Protein-polymer nano-machines. Towards synthetic control of biological processes
© Pennadam et al; licensee BioMed Central Ltd. 2004
Received: 13 May 2004
Accepted: 06 September 2004
Published: 06 September 2004
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 [1, 2]. have been focused in attempts to develop these systems. as machines capable of functioning at the low sub-micron and nanometre length-scales . 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 .
1. Type I Restriction-Modification enzymes
2. A versatile molecular motor
As previously mentioned, the final step of the subunit assembly pathway of the Type I Restriction-Modification enzyme EcoR124I produces a weak endonuclease complex of stoichiometry R2M2S1. We have produced a hybrid HsdR subunit combining elements of the HsdR subunits of the EcoR124I and EcoprrI [23–25] Type I Restriction-Modification enzymes. This subunit has been shown to assemble with the EcoR124I 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 R1-complex. The availability of the hybrid REase produced from core MTase(R124I) and HsdR(prrI), which provides a stable R1-complex, also gives a useful molecular motor that will not cleave the DNA that it translocates.
3. Sub-cellular localisation of R-M enzymes
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.  detected that the subunits of the R-M enzyme were predominantly in the spheroplast extract. 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 .
4. Uses of the EcoR124I molecular motor: polymer-protein conjugates in nanobiotechnology
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. A key property of these materials is 'smart' behaviour, especially the ability to undergo conformational or phase changes in response to variations in temperature and/or pH. Synthetic polymers with these properties are being developed for applications ranging from microfluidic device formation,  through to pulsatile drug release [31–34], control of cell-surface interactions [35–39], as actuators  and, increasingly, as nanotechnology devices .
In the context of bio-nanotechnology we focus here on the uses of one particular subclass of smart materials, i.e. substituted polyacrylamides, but it should be noted that there are many more examples of synthetic polymers and engineered/modified biopolymers that exhibit responsive behaviour and new types and applications of smart materials are constantly being reported.
4.1 Soluble PNIPAm-biopolymer conjugates
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]. Polymer-biopolymer conjugates can be prepared as monodisperse single units, or as self-assembling ensembles depending on the chemistries used for attaching the synthetic component and on the associative properties of the polymer and/or biopolymer. 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 . 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. Of especial note have been the recent reports of a temperature and photochemically switchable endoglucanase, which displayed varying and opposite activities depending on whether temperature or UV/Vis illumination was used as the switch .
4.2. Controllable DNA packaging and compartmentalization devices
We are currently developing responsive polymers as a switch to control the EcoR124I motor function and are investigating this polymer-motor conjugate as part of an active drug delivery system. We aim for the practical demonstration of a nano-scale DNA packaging/separation and delivery system uniting the optimal features of both natural and synthetic molecules. 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 EcoR124I 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].
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.
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.g. gene therapy), but also where protection of a therapeutic is required under one biological regime and release in another (e.g. prodrugs conjugated to DNA which can be released by nuclease-mediated degradation at the site of action). 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
This work is supported by a Wellcome Trust Showcase grant (Grant Reference 067484) and Institute of Biomedical and Biomolecular Sciences, University of Portsmouth.
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