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25 Staudt, L. et al. (2000) Identifying distinct sets of genes with similar expression patterns via ‘Gene Shaving’. Genome Biol. 1, 1–21 26 Herwig, R. et al. (1999) Large-scale clustering of cDNA-fingerprinting data. Genome Res. 9, 1093–1105 27 Heyer, L.J. et al. (1999) Exploring expression data: identification and analysis of co-expressed genes. Genome Res. 9, 1106–1115 28 Sherlock, G. (2000) Analysis of large-scale gene expression data. Curr. Opin. Immunol. 12, 201–205 29 Alter, O. et al. (2000) Singular value decomposition for genome-wide expression data processing and modeling. Proc. Natl. Acad. Sci.
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U. S. A. 97, 10101–10106 30 Herzel, H. et al. (2001) Extracting information from cDNA arrays. Chaos 11, 98–107 31 Michaels, G.S. et al. (1998) Cluster analysis and data visualization of large-scale gene expression data. Proc. Pac. Symp. Biocomp. 3, 42–53 32 Raychaudhuri, S. et al. (2000) Principal components analysis to summarize microarray experiments: Application to sporulation time series. Proc. Pac. Symp. Biocomp. 5, 452–463 33 Aach, J. et al. (2000) Systematic management and analysis of yeast gene expression data. Genome Res. 10, 431–445
34 Brazma, A. et al. (2000) One-stop shop for microarray data: Is a universal, public DNA-microarray database a realistic goal? Nature 403, 699–700 35 Ermolaeva, O. et al. (1998) Data management and analysis for gene expression arrays. Nat. Genet. 20, 19–23 36 Kellam, P. (2001) Microarray gene expression database: progress towards an international repository of gene expression data. Genome Biol. 2, 4011.1–4011.3 37 Bustin, S.A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193
Manipulating redox systems: application to nanotechnology Gianfranco Gilardi and Andrea Fantuzzi Redox proteins and enzymes are attractive targets for nanobiotechnology. The theoretical framework of biological electron transfer is increasingly well-understood, and several properties make redox centres good systems for exploitation: many can be detected both electrochemically and optically; they can perform specific reactions; they are capable of self-assembly; and their dimensions are in the nanoscale. Great progress has been made with the two main approaches of protein engineering: rational design and combinatorial synthesis. Rational design has put our understanding of the structure–function relationship to the test, whereas combinatorial synthesis has generated new molecules of interest. This article provides selected examples of novel approaches where redox proteins are ‘wired up’ in efficient electron-transfer chains, are ‘assembled’ in artificial multidomain structures (molecular Lego), are ‘linked’ to surfaces in nanodevices for biosensing and nanobiotechnological applications.
As the understanding of the structure–function relationship of redox proteins and enzymes is increasingly understood and new means are developed to tailor their properties for nanodevices, a new era is approaching in nanobiotechnology for exploiting these systems. The state-of-the-art research in this field is at a stage when domains that traditionally belong to the physical sciences (atomic-scale microscopy), chemistry (electrochemistry and electron transfer) and biology (enzymology, protein design and molecular biology) are coming together to offer new synergetic opportunities for nanobiotechnology1. Gianfranco Gilardi* Andrea Fantuzzi Dept of Biological Sciences, Imperial College of Science, Technology and Medicine, London, UK SW7 2AY. *e-mail: [email protected]
Redox proteins in nanobiotechnology
Redox proteins and enzymes carry out many key reactions of biological and technological importance (Fig. 1). The underlying process essential for these reactions is electron transfer. Protein-mediated electron transfer is a key phenomenon, not only in cellular http://tibtech.trends.com
processes (e.g. respiration and photosynthesis), but also in reactions of biotechnological interest (e.g. degradation of pollutants and biomass, and drug and food processing). Much progress has been made over the past ten years in understanding how the protein matrix finely tunes the parameters that are central to the regulation of biological electron transfer. R.A. Marcus’s theory of biological electron transfer (Box 1) gained him the 1992 Nobel Prize in Chemistry and fuelled many studies that attempted to unravel the details of key biological functions2–5. Undoubtedly, the protein matrix has a key role in regulating redox functions – indeed, few cofactors can perform the plethora of functions ascribed to redox proteins and enzymes. Even in simple electrontransfer proteins, such as b-type and c-type cytochromes that contain the same haem iron, a relatively simple parameter such as the redox potential varies over a range of 800 mV, from −400 mV for cytochrome c3 to +400 mV for cytochrome b559. This range highlights the power of the protein matrix in tuning function. More complex systems, such as the cytochrome P450 enzymes, gate electron transfer through a spin-state change associated with substrate binding. Such a mechanism allows variations of >100 mV to occur within the same protein and to allow the flow of electrons from the reductase only in the presence of the substrate. Much research has focused on ‘analytical protein engineering’, which involves the mutation of existing redox proteins to test our understanding of protein structure and function. A more recent trend is the so-called ‘de novo protein engineering’, which aims to create novel redox proteins from first principles.
0167-7799/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(01)01769-3
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of redox proteins promises the ability to engineer increasingly complex synthetic proteins.
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Proteins can now be created from first principles with the desired redox functionalities6. There are limitations related to the level of complexity of the desired target. For example, β-structures are more difficult to build owing to the complex interchain pattern of hydrogen bonding in different directions. However, the four-helix bundles have been a popular and successful fold for the de novo design of model cytochromes because of the relative simplicity of their intrachain hydrogen bonding pattern and because their assembly is driven by hydrophobic effects.
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Fig. 1. Use of redox proteins in nanotechnology. (a) Electrochemical device sensing the binding of a ligand based on changes of the redox properties, measured for example by differential pulse voltammetry (broken line, no ligand; unbroken line, with ligand)40. (b) Optical device sensing changes in optical properties of the redox centre upon ligand binding (broken line, no substrate; unbroken line, with substrate)43. (c) Detection of enzymatic activity through measurement of catalytic current. The same device can be used to ‘drive’ enzymatic reactions in nano-reactors42. (d) Example of engineered protein with multi-redox centres, immobilized on an electrode surface for the development of bio-electronic devices48.
Biomolecules have much to offer the field of nanobiotechnology: their dimensions are intrinsically in the nanoscale, they are formed by precise scaffolds where specific and often coupled functions are encoded, such as binding, stereo-specific catalysis, pumping and self-assembling. All these functions are increasingly better understood to the point where new biomolecules can be built from scratch. Presently, the great advances in drug design and the understanding of the structure–function relationship http://tibtech.trends.com
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Rational design of α-helical bundles, with added ability to incorporate haem, using solid-phase synthesis is a well-studied field of protein engineering. Functional studies on proteins designed from first principles offer the opportunity to test how much we really understand about redox proteins. A series of synthetic α-helical bundles has been used to assess the contribution of several factors to the redox potential (e.g. haem peripheral substitutions and electrostatic interactions with charged amino acids in the proximity of the haem, and protonation and/or deprotonation of neighbouring amino acids)7. A well-characterized four-helix bundle containing a 31-amino-acid repeated helix was used to show that electron-withdrawing or -donating ironporphyrin peripheral substituents had the largest effect on the redox potential, shifting it over a 225 mV range. The second largest effect was attributed to the pH of the solution; irrespective of the metalloporphyrin studied, a 160 mV shift was observed when the pH was varied between 4.0 and 8.5. The protonation state of both Glu and Lys residues was responsible for the electrostatic stabilization of the ferric porphyrin. Furthermore, the incorporation of the metalloporphyrin in the hydrophobic core modulated the redox potential. For all the metalloproteins studied, incorporation in the hydrophobic core increased the redox potential. Moreover, substitution of local amino acids led to a shift from +50 mV to −42 mV. However, it was difficult to predict the redox potentials based on changes of local, charged amino acids. Combinatorial synthesis
Combinatorial methods allow the exploration of possibilities that might not be obvious to rational design. Therefore, these methods might uncover unexpected findings that promise to extend our knowledge of structure–function relationships of redox proteins and of proteins in general. Combinatorial methods include two types of synthetic approach: chemical solid-phase synthesis and biological biosynthesis by recombinant DNA. Biological biosynthesis exploits the possibility of generating a library of mutants at the DNA level. All
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Box 1. Protein-mediated electron transfer According to the Marcus theorya – the theoretical framework that describes biological, protein-mediated electron transfer – the rate at which an electron is transferred from a donor to an acceptor (ket) in the non-adiabatic limit is given by Eqn 1, where V21,2 is the square of the electronic coupling of the donor and acceptor states and FC is the nuclear Franck-Condon factor. V21,2 relates to the exponential coefficient β and to distance R (Eqn 2), and FC relates to the driving force ∆G° and the reorganizational free energy λ (Eqn 3). ket = (2π/h)V21,2 FC V21,2 ∝ exp(−βR) FC = (4πλkBT)−1/2exp −[(∆G° + λ)2/4λkBT]
[1] [2] [3]
The nuclear motions around the donor and acceptor can be described by different nuclear wave functions, which can be approximated to a harmonic oscillator. This can be graphically represented by a potential well (Fig. I), in which the bottom of the parabola represents the equilibrium geometry. The curve of the product (right) has the same shape as that of the reactant (left) but it is shifted on the energy axis by an amount equal to the free energy of the reaction ∆G°. The reorganization energy λ is the energy that must be added to the reactant to move it from its equilibrium geometry to that of the product, while remaining on the reactant surface, without transferring an electron. The activation energy (∆E‡) in the nonadiabatic case corresponds to the height of the intersection between the parabola of the reactant with that of the product, and its relationship with ∆G° and λ is given by Eqn 4: ∆E‡ = (∆G° + λ)2/4λ
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combinatorial approaches rely on developing a screening strategy that can select mutants with the desired properties from a large combinatorial library easily and efficiently. Here, the biological biosynthetic approach has the advantage that biology per se is amenable to mutagenesis, amplification and expression of the polypeptidic chain. This eliminates ab initio the poorly folded mutants, allowing the display of the active mutants. In this case, libraries of random mutants can be generated on synthetic genes or on the cloned gene of a natural protein. An example of the latter is the application of combinatorial methods to the study of redox properties of the natural α-helix bundle of cytochrome b562 (Ref. 8). The gene encoding cytochrome b562 was randomized at Phe61 and Phe65 by combinatorial cassette PCR mutagenesis and was screened for active and correctly folded b562 mutants based on the colour assay of the periplasmic fractions indicating the correct incorporation of haem9. A mini-library of 29 http://tibtech.trends.com
[4]
As shown in the inset of Fig. I, three possibilities could arise: (1) when −∆G° = λ, the activation energy is equal to 0 (∆E‡ = 0); (2) when −∆G° < λ, the conditions for the so-called normal region are satisfied (increase of electron-transfer rates with increase in driving force); and (3) when −∆G° > λ, the conditions for the so-called Marcus-inverted region are satisfied (decrease of electron-transfer rates with increase in driving force). Reference a Marcus, R.A. and Sutin, N. (1985) Electron transfers in chemistry and biology. Biochim. Biophys. Acta 811, 265–322 Fig. I. Key parameters for biological electron transfer. The nuclear motions of the reactant (broken) and product (unbroken) are illustrated by the two parabolic wave functions, where the equilibrium geometry of the reactant (QR) and product (QP) is indicated on the reaction coordinate. The driving force ∆G° and the reorganizational free energy λ are shown graphically. The inset shows the relative positions of the parabolas in the three possible conditions (−∆G° < λ), (−∆G° = λ) and (−∆G° > λ).
mutants was generated and their redox potentials were found to have a 105 mV range, with the wild type at the upper end of the scale8. A good example of a combinatorial approach on a library of synthetic genes is the binary pattern of polar and non-polar amino acids in which the character of the residue is specified but the exact side-chain is varied randomly10. This was achieved by constructing a library of synthetic genes containing patterns of degenerate NAN codons (in which N denotes any base) where polar amino acids are required (Lys, His, Glu, Gln, Asp, Asn), and NTN where apolar amino acids are specified (Met, Leu, Ile, Val, Phe). The degenerate library of genes was expressed using a vector under the control of the T7 promoter, and 108 clones were obtained, 48 of which had the required pattern without errors and coded for the correct proteins10. Next, the cloned proteins were tested for their ability to bind haem; out of the 30 samples tested, 15 were found to bind haem with different degrees of stability
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and redox properties11, illustrating the relatively high rate of success in creating a haem-binding site using random approaches. However, this might not equate to creating a haem-binding pocket with the requisites for controlled catalysis. Interestingly, when the same library was screened for peroxidase activity12, four clones were found to be peroxidases with turnovers in the range of 5600–17 000 min−1, which although not as good as that of horseradish peroxidase (60 000 min−1), is much faster than that of microperoxidases (1260 min−1). This work illustrates the great potential offered by combinatorial methods, where the same random library could contain different members with the desired properties (such as fold, haem-binding and peroxidase activity), provided that a suitable screening method is devised to identify positive results efficiently. Another interesting example of the combinatorial synthetic approach is the synthesis of a library of fourhelix bundles on a modified cellulose13. A total of 462 proteins was synthesized and the haem incorporation was achieved in the solid phase. The midpoint potentials were estimated from the fraction of the reduced protein at an ambient potential of −95 mV. The library covered a range from −90 to −150 mV. As in the aforementioned case of Dutton and co-workers7, a relationship between the midpoint potentials and the amino acid variations was difficult to establish13. This might be because of the flexibility of the side chains, which might take unexpected orientations and/or interact with the solvent. When detailed structural information on these proteins is available, a clearer link between structure and function will be possible. Wiring up redox proteins
A new area of research is the use of biomolecules as electron-conductive materials for bioelectronics or for the construction of amperometric devices. Progress in the field of biomolecular electronics depends strongly on the availability of efficient, tailored molecular wires. Molecular wires are electron-conductive chains with an extended and efficient electronic conjugation that promotes strong coupling between the two groups attached to their ends. In the case of redox proteins, the two groups might be the redox centres of two proteins, or a protein and an electrode. A relatively large number of molecular wires has been developed over the past 20 years to achieve better contact between enzymes of bioanalytical interest and the electrode surface in amperometric devices. A detailed characterization of bridge-mediated electron transfer is given by Creager et al.14 who used AC voltammetry to study electron-transfer rates of ferrocene linked to a gold electrode by 3–6 repeats of phenylethynyl bridging groups. The exponential distance dependence (22–43 Å) of electron-transfer rates was characterized by a β value of 0.36 Å−1. Only recently have these approaches, aimed at achieving long-range electron transfer, been coupled to the ability of tailoring redox proteins by mutagenesis. One example of this approach is the study of the http://tibtech.trends.com
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feasibility of forming a complex between engineered azurin and thienoviologens hot wires. Thienoviologens consist of two pyridine or pyridinium groups linked by thiophene spacers of variable lengths. The engineered azurin used in this work is the mutant His117→Gly, in which one of the copper ligands, His117, is replaced by the small amino acid Gly, resulting in direct accessibility of external ligands to the copper ion that is 7 Å below the protein surface15,16. This gives access to external ligands linked to spacers of various lengths that provide a covalent link to either electrode surfaces17 or to another electron-transfer protein18. In the latter case, homo- and heterodimers have been built by constructing molecular wires from a spacer arm ending with two imidazoles (azurin His117→Gly homodimers, Fig. 2b), or from one imidazole and one FMN derivative (azurin His117→Gly–apoflavodoxin heterodimers). The choice of different spacer arms of different lengths allows controlled variation of the distance between the two redox centres, providing a good system for electron-transfer studies18. Assembling redox systems: modules and molecular Lego
A concept linking much of the ongoing work in the area of ‘building’ molecular tools for nanobiotechnology is the ‘module’ (Fig. 2) – used here to identify molecular elements of different levels of complexity. Starting with the simplest case, a module could be a ‘tag’, specifically attached to a redox protein to confer, for example, additional electron-transfer and/or electrochemical properties. The use of ruthenium derivatives bound to specific His residues on the surface of wild-type and engineered redox proteins has provided useful models with which to study the influence of the protein structure over electron-transfer rates19. Recently, the same approach was used to tag de novo designed three-helix bundle motifs, in which predicted electron-transfer rates were confirmed by measurements on structures designed from first principles20. Hill and co-workers21 also used small redox-active tags. A ferrocene derivative, the N-(2-ferroceneethyl)maleimide, was used as an electroactive label to derivatize the cysteines residues of glutathione and cytochrome P450cam to introduce novel electrochemical properties into these molecules21. The electrochemistry of the labelled P450cam showed signals from the haem and the ferrocene moieties, with a positive shift of the haem’s redox potential from −380 mV for the substrate-bound wild type to −280 mV for the modified protein. This work was further completed by the determination of the 3D structure of the ferrocene-derivatized P450cam, showing that not only the solvent-exposed Cys136 was labelled by the ferrocene but that Cys85 also was. The Cys85 residue was responsible for the positioning of the ferrocene rings in the camphor-binding pocket of the enzyme22. A module can also be an element that allows the study of a molecule. Hill’s group studied the waterinsoluble fullerenes electrochemically by linking a
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Fig. 2. ‘Modular’ approaches for the creation of new functional redox proteins. (a) Repeats of α-helical structures (cylinders) used to generate four helix bundles able to incorporate haem (red)6,24. (b) Linkage of two azurin homodimers (mutant H117G) through a ‘hot wire’ (red) connecting the two copper centres (blue)18. (c) ‘Molecular Lego’ approach to generate multidomain proteins with the desired electrontransfer properties (red, haem domain of cytochrome P450 BM3; blue, flavodoxin; green, connecting loop)25.
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thiol-selective fuellerenomaleimide to a unique surface-Cys mutant of azurin, S118C (Ref. 23). This enabled the electrochemical study of the fullerene moiety, in addition to the investigation into the effects of the label on the redox potential of the modified azurin (−41 mV shift). At a higher level of complexity, the ‘module’ can be a whole element of protein secondary structure (Fig. 2a). For example, a 16-residue peptide α1-B has been used as a repetitive building block for the assembly of many haem-binding four-helix bundles6,24. Another example is where the modules are helices with different combinations of selected amino acids at nine chosen positions, allowing haem binding13. In a more general context, a module can be a whole domain of a redox protein that is fused at the DNA level to generate a multidomain system with the desired http://tibtech.trends.com
properties (Fig. 2c). This approach has been developed in our laboratory and named molecular Lego25. The aim of molecular Lego is to generate artificial redox chains by assembling genes of wellcharacterized redox proteins and enzymes as prototypes for engineering systems that can be exploited by bioelectrochemistry. Many proteins, especially those unique to vertebrates, have mosaic structures in which various segments appear to have different origins. The DNA shuffling of introns and exons generates multidomain proteins assembled from building blocks. The molecular Lego approach selects key domains, or building blocks, to assemble artificial redox chains with the desired properties, ultimately capable of communicating with electrode surfaces. In spite of the large number of reactions carried out simultaneously by many redox enzymes on the numerous substrates in the cell, the living systems exhibit highly efficient redox chains, in which electrons are tunnelled in specific directions to sustain life. This success is the result of the slow process of evolution that can now be speeded up and mimicked by the protein engineer towards targets that benefit bioelectrochemistry. The link between protein domains can be achieved using various methods: for example, by genetically engineering a peptide linker between the C-terminus of one domain and the N-terminus of the other; or by engineering a disulfide bridge between the two domains. Electrontransfer efficiency might be expected to be enhanced by linking the domains, thus enhancing the local concentration of one partner with respect to the other. To ensure efficient electron transfer between the two domains and ultimately with the electrode, the position and length of the linkers should be chosen such that association complexes are favoured, allowing optimal electron transfer. Both linking methods have been used in the construction of homoand heterodimers of well-characterized redox proteins such as cytochrome c553 (c553) and flavodoxin (fld) from Desulfovibrio vulgaris, and the haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP)25,26. The disulfide-bridge linkage provided suitable models to study the influence of the inter-protein-complex formation in c553 homodimers. The gene-fusion approach generated interesting results for fld–BMP fusion25. The molecular assembly of fld–BMP allowed improved electrochemical accessibility of the catalytic BMP domain. Interestingly, this approach can also be extended to non-redox proteins such as the Escherichia coli repressor of primer (rop). Rop was successfully used as a module to insert novel redox properties27. Linking redox proteins to surfaces
The electronic coupling of the redox protein to the electrode and/or its availability to the probing light in optical-based sensors is of fundamental importance in the design of nanodevices. A key factor is the orientation of the redox protein on the surface of the sensor. For this
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Box 2. Methods for the immobilization of redox proteins Immobilization is an important aspect in the construction of nanodevices because sensitivity might strongly depend on concentration at the surface and specific orientation of the sensing biomolecule. These two requirements can be achieved in several ways depending on the nature of the support, properties and stability of the biomolecule. Both electrochemical and optical properties of redox proteins can be exploited for detection. Figure I illustrates several ways to achieve immobilizationa. Physical adsorption This can be achieved on the surface of metal oxides, carbon electrodes and silica oxides. Usually, this method leads to the formation of a randomly oriented layer, either on the surface of an electrode (Fig. Ia) or into the cavities formed by the porosity of the matrix. Although physical adsorption is relatively straightforward to achieve, it has some disadvantages: the protein might denature because of multiple contacts and interactions with the surface; binding of ligands might be affected; and unspecific
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multilayers might originate and prevent substrate accessibility. However, the chemical modification of the surface of either the electrode or the protein might increase the stability of the protein and introduce the possibility of controlling the density and environment of the immobilized species. This results in a higher level of the orientation in the self-assembled monolayer (SAM). Inclusion in polyelectrolytes or conducting polymers In this case, the proteins are trapped either in a polyelectrolyte or in a conducting polymer that is directly adsorbed or linked to the surface. This results in a non-oriented multilayer film (Fig. Ib). Inclusion in SAM This can be achieved, for example, using alkane-thiol or other thiol-terminated chains immobilized on the surface of a noble metal. The left part of Fig. Ic shows a monolayer of thio-lipids forming a membrane-like structure in which the proteins are immersed in different orientations. The right-hand part of Fig. Ic shows a SAM of alkane-thiol chains of different length allowing the formation of depressions on the surface that can accommodate the protein molecules. Non-oriented attachment to SAM Here, the alkane-thiol or other thiol-terminated chains are covalently bound to the surface of a noble metal. The other end presents a group that interacts with sites on the surface of the protein. This interaction is not specific, preventing control over orientation (Fig. Id).
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Oriented attachment to SAM These are achieved with the same strategy described above, but here, the reactive function of the SAM can interact specifically with a unique group on the protein surface (Fig. Ie). This unique site can be made either by chemical modification of an existing surface residue or by a genetic engineering approach.
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Fig. I. Graphical representation of some methods used to achieve protein immobilization. (a) Physical adsorption; (b) inclusion in polyelectrolytes/conducting polymers; (c) inclusion in self-assembled monolayers (SAMs); (d) non-oriented attachment to SAM; (e) oriented attachment to SAM; (f) direct site-specific attachment to gold.
Direct site-specific attachment to gold This can be achieved on gold by genetically engineering a unique Cys residue onto the surface of the protein, leading to a specific monolayer with high control over orientation and packing (Fig. If). Reference a Ferretti, S. et al. (2000) Self-assembled monolayers: a versatile tool for the formulation of bio-surfaces. Trends Anal. Chem. 19, 530–540
reason, much work has been dedicated to achieving controlled immobilization of biomolecules on solid surfaces. The selection of the materials and fabrication techniques is crucial for the adequate function, stability and performance, and is occasionally limited by the poor biocompatibility of the available materials and techniques of immobilization28. Examples of immobilization methods are given in Box 2. The stability of the biological elements is of fundamental importance to their correct functionality. Studies on the influence of sol–gel synthesis conditions on the stability, chemical function and enzymatic activity for horse-heart cytochrome c and bovine-liver catalase showed that http://tibtech.trends.com
the stability of the protein in solution is the limiting factor for encapsulating these molecules in an active form. However, in some instances, the resulting immobilized protein displayed enhanced stability towards destabilizing agents, such as extreme pH and hydrophobic solvents29. As a result, it has been shown that genetically engineered proteins with improved stability can maximize the entrapment and the long-term stability of the encapsulated protein30. Important advantages are gained if proteins can be immobilized at the electrode surface, ideally giving an electroactive monolayer film. This so-called ‘protein film voltammetry’ has contributed to the electrochemical characterization of these systems and
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facilitated a better understanding of the kinetics, energetics and mechanism of redox reactions in several proteins31. A higher level of control is obtained in the direct immobilization of proteins by sulfur–gold bonding because this results in a monolayer of molecules with determined orientation. This has been achieved, for example, on an engineed azurin, containing a Cys residue on the surface32 or by using a natural disulfide bond at the N-terminal of the protein33. Direct immobilization on gold was also achieved on cytochrome P450cam in which a unique surfaceexposed Cys was engineered to allow controlled immobilization34. This opened the possibility of having active redox enzymes in direct electric contact with the electrode surface and with defined orientation and properties, providing the basis for highly specific and selective biosensing and future bioreactor devices. This is particularly relevant when considering the expertise developed in tailoring the catalytic properties of this enzyme35. It is well-established that there are many factors that might affect the activity of a redox protein on a surface; for example, conformational changes, accessibility of binding, orientation of the bound molecule. However, another interesting factor is the interference from the interfacial force-field on the protein–ligand potential36. The importance of the electrostatic interactions was investigated on the binding of cytochrome b5 to cytochrome c. This was achieved using a streptavidin-modified electrode to immobilize either one or the other protein. This showed that the binding affinity for the soluble partner was substantially altered by the electrostatic environment, which emphasizes the significant influence of nonspecific, interfacial force-fields over molecular recognition at interfaces. Remarkably, these influences are present in spite of the preservation of the protein structure and the control of the orientation of the immobilized species36. Electronic transduction of enzyme–substrate interactions provides a general analytical means of detecting the respective substrate. The high specificity of enzyme–substrate interactions and the usually high turnover rates of biocatalysis, open the way to tailor sensitive and specific enzyme-based biosensor devices. Activation of enzymes at conductive surfaces allows the application of biocatalysts in electrically driven biotransformations (electrobiosynthesis). Recent applications of photosensitive enzymes and proteins have shown their potential in optical memories, optical bioswitches, electronic gates and biological actimometers37. Sensing redox activity
Several redox enzymes have been used to detect analytes of medical or environmental interest37. The electronic coupling between the redox enzyme and the electrode to construct detection devices has been achieved in several ways38: (1) by the electroactivity of http://tibtech.trends.com
the enzyme substrate or product (first-generation biosensors); (2) by the help of redox mediators, either free in solution or immobilized with the biomolecule (second-generation biosensors); and (3) by direct electron transfer between the electrode material and the redox-active biomolecule (third-generation biosensors). The advantages of a redox sensor based on direct electron transfer is the modulation of the redox- and electron-transfer properties by chemical and/or mutagenic modification of the biomolecule38. Efficient direct electron-transfer reactions have been reported for a small number of redox enzymes. Recently, cytochrome c has been immobilized on carboxyl groups of a gold–alkane-thiolate monolayer and has been used as a mediatorless superoxide sensor39. By using alkane-thiolate of different structures, the thermodynamic potential of the immobilized cytochrome was affected by 100 mV. The electrodes modified with cytochrome c showed a sharp increase in anodic current in the presence of the superoxide anion. Different amplitudes of the current were observed depending on the thermodynamic properties of the immobilized protein. Another example of a third generation biosensor is that of haemoglobin incorporated in a DNA film on pyrolytic graphite as a reagentless nitric oxide biosensor. Direct electrochemistry of the protein immobilized in the film showed high reactivity and specificity for nitric oxide, and UV–Vis spectroscopy showed that the haemoglobin in the film retained its native state40. Examples of second-generation biosensors are wellrepresented in the literature. One example is a novel ferrocene-based lipid for the immobilization of ferritin, gold-labelled BSA and flavocytochrome b2 (Ref. 41). The modified lipids formed membrane-like films that were adsorbed onto edge-plane graphite electrodes. In particular, the electrochemical properties of the immobilized flavocytochrome b2 responded to the presence of L-(+)lactate, offering the possibility of exploiting this enzyme–lipid-modified electrode to detect L-(+)lactate41. A modular configuration approach has been used to construct reagentless biosensors with layer-by-layer polyelectrolyte selfdeposition on a negatively charged alkane-thiolated gold electrode surface, interfacing different biocatalytic layers with positively charged redox polymers based on osmium42. The self-deposition of fructose dehydrogenase, horseradish peroxidase (HRP) and HRP/alcohol oxidase resulted in an amperometric reagentless biosensor for fructose, hydrogen peroxide and methanol with sensitivity of 19.3, 58.1 and 10.6 mA M−1 cm−1, respectively. The optical properties of the redox centres in oxidoreductases allow them to be exploited in optical devices (Fig. 1b). In this case, the biological element is immobilized on optically transparent supports, such as sol–gel glasses or specific metal oxides. Two recent examples in this area are the use of cytochrome c′ as a nitric oxide biosensor and cytochrome cd1 nitrite reductase as a nitrite ion sensor. Both proteins were
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encapsulated in sol–gel matrices; in the case of the cytochrome c′, there was a slight conformational change and altered-binding affinity owing to encapsulation, but the protein remained selective for nitric oxide and a calibration curve was determined43. By contrast, cytochrome cd1 nitrite reductase did not show detectable conformational changes on encapsulation on a sol–gel monolith and it maintained full enzymatic activity44. Because the limits of detection of nitric oxide achieved in this work are below those set by the EU, this approach is proposed for the determination of nitrite anions in environmental waters44. As for the sol–gel-encapsulated protein films, titanium dioxide allows high protein load, enabling optical sensing of ligand binding. The electrical conductivity of the titanium dioxide film also allows the adsorbed redox protein to be cycled between their oxidized and reduced states on application of positive and negative potentials, without the addition of mediators45. The viability of the combination of electrochemical control with optical probe was demonstrated using adsorbed haemoglobin to titanium dioxide films46. Repetitive cycling of the immobilized haemoglobin between Fe2+ and Fe3+ enabled the sensing ability for carbon monoxide to be tested and the respective binding curve to be determined. Nanobiocatalysis and bioelectronics
Acknowledgements We acknowledge financial support from the BBSRC (Engineering Biological Systems, grant E09561) and the Human Frontiers Science Programme (grant 0044/98). G. Gilardi would like to thank P. Senserini for her friendship and many stimulating discussions.
An attractive application of immobilized redox proteins originates from their ability to drive biotransformations (Fig. 1c). This can be useful in nanobiocatalysts that can generate a specific product of interest or transform a toxic substrate into a harmless product. One recent example of toxicsubstrate transformation is the electrode-driven oxidation of phenol, catalysed by a catalase immobilized onto graphite in the presence of soot47. The authors demonstrated the coupling of the reaction between the enzyme and substrate (biocatalytic process) with the electron-transfer process between the electrode and the enzyme (electrochemical process). Another application of biocatalytic electrodes is the development of biofuel cell elements. The biofuel cell uses biocatalysts to convert chemical energy into electrical energy. Given that many organic substrates are oxidized with a release of energy, the biocatalysed oxidation of the organic material at two compartmentalized electrode interfaces could lead to the conversion of chemical energy into electrical energy37. A biofuel cell has been proposed, comprising glucose as the fuel, hydrogen peroxide as the oxidizing agent, an anode functionalized with glucose oxidase reconstituted with PQQ and FAD, and a catode functionalized with microperoxidase-11 (Ref. 37). The system generated a maximum power of 32 µW at an external load of 3 kΩ (Ref. 37). Only recently has the use of redox proteins as biomaterials for electronic devices been investigated (Fig. 1d). Redox proteins present advantages for applications in electronic devices because their http://tibtech.trends.com
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intrinsic dimensions are in the nanoscale range and they are capable of self-assembly. These characteristics make redox proteins an attractive alternative to optical lithographic approaches. The microscale surface patterning of these biomolecules is an exciting novel field in which a few research groups have started to make the first steps. An interesting contribution in this area, is the synthesis of a four-helix bundle assembled as a monolayer onto a gold electrode48. The protein incorporated two haems displaying two different midpoint potentials of −184 mV and −114 mV, enabling this de novo protein to act as a rectifier element in which rapid vectorial electron transfer originated from the potential gradient48. Another interesting development concerns conducting polymers, such as poly(aniline) or poly(3-methyl-thiophene), which can change their conductivity by many orders of magnitude on oxidation and reduction49. These systems can fabricate microelectrochemical transistors, electrochemical devices that behave like solid-state junction field-effect transistors. The combination of these polymers with the appropriate redox enzyme allows the construction of microelectrochemical enzyme transistors with significant advantages, such as spatial control and high sensitivity, and not least the possibility of digital processing of information, making logical operations possible49. Finally, physiological functions of natural electrontransfer proteins can be optimized towards specific technological targets. For example, the lighttransducing properties of bacteriorhodopsin were exploited for applications in holographic-associative memories and branched-photocycle 3D-optical memories50; bacteriorhodopsin was immobilized on thin films of gelatine of polyvinyl alcohol, and the resulting device performed parallel write/read/erase operations using sequential multiphoton processes, without disturbing the data outside the doubly irradiated 3D elements. As the information storage depended crucially on the lifetimes of the excited states, protein engineering and chemical modifications on the chromophore were used to optimize the lifetimes of the photoproducts50. Future prospects
There are striking advances being made in designing and manipulating redox proteins and enzymes, both by synthetic and DNA-recombinant methods. These are able to create new biological tools. Great steps are also being made towards handling and linking proteins at interfaces with new ‘protein-friendly’ materials, where the properties are tailored for increased stability, controlled orientation and increased sensitivity. As the two streams of research come together in collaborative and multidisciplinary research, the future holds great promise. The interface between biology, chemistry and physics is crucial and much of its success will depend on a better understanding of the biological component.
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25 Staudt, L. et al. (2000) Identifying distinct sets of genes with similar expression patterns via ‘Gene Shaving’. Genome Biol. 1, 1–21 26 Herwig, R. et al. (1999) Large-scale clustering of cDNA-fingerprinting data. Genome Res. 9, 1093–1105 27 Heyer, L.J. et al. (1999) Exploring expression data: identification and analysis of co-expressed genes. Genome Res. 9, 1106–1115 28 Sherlock, G. (2000) Analysis of large-scale gene expression data. Curr. Opin. Immunol. 12, 201–205 29 Alter, O. et al. (2000) Singular value decomposition for genome-wide expression data processing and modeling. Proc. Natl. Acad. Sci.
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34 Brazma, A. et al. (2000) One-stop shop for microarray data: Is a universal, public DNA-microarray database a realistic goal? Nature 403, 699–700 35 Ermolaeva, O. et al. (1998) Data management and analysis for gene expression arrays. Nat. Genet. 20, 19–23 36 Kellam, P. (2001) Microarray gene expression database: progress towards an international repository of gene expression data. Genome Biol. 2, 4011.1–4011.3 37 Bustin, S.A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193
Manipulating redox systems: application to nanotechnology Gianfranco Gilardi and Andrea Fantuzzi Redox proteins and enzymes are attractive targets for nanobiotechnology. The theoretical framework of biological electron transfer is increasingly well-understood, and several properties make redox centres good systems for exploitation: many can be detected both electrochemically and optically; they can perform specific reactions; they are capable of self-assembly; and their dimensions are in the nanoscale. Great progress has been made with the two main approaches of protein engineering: rational design and combinatorial synthesis. Rational design has put our understanding of the structure–function relationship to the test, whereas combinatorial synthesis has generated new molecules of interest. This article provides selected examples of novel approaches where redox proteins are ‘wired up’ in efficient electron-transfer chains, are ‘assembled’ in artificial multidomain structures (molecular Lego), are ‘linked’ to surfaces in nanodevices for biosensing and nanobiotechnological applications.
As the understanding of the structure–function relationship of redox proteins and enzymes is increasingly understood and new means are developed to tailor their properties for nanodevices, a new era is approaching in nanobiotechnology for exploiting these systems. The state-of-the-art research in this field is at a stage when domains that traditionally belong to the physical sciences (atomic-scale microscopy), chemistry (electrochemistry and electron transfer) and biology (enzymology, protein design and molecular biology) are coming together to offer new synergetic opportunities for nanobiotechnology1. Gianfranco Gilardi* Andrea Fantuzzi Dept of Biological Sciences, Imperial College of Science, Technology and Medicine, London, UK SW7 2AY. *e-mail: [email protected]
Redox proteins in nanobiotechnology
Redox proteins and enzymes carry out many key reactions of biological and technological importance (Fig. 1). The underlying process essential for these reactions is electron transfer. Protein-mediated electron transfer is a key phenomenon, not only in cellular http://tibtech.trends.com
processes (e.g. respiration and photosynthesis), but also in reactions of biotechnological interest (e.g. degradation of pollutants and biomass, and drug and food processing). Much progress has been made over the past ten years in understanding how the protein matrix finely tunes the parameters that are central to the regulation of biological electron transfer. R.A. Marcus’s theory of biological electron transfer (Box 1) gained him the 1992 Nobel Prize in Chemistry and fuelled many studies that attempted to unravel the details of key biological functions2–5. Undoubtedly, the protein matrix has a key role in regulating redox functions – indeed, few cofactors can perform the plethora of functions ascribed to redox proteins and enzymes. Even in simple electrontransfer proteins, such as b-type and c-type cytochromes that contain the same haem iron, a relatively simple parameter such as the redox potential varies over a range of 800 mV, from −400 mV for cytochrome c3 to +400 mV for cytochrome b559. This range highlights the power of the protein matrix in tuning function. More complex systems, such as the cytochrome P450 enzymes, gate electron transfer through a spin-state change associated with substrate binding. Such a mechanism allows variations of >100 mV to occur within the same protein and to allow the flow of electrons from the reductase only in the presence of the substrate. Much research has focused on ‘analytical protein engineering’, which involves the mutation of existing redox proteins to test our understanding of protein structure and function. A more recent trend is the so-called ‘de novo protein engineering’, which aims to create novel redox proteins from first principles.
0167-7799/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(01)01769-3
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of redox proteins promises the ability to engineer increasingly complex synthetic proteins.
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Proteins can now be created from first principles with the desired redox functionalities6. There are limitations related to the level of complexity of the desired target. For example, β-structures are more difficult to build owing to the complex interchain pattern of hydrogen bonding in different directions. However, the four-helix bundles have been a popular and successful fold for the de novo design of model cytochromes because of the relative simplicity of their intrachain hydrogen bonding pattern and because their assembly is driven by hydrophobic effects.
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Fig. 1. Use of redox proteins in nanotechnology. (a) Electrochemical device sensing the binding of a ligand based on changes of the redox properties, measured for example by differential pulse voltammetry (broken line, no ligand; unbroken line, with ligand)40. (b) Optical device sensing changes in optical properties of the redox centre upon ligand binding (broken line, no substrate; unbroken line, with substrate)43. (c) Detection of enzymatic activity through measurement of catalytic current. The same device can be used to ‘drive’ enzymatic reactions in nano-reactors42. (d) Example of engineered protein with multi-redox centres, immobilized on an electrode surface for the development of bio-electronic devices48.
Biomolecules have much to offer the field of nanobiotechnology: their dimensions are intrinsically in the nanoscale, they are formed by precise scaffolds where specific and often coupled functions are encoded, such as binding, stereo-specific catalysis, pumping and self-assembling. All these functions are increasingly better understood to the point where new biomolecules can be built from scratch. Presently, the great advances in drug design and the understanding of the structure–function relationship http://tibtech.trends.com
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Rational design of α-helical bundles, with added ability to incorporate haem, using solid-phase synthesis is a well-studied field of protein engineering. Functional studies on proteins designed from first principles offer the opportunity to test how much we really understand about redox proteins. A series of synthetic α-helical bundles has been used to assess the contribution of several factors to the redox potential (e.g. haem peripheral substitutions and electrostatic interactions with charged amino acids in the proximity of the haem, and protonation and/or deprotonation of neighbouring amino acids)7. A well-characterized four-helix bundle containing a 31-amino-acid repeated helix was used to show that electron-withdrawing or -donating ironporphyrin peripheral substituents had the largest effect on the redox potential, shifting it over a 225 mV range. The second largest effect was attributed to the pH of the solution; irrespective of the metalloporphyrin studied, a 160 mV shift was observed when the pH was varied between 4.0 and 8.5. The protonation state of both Glu and Lys residues was responsible for the electrostatic stabilization of the ferric porphyrin. Furthermore, the incorporation of the metalloporphyrin in the hydrophobic core modulated the redox potential. For all the metalloproteins studied, incorporation in the hydrophobic core increased the redox potential. Moreover, substitution of local amino acids led to a shift from +50 mV to −42 mV. However, it was difficult to predict the redox potentials based on changes of local, charged amino acids. Combinatorial synthesis
Combinatorial methods allow the exploration of possibilities that might not be obvious to rational design. Therefore, these methods might uncover unexpected findings that promise to extend our knowledge of structure–function relationships of redox proteins and of proteins in general. Combinatorial methods include two types of synthetic approach: chemical solid-phase synthesis and biological biosynthesis by recombinant DNA. Biological biosynthesis exploits the possibility of generating a library of mutants at the DNA level. All
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Box 1. Protein-mediated electron transfer According to the Marcus theorya – the theoretical framework that describes biological, protein-mediated electron transfer – the rate at which an electron is transferred from a donor to an acceptor (ket) in the non-adiabatic limit is given by Eqn 1, where V21,2 is the square of the electronic coupling of the donor and acceptor states and FC is the nuclear Franck-Condon factor. V21,2 relates to the exponential coefficient β and to distance R (Eqn 2), and FC relates to the driving force ∆G° and the reorganizational free energy λ (Eqn 3). ket = (2π/h)V21,2 FC V21,2 ∝ exp(−βR) FC = (4πλkBT)−1/2exp −[(∆G° + λ)2/4λkBT]
[1] [2] [3]
The nuclear motions around the donor and acceptor can be described by different nuclear wave functions, which can be approximated to a harmonic oscillator. This can be graphically represented by a potential well (Fig. I), in which the bottom of the parabola represents the equilibrium geometry. The curve of the product (right) has the same shape as that of the reactant (left) but it is shifted on the energy axis by an amount equal to the free energy of the reaction ∆G°. The reorganization energy λ is the energy that must be added to the reactant to move it from its equilibrium geometry to that of the product, while remaining on the reactant surface, without transferring an electron. The activation energy (∆E‡) in the nonadiabatic case corresponds to the height of the intersection between the parabola of the reactant with that of the product, and its relationship with ∆G° and λ is given by Eqn 4: ∆E‡ = (∆G° + λ)2/4λ
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combinatorial approaches rely on developing a screening strategy that can select mutants with the desired properties from a large combinatorial library easily and efficiently. Here, the biological biosynthetic approach has the advantage that biology per se is amenable to mutagenesis, amplification and expression of the polypeptidic chain. This eliminates ab initio the poorly folded mutants, allowing the display of the active mutants. In this case, libraries of random mutants can be generated on synthetic genes or on the cloned gene of a natural protein. An example of the latter is the application of combinatorial methods to the study of redox properties of the natural α-helix bundle of cytochrome b562 (Ref. 8). The gene encoding cytochrome b562 was randomized at Phe61 and Phe65 by combinatorial cassette PCR mutagenesis and was screened for active and correctly folded b562 mutants based on the colour assay of the periplasmic fractions indicating the correct incorporation of haem9. A mini-library of 29 http://tibtech.trends.com
[4]
As shown in the inset of Fig. I, three possibilities could arise: (1) when −∆G° = λ, the activation energy is equal to 0 (∆E‡ = 0); (2) when −∆G° < λ, the conditions for the so-called normal region are satisfied (increase of electron-transfer rates with increase in driving force); and (3) when −∆G° > λ, the conditions for the so-called Marcus-inverted region are satisfied (decrease of electron-transfer rates with increase in driving force). Reference a Marcus, R.A. and Sutin, N. (1985) Electron transfers in chemistry and biology. Biochim. Biophys. Acta 811, 265–322 Fig. I. Key parameters for biological electron transfer. The nuclear motions of the reactant (broken) and product (unbroken) are illustrated by the two parabolic wave functions, where the equilibrium geometry of the reactant (QR) and product (QP) is indicated on the reaction coordinate. The driving force ∆G° and the reorganizational free energy λ are shown graphically. The inset shows the relative positions of the parabolas in the three possible conditions (−∆G° < λ), (−∆G° = λ) and (−∆G° > λ).
mutants was generated and their redox potentials were found to have a 105 mV range, with the wild type at the upper end of the scale8. A good example of a combinatorial approach on a library of synthetic genes is the binary pattern of polar and non-polar amino acids in which the character of the residue is specified but the exact side-chain is varied randomly10. This was achieved by constructing a library of synthetic genes containing patterns of degenerate NAN codons (in which N denotes any base) where polar amino acids are required (Lys, His, Glu, Gln, Asp, Asn), and NTN where apolar amino acids are specified (Met, Leu, Ile, Val, Phe). The degenerate library of genes was expressed using a vector under the control of the T7 promoter, and 108 clones were obtained, 48 of which had the required pattern without errors and coded for the correct proteins10. Next, the cloned proteins were tested for their ability to bind haem; out of the 30 samples tested, 15 were found to bind haem with different degrees of stability
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and redox properties11, illustrating the relatively high rate of success in creating a haem-binding site using random approaches. However, this might not equate to creating a haem-binding pocket with the requisites for controlled catalysis. Interestingly, when the same library was screened for peroxidase activity12, four clones were found to be peroxidases with turnovers in the range of 5600–17 000 min−1, which although not as good as that of horseradish peroxidase (60 000 min−1), is much faster than that of microperoxidases (1260 min−1). This work illustrates the great potential offered by combinatorial methods, where the same random library could contain different members with the desired properties (such as fold, haem-binding and peroxidase activity), provided that a suitable screening method is devised to identify positive results efficiently. Another interesting example of the combinatorial synthetic approach is the synthesis of a library of fourhelix bundles on a modified cellulose13. A total of 462 proteins was synthesized and the haem incorporation was achieved in the solid phase. The midpoint potentials were estimated from the fraction of the reduced protein at an ambient potential of −95 mV. The library covered a range from −90 to −150 mV. As in the aforementioned case of Dutton and co-workers7, a relationship between the midpoint potentials and the amino acid variations was difficult to establish13. This might be because of the flexibility of the side chains, which might take unexpected orientations and/or interact with the solvent. When detailed structural information on these proteins is available, a clearer link between structure and function will be possible. Wiring up redox proteins
A new area of research is the use of biomolecules as electron-conductive materials for bioelectronics or for the construction of amperometric devices. Progress in the field of biomolecular electronics depends strongly on the availability of efficient, tailored molecular wires. Molecular wires are electron-conductive chains with an extended and efficient electronic conjugation that promotes strong coupling between the two groups attached to their ends. In the case of redox proteins, the two groups might be the redox centres of two proteins, or a protein and an electrode. A relatively large number of molecular wires has been developed over the past 20 years to achieve better contact between enzymes of bioanalytical interest and the electrode surface in amperometric devices. A detailed characterization of bridge-mediated electron transfer is given by Creager et al.14 who used AC voltammetry to study electron-transfer rates of ferrocene linked to a gold electrode by 3–6 repeats of phenylethynyl bridging groups. The exponential distance dependence (22–43 Å) of electron-transfer rates was characterized by a β value of 0.36 Å−1. Only recently have these approaches, aimed at achieving long-range electron transfer, been coupled to the ability of tailoring redox proteins by mutagenesis. One example of this approach is the study of the http://tibtech.trends.com
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feasibility of forming a complex between engineered azurin and thienoviologens hot wires. Thienoviologens consist of two pyridine or pyridinium groups linked by thiophene spacers of variable lengths. The engineered azurin used in this work is the mutant His117→Gly, in which one of the copper ligands, His117, is replaced by the small amino acid Gly, resulting in direct accessibility of external ligands to the copper ion that is 7 Å below the protein surface15,16. This gives access to external ligands linked to spacers of various lengths that provide a covalent link to either electrode surfaces17 or to another electron-transfer protein18. In the latter case, homo- and heterodimers have been built by constructing molecular wires from a spacer arm ending with two imidazoles (azurin His117→Gly homodimers, Fig. 2b), or from one imidazole and one FMN derivative (azurin His117→Gly–apoflavodoxin heterodimers). The choice of different spacer arms of different lengths allows controlled variation of the distance between the two redox centres, providing a good system for electron-transfer studies18. Assembling redox systems: modules and molecular Lego
A concept linking much of the ongoing work in the area of ‘building’ molecular tools for nanobiotechnology is the ‘module’ (Fig. 2) – used here to identify molecular elements of different levels of complexity. Starting with the simplest case, a module could be a ‘tag’, specifically attached to a redox protein to confer, for example, additional electron-transfer and/or electrochemical properties. The use of ruthenium derivatives bound to specific His residues on the surface of wild-type and engineered redox proteins has provided useful models with which to study the influence of the protein structure over electron-transfer rates19. Recently, the same approach was used to tag de novo designed three-helix bundle motifs, in which predicted electron-transfer rates were confirmed by measurements on structures designed from first principles20. Hill and co-workers21 also used small redox-active tags. A ferrocene derivative, the N-(2-ferroceneethyl)maleimide, was used as an electroactive label to derivatize the cysteines residues of glutathione and cytochrome P450cam to introduce novel electrochemical properties into these molecules21. The electrochemistry of the labelled P450cam showed signals from the haem and the ferrocene moieties, with a positive shift of the haem’s redox potential from −380 mV for the substrate-bound wild type to −280 mV for the modified protein. This work was further completed by the determination of the 3D structure of the ferrocene-derivatized P450cam, showing that not only the solvent-exposed Cys136 was labelled by the ferrocene but that Cys85 also was. The Cys85 residue was responsible for the positioning of the ferrocene rings in the camphor-binding pocket of the enzyme22. A module can also be an element that allows the study of a molecule. Hill’s group studied the waterinsoluble fullerenes electrochemically by linking a
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Fig. 2. ‘Modular’ approaches for the creation of new functional redox proteins. (a) Repeats of α-helical structures (cylinders) used to generate four helix bundles able to incorporate haem (red)6,24. (b) Linkage of two azurin homodimers (mutant H117G) through a ‘hot wire’ (red) connecting the two copper centres (blue)18. (c) ‘Molecular Lego’ approach to generate multidomain proteins with the desired electrontransfer properties (red, haem domain of cytochrome P450 BM3; blue, flavodoxin; green, connecting loop)25.
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thiol-selective fuellerenomaleimide to a unique surface-Cys mutant of azurin, S118C (Ref. 23). This enabled the electrochemical study of the fullerene moiety, in addition to the investigation into the effects of the label on the redox potential of the modified azurin (−41 mV shift). At a higher level of complexity, the ‘module’ can be a whole element of protein secondary structure (Fig. 2a). For example, a 16-residue peptide α1-B has been used as a repetitive building block for the assembly of many haem-binding four-helix bundles6,24. Another example is where the modules are helices with different combinations of selected amino acids at nine chosen positions, allowing haem binding13. In a more general context, a module can be a whole domain of a redox protein that is fused at the DNA level to generate a multidomain system with the desired http://tibtech.trends.com
properties (Fig. 2c). This approach has been developed in our laboratory and named molecular Lego25. The aim of molecular Lego is to generate artificial redox chains by assembling genes of wellcharacterized redox proteins and enzymes as prototypes for engineering systems that can be exploited by bioelectrochemistry. Many proteins, especially those unique to vertebrates, have mosaic structures in which various segments appear to have different origins. The DNA shuffling of introns and exons generates multidomain proteins assembled from building blocks. The molecular Lego approach selects key domains, or building blocks, to assemble artificial redox chains with the desired properties, ultimately capable of communicating with electrode surfaces. In spite of the large number of reactions carried out simultaneously by many redox enzymes on the numerous substrates in the cell, the living systems exhibit highly efficient redox chains, in which electrons are tunnelled in specific directions to sustain life. This success is the result of the slow process of evolution that can now be speeded up and mimicked by the protein engineer towards targets that benefit bioelectrochemistry. The link between protein domains can be achieved using various methods: for example, by genetically engineering a peptide linker between the C-terminus of one domain and the N-terminus of the other; or by engineering a disulfide bridge between the two domains. Electrontransfer efficiency might be expected to be enhanced by linking the domains, thus enhancing the local concentration of one partner with respect to the other. To ensure efficient electron transfer between the two domains and ultimately with the electrode, the position and length of the linkers should be chosen such that association complexes are favoured, allowing optimal electron transfer. Both linking methods have been used in the construction of homoand heterodimers of well-characterized redox proteins such as cytochrome c553 (c553) and flavodoxin (fld) from Desulfovibrio vulgaris, and the haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP)25,26. The disulfide-bridge linkage provided suitable models to study the influence of the inter-protein-complex formation in c553 homodimers. The gene-fusion approach generated interesting results for fld–BMP fusion25. The molecular assembly of fld–BMP allowed improved electrochemical accessibility of the catalytic BMP domain. Interestingly, this approach can also be extended to non-redox proteins such as the Escherichia coli repressor of primer (rop). Rop was successfully used as a module to insert novel redox properties27. Linking redox proteins to surfaces
The electronic coupling of the redox protein to the electrode and/or its availability to the probing light in optical-based sensors is of fundamental importance in the design of nanodevices. A key factor is the orientation of the redox protein on the surface of the sensor. For this
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Box 2. Methods for the immobilization of redox proteins Immobilization is an important aspect in the construction of nanodevices because sensitivity might strongly depend on concentration at the surface and specific orientation of the sensing biomolecule. These two requirements can be achieved in several ways depending on the nature of the support, properties and stability of the biomolecule. Both electrochemical and optical properties of redox proteins can be exploited for detection. Figure I illustrates several ways to achieve immobilizationa. Physical adsorption This can be achieved on the surface of metal oxides, carbon electrodes and silica oxides. Usually, this method leads to the formation of a randomly oriented layer, either on the surface of an electrode (Fig. Ia) or into the cavities formed by the porosity of the matrix. Although physical adsorption is relatively straightforward to achieve, it has some disadvantages: the protein might denature because of multiple contacts and interactions with the surface; binding of ligands might be affected; and unspecific
(a)
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multilayers might originate and prevent substrate accessibility. However, the chemical modification of the surface of either the electrode or the protein might increase the stability of the protein and introduce the possibility of controlling the density and environment of the immobilized species. This results in a higher level of the orientation in the self-assembled monolayer (SAM). Inclusion in polyelectrolytes or conducting polymers In this case, the proteins are trapped either in a polyelectrolyte or in a conducting polymer that is directly adsorbed or linked to the surface. This results in a non-oriented multilayer film (Fig. Ib). Inclusion in SAM This can be achieved, for example, using alkane-thiol or other thiol-terminated chains immobilized on the surface of a noble metal. The left part of Fig. Ic shows a monolayer of thio-lipids forming a membrane-like structure in which the proteins are immersed in different orientations. The right-hand part of Fig. Ic shows a SAM of alkane-thiol chains of different length allowing the formation of depressions on the surface that can accommodate the protein molecules. Non-oriented attachment to SAM Here, the alkane-thiol or other thiol-terminated chains are covalently bound to the surface of a noble metal. The other end presents a group that interacts with sites on the surface of the protein. This interaction is not specific, preventing control over orientation (Fig. Id).
(d)
Oriented attachment to SAM These are achieved with the same strategy described above, but here, the reactive function of the SAM can interact specifically with a unique group on the protein surface (Fig. Ie). This unique site can be made either by chemical modification of an existing surface residue or by a genetic engineering approach.
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Fig. I. Graphical representation of some methods used to achieve protein immobilization. (a) Physical adsorption; (b) inclusion in polyelectrolytes/conducting polymers; (c) inclusion in self-assembled monolayers (SAMs); (d) non-oriented attachment to SAM; (e) oriented attachment to SAM; (f) direct site-specific attachment to gold.
Direct site-specific attachment to gold This can be achieved on gold by genetically engineering a unique Cys residue onto the surface of the protein, leading to a specific monolayer with high control over orientation and packing (Fig. If). Reference a Ferretti, S. et al. (2000) Self-assembled monolayers: a versatile tool for the formulation of bio-surfaces. Trends Anal. Chem. 19, 530–540
reason, much work has been dedicated to achieving controlled immobilization of biomolecules on solid surfaces. The selection of the materials and fabrication techniques is crucial for the adequate function, stability and performance, and is occasionally limited by the poor biocompatibility of the available materials and techniques of immobilization28. Examples of immobilization methods are given in Box 2. The stability of the biological elements is of fundamental importance to their correct functionality. Studies on the influence of sol–gel synthesis conditions on the stability, chemical function and enzymatic activity for horse-heart cytochrome c and bovine-liver catalase showed that http://tibtech.trends.com
the stability of the protein in solution is the limiting factor for encapsulating these molecules in an active form. However, in some instances, the resulting immobilized protein displayed enhanced stability towards destabilizing agents, such as extreme pH and hydrophobic solvents29. As a result, it has been shown that genetically engineered proteins with improved stability can maximize the entrapment and the long-term stability of the encapsulated protein30. Important advantages are gained if proteins can be immobilized at the electrode surface, ideally giving an electroactive monolayer film. This so-called ‘protein film voltammetry’ has contributed to the electrochemical characterization of these systems and
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facilitated a better understanding of the kinetics, energetics and mechanism of redox reactions in several proteins31. A higher level of control is obtained in the direct immobilization of proteins by sulfur–gold bonding because this results in a monolayer of molecules with determined orientation. This has been achieved, for example, on an engineed azurin, containing a Cys residue on the surface32 or by using a natural disulfide bond at the N-terminal of the protein33. Direct immobilization on gold was also achieved on cytochrome P450cam in which a unique surfaceexposed Cys was engineered to allow controlled immobilization34. This opened the possibility of having active redox enzymes in direct electric contact with the electrode surface and with defined orientation and properties, providing the basis for highly specific and selective biosensing and future bioreactor devices. This is particularly relevant when considering the expertise developed in tailoring the catalytic properties of this enzyme35. It is well-established that there are many factors that might affect the activity of a redox protein on a surface; for example, conformational changes, accessibility of binding, orientation of the bound molecule. However, another interesting factor is the interference from the interfacial force-field on the protein–ligand potential36. The importance of the electrostatic interactions was investigated on the binding of cytochrome b5 to cytochrome c. This was achieved using a streptavidin-modified electrode to immobilize either one or the other protein. This showed that the binding affinity for the soluble partner was substantially altered by the electrostatic environment, which emphasizes the significant influence of nonspecific, interfacial force-fields over molecular recognition at interfaces. Remarkably, these influences are present in spite of the preservation of the protein structure and the control of the orientation of the immobilized species36. Electronic transduction of enzyme–substrate interactions provides a general analytical means of detecting the respective substrate. The high specificity of enzyme–substrate interactions and the usually high turnover rates of biocatalysis, open the way to tailor sensitive and specific enzyme-based biosensor devices. Activation of enzymes at conductive surfaces allows the application of biocatalysts in electrically driven biotransformations (electrobiosynthesis). Recent applications of photosensitive enzymes and proteins have shown their potential in optical memories, optical bioswitches, electronic gates and biological actimometers37. Sensing redox activity
Several redox enzymes have been used to detect analytes of medical or environmental interest37. The electronic coupling between the redox enzyme and the electrode to construct detection devices has been achieved in several ways38: (1) by the electroactivity of http://tibtech.trends.com
the enzyme substrate or product (first-generation biosensors); (2) by the help of redox mediators, either free in solution or immobilized with the biomolecule (second-generation biosensors); and (3) by direct electron transfer between the electrode material and the redox-active biomolecule (third-generation biosensors). The advantages of a redox sensor based on direct electron transfer is the modulation of the redox- and electron-transfer properties by chemical and/or mutagenic modification of the biomolecule38. Efficient direct electron-transfer reactions have been reported for a small number of redox enzymes. Recently, cytochrome c has been immobilized on carboxyl groups of a gold–alkane-thiolate monolayer and has been used as a mediatorless superoxide sensor39. By using alkane-thiolate of different structures, the thermodynamic potential of the immobilized cytochrome was affected by 100 mV. The electrodes modified with cytochrome c showed a sharp increase in anodic current in the presence of the superoxide anion. Different amplitudes of the current were observed depending on the thermodynamic properties of the immobilized protein. Another example of a third generation biosensor is that of haemoglobin incorporated in a DNA film on pyrolytic graphite as a reagentless nitric oxide biosensor. Direct electrochemistry of the protein immobilized in the film showed high reactivity and specificity for nitric oxide, and UV–Vis spectroscopy showed that the haemoglobin in the film retained its native state40. Examples of second-generation biosensors are wellrepresented in the literature. One example is a novel ferrocene-based lipid for the immobilization of ferritin, gold-labelled BSA and flavocytochrome b2 (Ref. 41). The modified lipids formed membrane-like films that were adsorbed onto edge-plane graphite electrodes. In particular, the electrochemical properties of the immobilized flavocytochrome b2 responded to the presence of L-(+)lactate, offering the possibility of exploiting this enzyme–lipid-modified electrode to detect L-(+)lactate41. A modular configuration approach has been used to construct reagentless biosensors with layer-by-layer polyelectrolyte selfdeposition on a negatively charged alkane-thiolated gold electrode surface, interfacing different biocatalytic layers with positively charged redox polymers based on osmium42. The self-deposition of fructose dehydrogenase, horseradish peroxidase (HRP) and HRP/alcohol oxidase resulted in an amperometric reagentless biosensor for fructose, hydrogen peroxide and methanol with sensitivity of 19.3, 58.1 and 10.6 mA M−1 cm−1, respectively. The optical properties of the redox centres in oxidoreductases allow them to be exploited in optical devices (Fig. 1b). In this case, the biological element is immobilized on optically transparent supports, such as sol–gel glasses or specific metal oxides. Two recent examples in this area are the use of cytochrome c′ as a nitric oxide biosensor and cytochrome cd1 nitrite reductase as a nitrite ion sensor. Both proteins were
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encapsulated in sol–gel matrices; in the case of the cytochrome c′, there was a slight conformational change and altered-binding affinity owing to encapsulation, but the protein remained selective for nitric oxide and a calibration curve was determined43. By contrast, cytochrome cd1 nitrite reductase did not show detectable conformational changes on encapsulation on a sol–gel monolith and it maintained full enzymatic activity44. Because the limits of detection of nitric oxide achieved in this work are below those set by the EU, this approach is proposed for the determination of nitrite anions in environmental waters44. As for the sol–gel-encapsulated protein films, titanium dioxide allows high protein load, enabling optical sensing of ligand binding. The electrical conductivity of the titanium dioxide film also allows the adsorbed redox protein to be cycled between their oxidized and reduced states on application of positive and negative potentials, without the addition of mediators45. The viability of the combination of electrochemical control with optical probe was demonstrated using adsorbed haemoglobin to titanium dioxide films46. Repetitive cycling of the immobilized haemoglobin between Fe2+ and Fe3+ enabled the sensing ability for carbon monoxide to be tested and the respective binding curve to be determined. Nanobiocatalysis and bioelectronics
Acknowledgements We acknowledge financial support from the BBSRC (Engineering Biological Systems, grant E09561) and the Human Frontiers Science Programme (grant 0044/98). G. Gilardi would like to thank P. Senserini for her friendship and many stimulating discussions.
An attractive application of immobilized redox proteins originates from their ability to drive biotransformations (Fig. 1c). This can be useful in nanobiocatalysts that can generate a specific product of interest or transform a toxic substrate into a harmless product. One recent example of toxicsubstrate transformation is the electrode-driven oxidation of phenol, catalysed by a catalase immobilized onto graphite in the presence of soot47. The authors demonstrated the coupling of the reaction between the enzyme and substrate (biocatalytic process) with the electron-transfer process between the electrode and the enzyme (electrochemical process). Another application of biocatalytic electrodes is the development of biofuel cell elements. The biofuel cell uses biocatalysts to convert chemical energy into electrical energy. Given that many organic substrates are oxidized with a release of energy, the biocatalysed oxidation of the organic material at two compartmentalized electrode interfaces could lead to the conversion of chemical energy into electrical energy37. A biofuel cell has been proposed, comprising glucose as the fuel, hydrogen peroxide as the oxidizing agent, an anode functionalized with glucose oxidase reconstituted with PQQ and FAD, and a catode functionalized with microperoxidase-11 (Ref. 37). The system generated a maximum power of 32 µW at an external load of 3 kΩ (Ref. 37). Only recently has the use of redox proteins as biomaterials for electronic devices been investigated (Fig. 1d). Redox proteins present advantages for applications in electronic devices because their http://tibtech.trends.com
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intrinsic dimensions are in the nanoscale range and they are capable of self-assembly. These characteristics make redox proteins an attractive alternative to optical lithographic approaches. The microscale surface patterning of these biomolecules is an exciting novel field in which a few research groups have started to make the first steps. An interesting contribution in this area, is the synthesis of a four-helix bundle assembled as a monolayer onto a gold electrode48. The protein incorporated two haems displaying two different midpoint potentials of −184 mV and −114 mV, enabling this de novo protein to act as a rectifier element in which rapid vectorial electron transfer originated from the potential gradient48. Another interesting development concerns conducting polymers, such as poly(aniline) or poly(3-methyl-thiophene), which can change their conductivity by many orders of magnitude on oxidation and reduction49. These systems can fabricate microelectrochemical transistors, electrochemical devices that behave like solid-state junction field-effect transistors. The combination of these polymers with the appropriate redox enzyme allows the construction of microelectrochemical enzyme transistors with significant advantages, such as spatial control and high sensitivity, and not least the possibility of digital processing of information, making logical operations possible49. Finally, physiological functions of natural electrontransfer proteins can be optimized towards specific technological targets. For example, the lighttransducing properties of bacteriorhodopsin were exploited for applications in holographic-associative memories and branched-photocycle 3D-optical memories50; bacteriorhodopsin was immobilized on thin films of gelatine of polyvinyl alcohol, and the resulting device performed parallel write/read/erase operations using sequential multiphoton processes, without disturbing the data outside the doubly irradiated 3D elements. As the information storage depended crucially on the lifetimes of the excited states, protein engineering and chemical modifications on the chromophore were used to optimize the lifetimes of the photoproducts50. Future prospects
There are striking advances being made in designing and manipulating redox proteins and enzymes, both by synthetic and DNA-recombinant methods. These are able to create new biological tools. Great steps are also being made towards handling and linking proteins at interfaces with new ‘protein-friendly’ materials, where the properties are tailored for increased stability, controlled orientation and increased sensitivity. As the two streams of research come together in collaborative and multidisciplinary research, the future holds great promise. The interface between biology, chemistry and physics is crucial and much of its success will depend on a better understanding of the biological component.
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