achieved more simply than the ordering of objects in a plane or in a three-dimensional space.
However, proteins are three-dimensional objects, and three-dimensionality is essential for the function of most of them. However, the primary structure of proteins is linear . Proteins are always constructed by a linear coupling of their building units, the amino acids (Figure 3). The sequence of amino acids, i.e., their linear order, determines the properties and functions of the protein. It can be understood as a linear code and can be described by a sequence of symbols or letters. The linear character of the primary structure of proteins is reflected by the linear character of the chemical structure that is attributable to the topology of covalent bonds between all included atoms. The chemical construction of proteins through the peptide bonds between the amino acids is strictly one dimensional.
This one-dimensional primary structure of proteins means an ultimate reduction of complexity and a restriction of the simplest possibility of linking of the chemical building units. Despite this strict simplification, the variability in the primary structure is very high, because the k=20 different types of building units allow a very high variability z in the information content, which strongly increases with the chain lengths N: z=kN. This number z is much larger than all the possibilities of realization in the universe for a typical size of a protein with a chain length N of 300 (Figure 3). It amounts to about 20300∼2*10390, which is very high in comparison with the estimated number of elementary process steps in the universe of 10120, which is also a very high number but can be neglected in comparison with the combinatorial variability of amino acids in a protein.
The transition between this apparent disproportionately high number of possibilities and the real number of realized proteins is the transition between the one-dimensional coupling of building units, on the one hand, and the three-dimensional functional structures, on the other hand . The ultimate selection criterion is the spontaneous but well-defined self-controlled folding of the primarily formed linear molecule into a stable three-dimensional structure under certain boundary conditions [50–56]. Only a very small part of all possible amino acid sequences seems to fulfill this condition and can be used by living beings for constructing cell tissues and organs (Figure 3). The number of usable sequences for functional protein structures is further restricted by the surface affinity properties in the folded states, by electrical charging, by the tendency of uncontrolled aggregation, and by interaction with solvents. However, this number is high enough to generate the unbelievable multiplicity of life. In summary, the restriction to one dimension in the primary structure is very efficient because it can be combined with a mechanism of transition of the linear structure into three-dimensional functional nanoarchitectures.
At the same time, this mechanism allows the reduction of a superastronomic number of possibilities to a much smaller set. This reduction mechanism means the introduction of very strict criteria of selection for achieving the maximum self-organization potential, reliability, and integrability for higher levels of arrangement of hierarchically organized molecular nanosystems .
5 Serial processing
The restriction to one dimension is not only of interest from the point of view of complexity in the organization of building units, but also from the point of view of fabrication and processing. The one-dimensional character is typical for all objects that are generated or translated by a serial process. Technologies are full of examples of such serial operations, which correspond to the generation or operation in time, e.g., in the case of serial printing or drawing, in the main structure of computer programs, or in automated manufacturing. One-dimensional, serial fabrication processes can be organized much more regularly and well defined than complex three-dimensional processes. Standardization and automation, quality control, and correction of mistakes can be more easily included in all fabrication processes.
The advantages of serial processing are not only found in conventional fabrication but also in micro- and nanotechnology. A typical example is the fast transfer of digital layout data into physical structures during the writing of masks by using electron beams. An analog serial mechanism is also found in the synthesis of proteins [58–62]. The linear primary structure of natural peptides is the direct result of a serial fabrication process. The so-called molecular transcription of the DNA sequence into an RNA sequence by the DNA-dependent RNA polymerase, as well as the so-called translation process from the RNA sequence information into the arrangements of amino acids in the peptide chain by ribosomes, are serial natural molecular manufacturing processes  that can be understood as a transfer from digital data (mRNA) into physical structures (protein). This perspective is supported by the fact that protein synthesis is organized by a strict clock cycle similar to that in macroscopic mechanical machines.
Serial processes are always connections between the temporal and the spatial order. Operational steps are translated into an object order in space or vice versa. Information coded in symbol sequences or object orders are translated into process orders. Furthermore, process orders are translated into arrangements of objects and linking or structuring steps.
The recent serial manufacturing in some parts of micro- and nanotechnologies corresponds completely to the serial work principle, e.g., in laser micromachining . The main problem is that self-organization and selection mechanisms are not consequently used; rather, micro- and nanofabrication have been organized recently like conventional fabrication processes. One important consequence is the recent requirement for macroscopic devices and large facilities for nanomanufacturing. These macroscopic facilities for serial manufacturing have to be substituted by micro- and nano-scale devices for serial processing. The biomolecular machines and mechanisms of replication and transcription of nucleic acids and, even more, of the translation process are models for such nanomachines.
6 Dualism of structural hierarchy and strength of single bonds
The restriction into one dimension for the connection of amino acids and the transition to three-dimensional functional molecular architectures are parts of a strict hierarchical organization of proteins. The structural hierarchy is based on a first level involving only a few atoms and can be observed up to the level of organisms, populations, and ecological communities. Here, the character of molecular organization at the atomic scale and in the nanometer range is of interest, in particular. The key issue is a dualism between structural hierarchy and bond strength, which means a stepwise reduction of the stability of single intramolecular bonds with increasing structural organization level.
Many proteins are parts of supermolecular assemblies consisting of a set of several single macromolecules. Functional units in terms of natural nanomachines are formed by a spontaneous assembly of a predetermined number of proteins of certain types, in many cases with the inclusion of additional non-protein molecules [65, 66]. The protein-synthesizing machineries, the ribosomes themselves, are the product of a molecular nanoassembling process [67, 68]. In general, this supermolecular organization level is realized by non-covalent intermolecular interactions. This level is called “quaternary structure”. Each of the involved single macromolecules must be equipped with a well-defined and matching surface topography and chemical surface functions in order to meet the requirements of highly specific molecular interactions.
A well-balanced combination between the defined three-dimensional arrangement of certain atom groups and a certain degree of freedom for other groups of protein molecules is an ultimate precondition for the assembly of single protein molecules into quaternary structures, as well as for their individual chemical and biochemical functions. All atoms of a single protein molecule are involved in one specific network of covalent bonds. However, this topology of bonds allows a very large number of different conformations. Only one of them meets the requirements for the protein function. The correct three-dimensional folding of the whole molecule through the correct rotational positions of all movable bonds leads to the predetermined conformation. This well-defined conformation is called the “tertiary structure” of the protein [69, 70]. It represents the second level below that of supermolecular assemblies.
Larger protein molecules are often subdivided into domains, which means a set of some subunits with tertiary structure with a compact internal structure and a higher mobility between these parts [71, 72]. This form of organization represents a submacromolecular tertiary structure and is an additional level in the hierarchical spatial organization of these proteins.
Each amino acid unit in the peptide chain contains two twistable bonds, and the variability in the tertiary structure of a protein is given by all possible angles formed by the rotation of these two bonds in each building unit. Two typical angle ranges are mostly found for both bonds due to the spontaneous minimization of conformational energy. However, the number of possible conformations is in the order of magnitude of (2*2)N, and therefore is very high if a typical chain length of about 300 is considered. It is too high for an efficient selection process for the identification of best-suited amino acids and the molecular organization of the peptide chain in space through an ongoing trial-and-error process. This fact is a simple mathematical reason for the existence of a further organization level in the hierarchical molecular structure of proteins between the tertiary structure and the amino acid. This level is called the “secondary structure”. It is marked by a smaller set of local conformational arrangements of groups of amino acids. The most stable and frequently used types [73, 74] are a helical arrangement (α-structure) and a folded plane (β-structure). Both types are stabilized by a regular and comparatively dense arrangement of cooperative hydrogen bridges. They represent a stable polyvalent bond similar to the regular stabilization of the DNA molecules in the double helix. Both structures are compact units that are comparatively robust against mechanical and chemical forces, and represent parts of the peptide chain that can be moved, in most cases, only as a whole.
The level below the secondary structure is characterized by the linear order of amino acids in the peptide chain. It is called the “primary structure”. It determines all higher levels by the specific space requirements and chemical properties of the different amino acid residues. The primary structure corresponds to the valence bond topology of all atoms of the proteins and is directly determined by the molecular information coded by the nucleic acid sequence segment that is responsible for the synthesis of the related protein.
To understand the essential function of the hierarchical organization of proteins and of the significance of this organization at the atomic level, it is very important to recognize that there is an additional organization level below the single amino acid (Figure 4). This level is marked by the stereotype-like standard construction of the proteinogenic amino acids: each of the 20 building units consists of three atomic modules of the exact same structure and only one variable part. Thus, each amino acid can be understood as being composed of four small functional units, each of them consisting only of a few atoms. The first one is the central C-H unit that represents a coupling unit between the other three components. The second and third elements are the COOH group and the NH2 group, which are two complementary functional units that are connected by a condensation reaction during the formation of peptides. Finally, the variable side chain represents the fourth part, the only one that distinguishes the amino acids from each other. Thus, the basic units below the level of primary structure are really represented by small atomic groups only: two atoms in the CH group, and three and four atoms in the amino and carboxylic groups, respectively.
From the strictly hierarchical organization of proteins as described above, it can be concluded that hierarchies with several levels and comparatively low knot strengths are well suited for spontaneous molecular organization. There are three organization levels between the atomic level and the construction of a protein of moderate size consisting of the order of magnitude of about 104 atoms forming secondary structure units in the order of magnitude of 20 and consisting of amino acids in the order of magnitude of 500.
Besides the structural principles of hierarchical organization itself, it is crucial to ask how this type of structure can be achieved by a spontaneous molecular arrangement. It is obvious that chemical processes must control the processes that lead to such structures. What are the general rules for these processes, and how could they be used for general nanotechnological strategies?
Probably the most fundamental issue in the organization of hierarchy levels is the character of the level-forming attractive forces, which means the types of chemical bonds. Strong covalent bonds determine the lower levels. All atoms in a single amino acid are connected by covalent bonds that are stable against hydrolysis. At this level, the connection between all atoms has maximum stability. Moderate changes of temperature or pH cannot destroy the elementary building units. They are also insensitive to an enzymatic attack by hydrolases such as proteases.
At the next level – the primary structure of proteins – the amino acids are also connected by covalent bonds. Each of these bonds is stable enough for connecting hundreds of amino acids in one long peptide chain. However, these bonds, the peptide couplings, are the result of a condensation process and can be split by a hydrolysis reaction. Such a hydrolytic splitting takes place, e.g., owing to the proteolytic activity of enzymes in human bodies and other living beings that receive amino acids from the strange proteins in food in order to construct their own specific protein molecules. This sensitivity to hydrolytic splitting is the differentiating factor between the lowest and the next higher level of chemical organization in proteins. The typical bonds of the higher level (primary structure) are less stable than the non-polar or low-polar covalent bonds at the lowest level (amino acids).
Groups of hydrogen bonds supply the typical attractive forces for the stabilization of secondary structures. A single hydrogen bond is too weak for achieving stable molecular architectures. The stability of α-helices and β-sheets is only achieved by the cooperative effect of several hydrogen bonds with the above-mentioned polyvalent character. Obviously, this cooperation of several directed weaker bonds is a very efficient strategy for the fixation of a certain conformation of a larger molecule. The regular structure of amino acids and the strict periodicity of peptide bonds in the peptide backbone support the formation of the required regular arrangements of several or dozens of hydrogen bridges.
The most important components of the stabilization of the tertiary structure are less directed and less stable than the groups of hydrogen bonds stabilizing the secondary structures if they are considered from the point of view of single bonds. Tertiary structures are mainly stabilized by hydrophobic forces; that means the dominance of van der Waals interactions between non-polar amino acid residues, which are mainly found in the internal parts of the folded proteins. These interactions and the formed three-dimensional structures are further supported by additional hydrogen bonds, electrostatic interactions, and – in the presence of metal ions to certain degree – by more or less weak coordinative bonds. In general, the tertiary structures also have the character of a polyvalent stabilization; however, this stabilization is, in most cases, less directed and less strictly localized in distinct single bonds than in the case of the hydrogen bonds stabilizing the secondary structure. Whereas the single components of the forces that stabilize the tertiary structures are weak and less directed, the total of these bonds and their spatial distribution are able to cause the highly specific character of three-dimensional folding of the macromolecule.
The cooperative effect of polyvalent interactions in proteins leads to a surprisingly high stability of these biomolecules despite the low energy of the involved single second-order interactions, such as hydrogen or van der Waals bonds. Nanopores formed by proteins can be stable enough for realizing nanomachines for a reliable serial identification of nucleotides in a DNA sequence . An astonishing stability is also observed in proteins for electron transfer processes, e.g., in mitochondria and chloroplasts. High tolerance against pH changes is required and realized, e.g., for the reliable function of voltage-gated ion channels, which are important for the action potentials in neurons .
In summary, it can be concluded that there is an increase in the number of bonds, but a decrease in the strength and localization of single bonds, with increasing organization level. It seems that the strategy of stepwise decreasing strength of characteristic single bonds with increasing hierarchical level and size of building units is a fundamental principle for using molecular self-organization to generate functional nanoarchitectures.
7 Time-scale management: combination of reversible and stable structures
Mechanical functions and different environmental conditions demand a combination of stable structural elements and mobility, as well as matching shapes and dynamic responses [77, 78]. How can nanoarchitectures be kept stable and mobile at the same time? Distinguishing between thermodynamic and kinetic stability is important: a molecular system can be considered stable if it is in thermodynamic equilibrium, i.e., in a state of a minimum of free energy. In this state, many microscopic processes can be occurring; however, these processes are ideally reversible and compensate each other, and the total of all processes results in the equilibrium state. The system is highly dynamic but remains in equilibrium as long as the environmental conditions – concentrations, temperature, and pressure – are not changed.
In case of kinetic stability, the global minimum of free energy can be much lower than that in the thermodynamic state. It is sufficient to have the system at a local minimum if the thresholds for activation are high enough to keep the system in the original state.
What is the fundamental difference between the two above-described cases of molecular stability? They differ in their relaxation behavior: in the case of thermodynamic stability, each change of condition will lead to a shift in the system state. The system can be understood as reversible or “ideal elastic”. After a change of conditions, it is quickly transformed into a modified state. The original state will quickly be achieved if the original environmental conditions are reintroduced. In contrast, the state of the kinetically stable system will be unaffected by environmental changes as long as the critical threshold is not reached. However, after passing this threshold, the system will be converted into a new state. A reintroduction of the original environmental conditions does not return the system back to the original state as long as the activation threshold cannot be passed. The system response is more non-reversible or “switch-like”.
In general, small molecules and larger molecules with simple chemical structures can be either thermodynamically or kinetically stable; they can respond to a change of conditions either by an elastic response or by a switch-like chemical reaction . In contrast, molecules like folded proteins, with their sophisticated hierarchy of bond strengths, combine both types of response behavior. They involve highly dynamic structural features that are marked by low activation thresholds and fast equilibration [80–82], as well as comparatively kinetically stable structure elements that need a higher activation for switching from the original state into another state.
The hierarchy of bond strengths of proteins corresponds to a hierarchy of activation thresholds . This hierarchy of activation thresholds corresponds to a hierarchy of reaction probabilities or constants of lifetimes . A good approximation of the spectrum of reaction probabilities Pr and lifetime of states Ts is given by the product of the principal process probability (frequency) P0 and the probability of passing the activation threshold Pact corresponding to the Arrhenius equation:
The kinetic lifetime of a certain state Ts can be regarded as the reciprocal of Pr:
The Boltzmann constant k and the temperature T are globally valid. P0 is mainly determined by the activation entropy of the regarded system, which is strongly related to the specific local motion of atom groups. The remaining activation energy Ea is the key parameter for the regulation of the process. An enhancement of Ea leads to an increase of stability, higher lifetimes, and more switch-like behavior. A decrease means faster relaxation into the equilibrium. The position of Ea in the exponent is very important for the spectrum of the lifetime of molecular states in proteins . It means that moderate differences in the activation barriers result in very different time constants (Figure 4). Factors between 10 and 100, which are typical for the differences in the activation of covalent and hydrogen bonds, for example, result in lifetime ratios of many orders of magnitudes.
A tuning of activation barriers is also possible by enhancing the numbers of hydrogen bonds or other weak interactions that act cooperatively. In this way, a single protein molecule consists of structural elements with a wide spectrum of characteristic lifetime constants, elastic response and switchable units, and possesses a balanced mixture of stable and flexible elements.
This balanced combination of rigid and flexible structures, with high fluctuations due to low energy barriers and rare stochastic events due to higher energy barriers, is not only of interest for protein nanostructures, but should also be considered for the development of molecular nanomachines realized in other substance classes. It is imaginable that in the future, hybrid structures will be developed for nanodevices in which organic molecular hinges, springs, and bearings are connected to non-flexible nano-objects like clusters, rods, disks, tubes, and other inorganic or polymer nanoparticles, and conjugated systems such as graphene-like elements. This proposed heterogeneous composition of future nanodevices means, on the one hand, a certain violation of the concept of uniformity in the elementary modules and hierarchical organization of nanoarchitectures as they are realized in the world of proteins. On the other hand, the integration of diverse nanomaterials into molecular and supermolecular construction would open a wide spectrum of new devices and functions. The open question is, how can this diversity and the huge number of thinkable combinations of molecules and particles be managed? Here, it is assumed that we need for the particles to have a similar restriction to certain types of basic units and connection principles, as in the concept of molecular organization hierarchies. The expected convergence between the development of new molecular synthesis and assembling strategies and the development of nanomaterials would probably result in a complete dissolution of the borderline between solid and soft matter, between structure and function, and between material and device.
The interplay of motions with low activation barriers – which means low time constant and high fluctuation rates – with motions and reactions with higher activation barriers is well illustrated by Kramer’s theory of reaction rates . The transition between two states that are separated by a higher energetic threshold is rare because the fluctuations of the system are normally near the potential minima. The transition takes place only in a few cases when the fast fluctuations around the minimum reach an elongation, achieving the main threshold. In the typical case of a strongly damped system, the transition rate k can be expressed through the substitution of the prefactor in the Arrhenius equation [compare Eq. (1)] by the quotient of the product of the system frequencies of the potential minimum ωa and of the transition state ωb and the viscous damping 2πγ (Figure 5).
8 Limited mobility
The organization of some proteins in domains, the existence of compact and stable secondary structure units, and their connection by better movable molecular links is not only important for the hierarchical structure of the macromolecules, but also for their function. Proteins are, in general, characterized by a certain geometry; however, they are also characterized by a certain intramolecular mobility or by well-defined changes in molecular shape in the presence of mechanical or chemical stress. The extended lifetime spectrum of intramolecular units is directly related to a spectrum of intramolecular mobility. In analogy to the hierarchical organization of molecular building units and bond strengths, this spectrum is also hierarchically organized.
Most proteins combine rigid construction elements with movable linking parts. The constructions can roughly be understood to be like the fixed and movable parts of a macroscopic machine. Many textbook illustrations of proteins use this picture of machine-like molecular structures to explain molecular functions in relation to molecular geometry, which mostly deviates from X-ray diffraction and nuclear magnetic resonance investigations.
However, the model of a macroscopic machine is only partially true. We have to be aware that the involved atoms and groups of atoms are quantum objects, and all parts of the macromolecule undergo thermal activation, exchange of impulses, and permanent fluctuations. Therefore, the difference in mobility between the different parts of the macromolecule is mainly a differentiation in the time scales of the concerned motions . The dynamics of the protein and its response to impulses from outside or its interactions with external partners is determined by these different time scales, by the universally present mixture of frequent reversible relaxations into the equilibrium, and non-frequent environmental-dependent switching processes.
In the following, some molecular strategies for the management of limited mobility in proteins will be discussed. Important strategies for controlling local mobility are based on the specific properties of the amino acids or groups of them. The inclusion of the simplest amino acid, glycine, or still more oligo-glycine segments in the peptide chain will cause high mobility owing to the low space requirement of these building units [88, 89]. The inclusion of glycine is also very important for the molecular configuration because it is the only amino acid without optical activity. In contrast, groups of valine, leucine, and isoleucine units, and still more of the aromatic amino acid phenylalanine, will form more hydrophobic regions with comparatively high space requirements.
The rotatability in the backbone is most efficiently suppressed at positions with proline units . This is due to the ring structure that includes the nitrogen atom of the amino group and, correspondingly, the peptide group. Such proline incorporation is always connected to a rigid part of the peptide backbone.
Some other elements for lowering molecular mobility are constructed for a certain dependence on outside conditions. A very efficient stabilization method is the inclusion of disulfide bridges, i.e., a covalent coupling between cysteine units in different parts of a peptide chain, which is also sometimes used for the coupling of separate peptide molecules . The possibility of the formation of such bridges can be introduced by the inclusion of two cysteines at the required positions in the amino acid chain because their thiol groups are the essential functions for the covalent coupling by an oxidation step. The formation or splitting of these bonds is mainly controlled by enzymatic activity and by the redox potential and the availability of active hydrogen. Thus, molecular mobility is determined in this case by a sequence feature on the one side (the inclusion of cysteine), and by the dehydrogenation or hydrogenation depending on the environmental conditions on the other side.
A similar combination of a structural motif and outside conditions for controlling molecular mobility is found in the case of histidine units or groups of histidine [92–94]. This amino acid forms a stable coordinative bond with metal ions, in particular nickel ions. Thus, low mobility and a fixed molecular geometry are achieved in the presence of histidine units and nickel ions at the same time. The presence or absence of ions and the ion strength are important factors for the stabilization or destabilization of protein conformations and for the modulation of the molecular mobility, because the donating ability of nitrogen and oxygen atoms in the functional groups promotes the formation of coordinative bonds.
Besides the ion strength, pH is a very important factor for controlling the structure and mobility of protein molecules . High proton concentrations lead to a destruction of coordinative bonds and enhance the positive charge of the protein molecule through the protonation of amino groups. In contrast, an increase in pH causes a reduction of positive charge or an increase of negative charge through the deprotonation of carboxylic groups. These effects modulate not only the interaction of amino acid residues with metal ions but also the electrostatic interactions between different parts of the macromolecule. Therefore, pH is very important for the intramolecular mobility and for the stability of the native shape . The pH sensitivity of proteins can be adapted to extreme conditions, e.g., in extremophiles [97, 98]. Frequently, a complete loss of molecular shape and the order of three-dimensional structure are observed by means of an acidification-induced denaturation.
These examples illustrate that the hierarchically organized mobility of proteins is conferred by their structure and by the order and local arrangement of amino acids with different space requirements and chemical functions . Despite the pure sequence-dependent intramolecular mobility, the sensitivity of molecular mobility from the environmental conditions is also determined by the inclusion of certain amino acids at certain positions in the peptide sequence.
Limited mobility is required in many protein functions, namely enzymatic activities and, particularly, as molecular motors. Motor proteins such as the microtubulin/kinesin or the actin/myosin system as well as the membrane-fixed ATPase are composed of several components that form movable parts. The systems combine well-defined geometries of molecular components with a well-defined degree of freedom of motion. The activation threshold is overcome in all three cases by a coupling between the energy-supplying conversion of ATP into ADP with a motion step of the macromolecule. For example, a complete molecular turn of 360° of the rotor molecule in the ATPase complex is performed in three steps of 120°, in which one ATP molecule is consumed per step . The whole system can be understood as a molecular machine driven by ATP as an outside energy source (see Section 9).
The variability of structures makes proteins very interesting for molecular construction in the frame of “protein nanotechnology”. Therefore, the natural building block strategy of peptides can be used . Barrels  and cage-like structures [102, 103] are typical examples of this protein-based nanotechnological approach.
The concept of hierarchical molecular organization  is applied by the use of proteins for different nanotechnological fabrication approaches. The self-assembling property of peptides and proteins can be used for creating structures up to the micron range . The use of regular protein assemblies in cellular nanomotors is particularly promising for mechanical nanoactuation . Despite the use of lower levels of molecular building blocks, the application of naturally completed assemblies seems to be an attractive way for the development of new materials and devices, e.g., by using complete viruses . A special strategy is the use of biomacromolecules and their assemblies for making nanomaterials by templating for biomineralization .
9 Equilibria, energy dissipation, and entropy export
Chemical reactions and even molecular processes involving macromolecules can be simply understood in many cases as a relaxation into a new state of equilibrium after disturbing this equilibrium from the outside. This relaxation process is accompanied by a reduction of free energy and is mostly related to certain energy dissipation and/or an increase of entropy. The spontaneity of exothermic processes seems to be trivial. Also trivial is the spontaneity of entropy-producing processes. However, from this simple point of view, the biochemical processes in cells seem to run in contradiction with the second law of thermodynamics, because it demands a permanent increase of entropy in closed systems if irreversible processes like growth or differentiation are occurring, and increasing entropy means a loss of order or a loss of systemic information. The explanation for this apparent contradiction is given by the open thermodynamic character of cells: they are not closed in a thermodynamic sense but are able to exchange matter, energy, and entropy with the environment and keep themselves in an “isoentropic state” . The key issue is their ability to realize an entropy export, which means that they can release more entropy to the environment than the entropy they produce themselves.
The basic laws of thermodynamics and the principle of entropy export are not only valid for organisms and cells but also for molecules. The only difference consists in the much higher importance of fluctuations at the molecular level. The thermodynamic aspects of molecular functions of proteins must be discussed reconsidering this background.
There are two principal mechanisms of protein activities. The first type is in complete analogy to the activity of small molecules. In this case, a chemical reaction is driven directly by the free energy of the mixture of reaction partners . The protein may be involved as a direct reaction partner or in the form of a catalyst. The reaction is marked by a continuous reaction trajectory directed to the new chemical equilibrium. The protein can be regarded as an outside-driven tool in the whole process.
The second type is not driven by the free energy of the reaction partners directly  but by an additional process that enhances the activity of tools and that acts as a separate channel for the entropy export. This type of process is similar to a machine that is driven by an outside energy source like a steam engine or an electrical motor (Figure 6). The produced entropy is transferred to these energy-supplying systems – the conversion of the chemical power of wood or coal into heat, or of electrical current into heat. It is very typical that technical systems that standardize energy supplies and systems for entropy export are used as steam turbines or electrical power. The same is true for energizing systems at the molecular level in the case of the activation of proteins. The trick is always found in a coupling between a universal molecular energy supply and a specific energy-consuming and entropy-producing protein activity. A typical example, therefore, is the universal use of ATP for driving the muscles, or for driving the linear transport of particles by kinesin molecules on the supermolecular rails of tubulin, or by the active trans-membrane transport of ions against concentration gradients by the rotating ATPase molecule. Such biomolecular systems are also being used for model systems in molecular nanotechnology .
Driven nanotechnological systems need an energetically pumped environment. The coupling between energy resources in the environment and the nanosystems must involve the uptake of usable energy on the one side and the release of degraded energy on the other side. At the molecular level, it means the uptake of particles and photons with energies above the thermal equilibrium or the use of exploitable metastable chemical systems. The fundamental possibilities for this energy supply and entropy exchange are also demonstrated in nature, e.g., in photosynthesis and heterotrophic metabolism: either the nanosystem uses photons between the mid-infrared and mid-ultraviolet range for photochemical energy harvesting, or they have to earn energy through the collection and conversion of applicable substrates. A certain special form of chemical energy supply at the nano-level could also be realized through an electrochemical pumping. For nearly all cases, it has to be considered that the nanosystems would be operated in certain environments with specific physical and chemical conditions. The pumping and energy transfer systems should be adapted to the specific features of the operation environment; however, besides this consideration, they should be constructed as universally as possible.
It is an ultimate challenge for molecular nanotechnology to find new and efficient molecular systems with such a coupling between universal molecular or physical activation mechanisms and specific entropy-producing and energy-consuming operations. A promising step in this direction can be found recently in the use of such coupled biocatalytic processes in organic synthesis [113, 114].
It is assumed that this principle of coupling between very specific molecular operations and general mechanisms of molecular activations can be applied to a much larger extent. Therefore, we have to reconsider that our model, the proteins, can only be understood as a form of dualism between outside- or system-driven nano-scaled machines and thermally activated fluctuating quantum objects. They teach us that new nano-sized tools for molecular nanotechnology also have to be developed as technical devices that integrate, in such a dualistic sense, the properties of spontaneously behaving and fluctuating chemical molecules and of well-determined externally driven automata.
Besides the above-mentioned general statement, it would helpful to consider some general aspects for the construction of nano-scaled active devices in future molecular nanotechnology. An important part of these aspects can be summarized in the form of seven rules that are derived from the analysis of the construction and function of proteins:
Molecular modularization: Functional nanosystems must have a modular construction. The building units should belong to a few or, better, only one class of molecules or atom groups with standardized functions. Their number should be restricted, and they should be selected by using a set of a few antagonistic or complementary properties.
Restriction of dimensions: The systems should not be directly assembled in two or three dimensions but should be generated as a linear object that is folded in a secondary step into a functional three-dimensional device.
Serial processing: The primarily formed linear molecular objects can be generated by a serial process. They can be transformed, transcripted, or translated by further serial processes. Strings, chains of electronic signals, or other serial codes can be directly used for implanting information into the molecules, transferred from one class of coding molecule to other classes, and back to electronic devices or other physical storage and information-processing systems.
Hierarchy of structures and bond strengths: The required nanotechnological functions can only be realized in macromolecules with a suited internal structure, which must be based on a hierarchy of structural elements and stepwise varied strengths of intrasystem interactions.
Hierarchy of time scales: The combination of stable and flexible elements, and that of reliability and specific responses demand a combination of near-equilibrium fluctuations with switch-like state transformations. These combinations have to be realized by the system-internal definition of characteristic lifetimes and relaxation times in a wide spectrum.
Limited mobility: Functional molecular nanosystems have to combine well-defined geometries and chemical functions with intramolecular mobility, which demands a mixture of stability and elastic response and a restricted number of degrees of motion freedom.
Entropy export management: Functional nanosystems have to combine non-energized responses with activations using external energy sources, which includes a general principle and a general tool for the storage and conversion of energy and for management for entropy export in order to enable molecular nanosystems to act as nano-scaled machines.
About the author
J. Michael Koehler is the head of the Department of Physical Chemistry and Microreaction Technology at the Technical University of Ilmenau (Germany) since 2001. He studied chemistry in Halle/Saale and Jena, where he also habilitated in General and Physical Chemistry (1992). He led a research department at the Institute of Physical High Technologies in Jena between 1991 and 2000. During this time, he also taught at the Universities of Wuppertal and Jena. Professor Koehler inter alia has edited books on microlithography, microsystem technology, and nanotechnology. His current research interests are focused on nanotechnology and on the application of droplet-based microfluidics in nanoparticle syntheses and bioscreenings.
I thank Otmar Asperger (Leipzig) and Stuart Lindsay (Tempe) for critical discussions and advice.
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