Electron cryomicroscopy of biological machines at subnanometer resolution

Advances in electron cryomicroscopy (cryo-EM) have made possible the structural determination of large biological machines in the resolution range of 6-9 angstroms. Rice dwarf virus and the acrosomal bundle represent two distinct types of machines amenable to cryo-EM investigations at subnanometer resolutions. However, calculating the density map is only the first step, and much analysis remains to extract structural insights and the mechanism of action in these machines. This paper will review the computational and visualization methodologies necessary for analysis (structure mining) of the computed cryo-EM maps of these machines. These steps include component segmentation, averaging based on local symmetry among components, density connectivity trace, incorporation of bioinformatics analysis, and fitting of high-resolution component data, if available. The consequences of these analyses can not only identify accurately some of the secondary structure elements of the molecular components in machines but also suggest structural mechanisms related to their biological functions.     

ATP-dependent proteases of bacteria: recognition logic and operating principles

ATP-powered AAA+ proteases degrade specific proteins in intracellular environments occupied by thousands of different proteins. These proteases operate as powerful molecular machines that unfold stable native proteins before degradation. Understanding how these enzymes choose the "right" protein substrates at the "right" time is key to understanding their biological function. Recently, proteomic approaches have identified numerous substrates for some bacterial enzymes and the sequence motifs responsible for recognition. Advances have also been made in elucidating the mechanism and impact of adaptor proteins in regulating substrate choice. Finally, recent biochemical dissection of the ATPase cycle and its coupling to protein unfolding has revealed fundamental operating principles of this important, ubiquitous family of molecular machines.     

Structural and organisational conditions for being a machine

Although the analogy between macroscopic machines and biological molecular devices plays an important role in the conceptual framework of both neo-mechanistic accounts and nanotechnology, it has recently been claimed that certain complex molecular devices (consisting of biological or synthetic macromolecular aggregates) cannot be considered machines since they are subject to physicochemical forces that are different from those of macroscopic machines. However, the structural and physicochemical conditions that allow both macroscopic machines and microscopic devices to work and perform new functions, through a combination of elemental functional parts, have not yet been examined. In order to fill this void, this paper has a threefold aim: first, to clarify the structural and organisational conditions of macroscopic machines and microscopic devices; second, to determine whether the machine-like analogy fits nanoscale devices; and third, to assess whether the machine-like analogy is appropriate for describing the behaviour of some biological macromolecules. Finally, the paper gives an account of ‘machine’ which, while acknowledging the physicochemical and organisational differences between man-made machines and biological microscopic devices, nevertheless identifies a common conceptual core that allows us to consider the latter ‘machines’.     

AAA+ ATPases: achieving diversity of function with conserved machinery

AAA+ adenosine triphosphatases (ATPases) are molecular machines that perform a wide variety of cellular functions. For instance, they can act in vesicle transport, organelle assembly, membrane dynamics and protein unfolding. In most cases, the ATPase domains of these proteins assemble into active ring-shaped hexamers. As AAA+ proteins have a common structure, a central issue is determining how they use conserved mechanistic principles to accomplish specific biological actions. Here, we review the features and motifs that partially define AAA+ domains, describe the cellular activities mediated by selected AAA+ proteins and discuss the recent work, suggesting that various AAA+ machines with very different activities employ a common core mechanism. The importance of this mechanism to human health is demonstrated by the number of genetic diseases caused by mutant AAA+ proteins.     

Biotechnology and molecular computing

Carbon-based molecular machines are in sight for the next-generation electronics industry. In that nature has been producing and maintaining molecular machines for millions of years, the biological sciences may hold the keys to tomorrow's computer science disciplines. This article reviews some contributions that natural systems may make, and some general concerns that have yet to be resolved in realizing molecule-based devices.     

Single molecule thermodynamics in biological motors

Biological molecular machines use thermal activation energy to carry out various functions. The process of thermal activation has the stochastic nature of output events that can be described according to the laws of thermodynamics. Recently developed single molecule detection techniques have allowed each distinct enzymatic event of single biological machines to be characterized providing clues to the underlying thermodynamics. In this study, the thermodynamic properties in the stepping movement of a biological molecular motor have been examined. A single molecule detection technique was used to measure the stepping movements at various loads and temperatures and a range of thermodynamic parameters associated with the production of each forward and backward step including free energy, enthalpy, entropy and characteristic distance were obtained. The results show that an asymmetry in entropy is a primary factor that controls the direction in which the motor will step. The investigation on single molecule thermodynamics has the potential to reveal dynamic properties underlying the mechanisms of how biological molecular machines work.     

The emergence of molecular machines as a prerequisite of the ancient RNA world evolution

The problem of the start of biological evolution in the ancient RNA world is considered. It is postulated that the appearance of catalytic RNAs — ribozymes — via spontaneous cis- and trans-rearrangements of polyribonucleotides in primordial Darwin ponds should not have been sufficient for the start of evolution, until a new class of functional RNA, namely energy-dependent molecular machines, arose. The proposed hypothesis is that the simplest and primary type of molecular machines could be nucleoside triphosphate-dependent RNA-based helicases, which were capable of unwinding the stable double-helical RNAs inevitably formed during RNA syntheses on complementary templates. Thereupon, unwinding RNA polymerases could appear as a result of association or fusion of helicases and polyribonucleotide-polymerizing ribozymes. The latter event provided the mechanism of RNA replication using the double-helical RNAs as a communal genofond (gene pool) of a Darwin pond, and thus initiated the fast evolution of the ancient RNA world.     

The sliding filament model of muscle contraction. I. Quantum mechanical formalism

The quantum mechanical analog of work is defined and discussed by using a simple hypothetical molecular machine, thus enabling the introduction of clearly defined ideas which are necessary for a molecular discussion of biological machines such as the contractile machinery in striated muscle. The problem of control of such quantum machines is discussed and shown to be possible using the concept of a stimulated transition. The problem of “reversibility” is also discussed and shown to have a satisfactory solution for the orders of magnitude of the forces and velocities involved in muscular contractile machinery.     

In defence of the high energy phosphate bond

The concept of the “high energy phosphate bond” has recently been strongly criticised by Banks &; Vernon. The criticisms were: (i) energy cannot be stored in molecules; let alone in bonds; (ii) a muscle is an open system, so that the free energy of hydrolysis of ATP is irrelevant; (iii) biological reactions cannot be at equilibrium, therefore they must be inefficient. It is argued that all of these criticisms arise because the timescale appropriate to molecular events has been left out; in particular “stored energy” needs to be defined relative to the machine which uses that energy. The criticisms may be answered using a conceptual framework which overcomes this deficiency and which was developed previously to extend classical thermodynamics to the molecular level. Criticism (i) is refuted by discussing in detail the chemiluminescent reaction of rubrene oxide, which can only be described as liberating internal energy stored in a single molecule; while it is true that entropy cannot be retained in a single molecule for long enough to do useful work with it, there is nothing which forbids internal energy from being so stored. Criticism (ii) would be true if, and only if, a muscle uses the same kind of mechanism as do the ordinary chemical machines with which we are familiar (batteries, etc). But this assumption is shown to be false; a muscle cannot use this type of mechanism. Criticism (iii) is answered by finding the conditions under which a biological machine could in fact approach 100% efficiency. These conditions are: (i) in metabolism ΔF = 0; the steps are in thermal equilibrium; (ii) in the molecular machines themselves ΔS = 0, so that ΔF = ΔH; the machines are mechanical. It is also shown that not only are these conditions perfectly conceivable, but also that there is good evidence that, over the course of evolution, they have actually been attained.     

Structural biology of cellular machines

Multi-component macromolecular machines contribute to all essential biological processes, from cell motility and signal transduction to information storage and processing. Structural analysis of assemblies at atomic resolution is emerging as the field of structural cell biology. Several recent studies, including those focused on the ribosome, the acrosomal bundle and bacterial flagella, have demonstrated the ability of a hybrid approach that combines imaging, crystallography and computational tools to generate testable atomic models of fundamental biological machines. A complete understanding of cellular and systems biology will require the detailed structural understanding of hundreds of biological machines. The realization of this goal demands a concerted effort to develop and apply new strategies for the systematic identification, isolation, structural characterization and mechanistic analysis of multi-component assemblies at all resolution ranges. The establishment of a database describing the structural and dynamic properties of protein assemblies will provide novel opportunities to define the molecular and atomic mechanisms controlling overall cell physiology.     

Critical analysis of objections against the biological molecular energy machines.

In the past, two important objections against McClare's idea of biological molecular energy machines were raised. One of the criticisms was concerned with the origin of energy gained in ATP cleavage and with an interpretation of McClare's "excited vibrational state." The former argument reveals a failure of the critics to comprehend McClare's approach. As to the excited vibrational state, it can be identified with nonequilibrium conformational states of the unit rather than with a single vibrational mode. The other criticism based on Brillouin's energy cost of measurement argued that reversible operation of biological molecular energy machines would be virtually impossible. Using propagation velocities of deformations of the unit's structure (instead of velocity of light), the objections against reversibility are invalidated even in the framework of the critic's approach. McClare's idea and relevant definitions are thus physically correct.     

Plausible view on the biological molecular energy machines.

A qualitative picture of operation modes of biological molecular energy machines is presented. It is suggested that there is mutual control between the flow of molecular energy stored in a biological molecular energy machine and the sequence of nonequilibrium conformational states through which the machine passes in doing work. If the structure of the conformational space is favorable, the set of trajectories in this space decomposes into two families, each of which accomplishes another task. This divergence of trajectories enables to distinguish molecular objects according to differences in interaction between the machine and the object, i.e., to perform a measurement on a molecular object and process the object according to the result of that measurement.     

Protein-polymer nano-machines. Towards synthetic control of biological processes

The exploitation of nature's machinery at length scales below the dimensions of a cell is an exciting challenge for biologists, chemists and physicists, while advances in our understanding of these biological motifs are now providing an opportunity to develop real single molecule devices for technological applications. Single molecule studies are already well advanced and biological molecular motors are being used to guide the design of nano-scale machines. However, controlling the specific functions of these devices in biological systems under changing conditions is difficult. In this review we describe the principles underlying the development of a molecular motor with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for control of the motor function. The molecular motor is a derivative of a TypeI Restriction-Modification (R-M) enzyme and the synthetic polymer is drawn from the class of materials that exhibit a temperature-dependent phase transition.The potential exploitation of single molecules as functional devices has been heralded as the dawn of new era in biotechnology and medicine. It is not surprising, therefore, that the efforts of numerous multidisciplinary teams 12. have been focused in attempts to develop these systems. as machines capable of functioning at the low sub-micron and nanometre length-scales 3. However, one of the obstacles for the practical application of single molecule devices is the lack of functional control methods in biological media, under changing conditions. In this review we describe the conceptual basis for a molecular motor (a derivative of a TypeI Restriction-Modification enzyme) with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for controlling the motor function 4.     

Self-organization versus watchmaker: molecular motors and protein translocation

Generation of directional movement at the molecular scale is a phenomenon crucial for biological organization and dynamics. It is traditionally described in mechanistic terms, in consistency with the conventional machine-like image of the cell. The designated and highly specialized protein machines and molecular motors are presumed to bring about most of cellular motion. A review of experimental data suggests, however, that uncritical adherence to mechanistic interpretations may limit the ability of researchers to comprehend and model biology. Specifically, this article illustrates that the interpretation of molecular motors and protein translocation in terms of stochasticity and self-organization appears to provide a more adequate and fruitful conceptual framework for understanding of biological organization at the molecular scale.     

Fractal Globules: A New Approach to Artificial Molecular Machines

The over-damped relaxation of elastic networks constructed by contact maps of hierarchically folded fractal (crumpled) polymer globules was investigated in detail. It was found that the relaxation dynamics of an anisotropic fractal globule is very similar to the behavior of biological molecular machines like motor proteins. When it is perturbed, the system quickly relaxes to a low-dimensional manifold, M, with a large basin of attraction and then slowly approaches equilibrium, not escaping M. Taking these properties into account, it is suggested that fractal globules, even those made by synthetic polymers, are artificial molecular machines that can transform perturbations into directed quasimechanical motion along a defined path.     

Models of higher-order structure: foldamers and beyond

Folding, an attribute common to biological macromolecules such as proteins and nucleic acids, enables the formation of complex three-dimensional structure and thus enables the function of these exquisite molecular machines. Chemists are exploring the folding of natural and artificial systems with increasing enthusiasm and boldness of molecular design. The most recent achievements in the area of artificial folding molecules are described in this review.     

A structural perspective on the dynamics of kinesin motors

Despite significant fluctuation under thermal noise, biological machines in cells perform their tasks with exquisite precision. Using molecular simulation of a coarse-grained model and theoretical arguments, we envisaged how kinesin, a prototype of biological machines, generates force and regulates its dynamics to sustain persistent motor action. A structure-based model, which can be versatile in adapting its structure to external stresses while maintaining its native fold, was employed to account for several features of kinesin dynamics along the biochemical cycle. This analysis complements our current understandings of kinesin dynamics and connections to experiments. We propose a thermodynamic cycle for kinesin that emphasizes the mechanical and regulatory role of the neck linker and clarify issues related to the motor directionality, and the difference between the external stalling force and the internal tension responsible for the head-head coordination. The comparison between the thermodynamic cycle of kinesin and macroscopic heat engines highlights the importance of structural change as the source of work production in biomolecular machines.     

Nanobiotechnology: the fabrication and applications of chemical and biological nanostructures

Biology can teach the physical world of electronics, computing, materials science and manufacturing how to assemble complex functional devices and systems that operate at the molecular level. Our present capability to fabricate simple molecular tools, devices, materials and machines is primitive compared with the sophistication of nature. Nevertheless, the nanomanufacturing of 'biomimetic' devices is moving ahead strongly. Recent developments have been made in the use of biological systems in molecular self-assembly, spatial positioning, microconstruction, biocomposite fabrication, nanomachines and biocomputing.     

Synthetic biology and its alternatives. Descartes,Kant and the idea of engineering biological machines

The engineering-based approach of synthetic biology is characterized by an assumption that ‘engineering by design’ enables the construction of ‘living machines’. These ‘machines’, as biological machines, are expected to display certain properties of life, such as adapting to changing environments and acting in a situated way. This paper proposes that a tension exists between the expectations placed on biological artefacts and the notion of producing such systems by means of engineering; this tension makes it seem implausible that biological systems, especially those with properties characteristic of living beings, can in fact be produced using the specific methods of engineering. We do not claim that engineering techniques have nothing to contribute to the biotechnological construction of biological artefacts. However, drawing on Descartes’s and Kant’s thinking on the relationship between the organism and the machine, we show that it is considerably more plausible to assume that distinctively biological artefacts emerge within a paradigm different from the paradigm of the Cartesian machine that underlies the engineering approach. We close by calling for increased attention to be paid to approaches within molecular biology and chemistry that rest on conceptions different from those of synthetic biology’s engineering paradigm.     

70% efficiency of bistate molecular machines explained by information theory,high dimensional geometry and evolutionary convergence

The relationship between information and energy is key to understanding biological systems. We can display the information in DNA sequences specifically bound by proteins by using sequence logos, and we can measure the corresponding binding energy. These can be compared by noting that one of the forms of the second law of thermodynamics defines the minimum energy dissipation required to gain one bit of information. Under the isothermal conditions that molecular machines function this is joules per bit ( is Boltzmann''s constant and T is the absolute temperature). Then an efficiency of binding can be computed by dividing the information in a logo by the free energy of binding after it has been converted to bits. The isothermal efficiencies of not only genetic control systems, but also visual pigments are near 70%. From information and coding theory, the theoretical efficiency limit for bistate molecular machines is ln 2 = 0.6931. Evolutionary convergence to maximum efficiency is limited by the constraint that molecular states must be distinct from each other. The result indicates that natural molecular machines operate close to their information processing maximum (the channel capacity), and implies that nanotechnology can attain this goal.