Braun, T. Bruce, A. Crane, D. Gibbons, M. Grigg, L. Hicks, D. Hollingsworth, R. In: P. Weingart, N. Klein, J. Leydesdorff, L. Malsch, I. Metzger, N.
Meyer, M. Some propositions about an emerging field between hype and path-dependency, Scientometrics , 70 3 [this issue]. Morillo, F. Porter, A. Price, D. Rinia, E. Scientometrics , — Roco, M. Shinn, T. Schild, I. Schliwa, M. Schummer, J. Van Leeuwen, T. Van Raan, A.
In fact, the enzyme is capable of two opposing functions restriction and modification , which are controlled enzymatically through an allosteric effector ATP and temporally through the assembly of the holoenzyme. In addition, the R-M enzyme has a powerful ATPase activity, which is associated with DNA translocation prior to cleavage; it is this translocation process that leads to random cleavage sites. The yellow block represents the recognition sequence for the enzyme. During translocation, an expanding loop is produced.
Type I R-M enzymes fall into families based on complementation grouping, protein sequence similarities, gene order and related biochemical characteristics [ 6 - 8 ]. Within one sub-type the IC family there are three well-described members, including EcoRI, which is the focus of our interest. Therefore, the HsdR subunit is the motor subunit of the enzyme and this subunit is associated with helicase activity [ 15 - 18 ]. However, the precise mechanism of DNA translocation is uncertain and the true nature of the motor function has yet to be fully determined but a number of important functional units — nuclease, helicase and assembly domains have been identified within the HsdR subunit [ 19 ].
Schematic of the motor subunits. Szczelkun et al. However, highly efficient cleavage of circular DNA carrying only a single recognition sites for the enzyme suggests collision-based cleavage is not the whole story [ 20 , 22 ]. Mechanism of DNA cleavage. The enzyme subunits are represented by: green ellipse — M2S complex, green box — HsdR subunit with ATPase and restrictase activities; C denoting cleavage site.
The black line represents DNA with the yellow box denoting the recognition sequence. Arrow shows direction of DNA translocation. For more details see text. DNA translocation has been assayed in bulk solution using protein-directed displacement of a DNA triplex and the kinetics of one-dimensional motion determined.
Molecular motor - Wikipedia
The data shows processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. Furthermore, this can only be explained by bi-directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. Motor activity of type I R-M Enzyme. The motor follows the helical thread of the DNA resulting in spinning of the DNA end illustrated by the rotation of the yellow cube.
As previously mentioned, the final step of the subunit assembly pathway of the Type I Restriction-Modification enzyme EcoRI produces a weak endonuclease complex of stoichiometry R 2 M 2 S 1. This subunit has been shown to assemble with the EcoRI DNA methyltransferase MTase to produce an active complex with low-level restriction activity. We have also assembled a hybrid REase and the data obtained show that the hybrid endonuclease REase containing only HsdR prrI is an extremely weak complex, producing primarily R 1 -complex.
As can be seen from the above, DNA cleavage by Type I restriction enzymes occurs by means of a very unusual, and highly energy-dependent, mechanism. Therefore, these enzymes are believed to be involved not only as a defence mechanism for the bacterial cell, but also in some types of specialised recombination system controlling the flow of genes between bacterial strains [ 26 , 27 ]. A periplasmic location would be well adapted for the restriction activity of R-M enzymes, but recombination requires a cytoplasmic location.
Restriction enzymes protect the cells by cutting foreign DNA and could be assumed to be located at the cell periphery. Using immunoblotting to analyse subcellular fractions, Holubova et al. The HsdR and HsdM subunits were found in the membrane fraction only when co-produced with HsdS and, therefore, part of a complex enzyme, either methylase or endonuclease. Further studies have shown that the R-M enzyme is bound to the membrane via the HsdS subunit and that for some enzymes this may involve DNA [ 29 ]. One of the major obstacles for the practical application of single molecule devices is the absence of control methods in biological media, where substrates or energy sources such as ATP are ubiquitous.
Synthetic polymers offer a robust and highly flexible means by which devices based on single biological molecules can be controlled. They can also be used to link individual biomacromolecules to surfaces, package them or to control their specific functions, thus expanding the applicability of the natural molecules outside conventional biological environments.
Moreover, a number of synthetic polymers have been recently developed that can potentially perform nanoscale operations in a manner identical to natural and engineered biopolymers. Synthetic polymers with these properties are being developed for applications ranging from microfluidic device formation, [ 30 ] through to pulsatile drug release [ 31 - 34 ], control of cell-surface interactions [ 35 - 39 ], as actuators [ 40 ] and, increasingly, as nanotechnology devices [ 41 ].
In the context of bio-nanotechnology we focus here on the uses of one particular subclass of smart materials, i. Poly N-isopropylacrylamide PNIPAm is the prototypical smart polymer and is both readily available and of well-understood properties [ 42 ]. Accordingly, the LCST of a given polymer can in principle be "tuned" as desired by variation in hydrophilic or hydrophobic co-monomer content.
Covalent attachment of single or multiple responsive polymer chains to biopolymers offers the possibility of exerting control over their biological activity as, in theory at least, the properties of the resultant polymer-biopolymer conjugate should be a simple additive function of those of the individual components. This principle is now being widely exploited in pharmaceutical development, as covalent attachment of, for example, PEG chains to therapeutic proteins has been shown to stabilize the proteins without losing their biological function [ 43 - 48 ].
Furthermore, by altering the response stimulus of the synthetic polymer, and how and where it is attached to the biopolymer, the activity of the overall conjugate can be very closely regulated. These chimeric systems can thus be considered as true molecular-scale devices. Pioneering work in this area has been carried out by Hoffman, Stayton and co-workers, who engineered a mutant of cytochrome b5 such that a single cysteine introduced via site-directed mutagenesis was accessible for reaction with maleimide end-functionalised PNIPAm [ 49 ].
Since the native cytochrome b5 does not contain any cysteine residues this substitution provided a unique attachment point for the polymer. The resultant polymer-protein conjugate displayed LCST behaviour and could be reversibly precipitated from solution by variation in temperature. This approach has proved to be very versatile and a large number of polymer-biopolymer conjugates have now been prepared, incorporating biological components as diverse as antibodies, protein A, streptavidin, proteases and hydrolases [ 50 , 51 , 50 , 51 ].
The biological functions or activities of these conjugate systems were all similar to their native counterparts, but were switched on or off as a result of thermally induced polymer phase transitions. We are currently developing responsive polymers as a switch to control the EcoRI motor function and are investigating this polymer-motor conjugate as part of an active drug delivery system. In essence, we assemble a supramolecular device containing the molecular motor capable of binding and directionally translocating DNA through an impermeable barrier.
To control the process of translocation in biological systems, where a constant supply of ATP is present, we have added to the motor subunit of EcoRI the thermoresponsive poly N-isopropylacrylamide PNIPAm , which, through its coil-globule transition, acts as a temperature-dependent switch controlling motor activity. PNIPAm copolymers with reactive end-groups are being attached to a preformed R subunit of the motor via coupling of a maleimide-tipped linker on the synthetic polymer terminus to a cysteine residue.
This residue has been selected, as it is both accessible and located close to the active centre on the R subunit of the motor. The protein-polymer conjugates are stable to extensive purification and, when combined with M2S complex, the activity of this conjugate motor system is similar to the native counterpart, but can be switched on or off as a result of thermally induced polymer phase transitions [ 53 , 54 ].
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Conversely, in another environment e. Schematic representation of the molecular motor function controlled by a thermoresponsive polymer switch. R, M and S denote the specific motor subunits. Chain-extension of the polymer below LCST provides a steric shield blocking the active site.
Chain collapse above LCST enables access to the active site and restoration of enzyme function. The conjugation of the motor with synthetic polymers brings additional advantages. One such benefit arises from the ability to functionalise the polymer side chains or terminus in a way that allows attachment of the entire complex to surfaces for sensing and device applications.
Therefore, although our hybrid polymer-protein conjugate was originally aimed at gene targeting as it has the potential to increase the delivery of intact DNA to cell nuclei and thereby increase gene expression this system may also be used in building automated nano-chip sensors, therapeutic and diagnostic devices, where DNA itself would be a target, or where DNA might be used as a 'conveyor-belt' for attached molecules. The strength of the molecular motor has proven sufficient to disrupt most protein-DNA interactions and thus numerous processes and applications where highly localised force is required can also be envisaged.
- Molecular Motors in Bionanotechnology (2013, Hardcover)!
- Molecular Motors in Bionanotechnology;
- Keith Firman (Author of Molecular Motors in Bionanotechnology)?
- Rotary molecular motors - Bionanotechnology.
- La Leçon d’Histoire de M. Kahn - Volume 1 (Zoo Academy - Français) (French Edition).
The use of synthetic polymers offers a number of possibilities, which otherwise could not be exploited or would be difficult to take advantage of, if purely biological systems were used. Moreover, the combination of the properties of molecular motors with "smart" polymers has hitherto been unexplored and represents a novel concept in nanotechnology, which could ultimately lead to a wholly new class of molecular devices. Nanoscale control of molecular transport in vitro and especially in vivo opens up a whole host of possibilities in medicine, including drug or DNA delivery e.
In addition, this system may allow the generation of switchable nanodevices and actuators, controllable by changes in the synthetic copolymer structure as well as ATP-mediated DNA motion and may pave the way for biofeedback-responsive nanosystems. It can be used for nano-scale isolation of various biochemical processes in separate compartments connected via a tightly controlled shuttle device.
In essence, this concept bridges the disciplines of chemistry and biology by using a biological motor to control chemistry and a synthetic polymer to regulate biological processes. KF conceived the idea of using the modified R-M enzyme as a molecular motor and carried out, with co-workers, the molecular studies of the motor components, SSP carried out the polymer synthesis, polymer-motor conjugations and functional studies, CA designed and participated in the synthesis of smart polymers and DCG conceived of the study.
All authors participated in study design and coordination as well as the reading and approval of the final manuscript. National Center for Biotechnology Information , U. Journal List J Nanobiotechnology v. J Nanobiotechnology. Published online Sep 6. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Sivanand S Pennadam: ku. Received May 13; Accepted Sep 6. This article has been cited by other articles in PMC. Abstract 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.
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