Integral membrane proteins are important biological macromolecules with structural features and functionalities that make them attractive targets for nanotechnology. I provide here a broad review of current activity in nanotechnology related to membrane proteins, including their application as nanoscale sensors, switches, components of optical devices and as templates for self-assembled arrays.

Introduction

Nanotechnology, the science of the very small, will have a big impact. Typically, nanotechnology concerns objects with at least one dimension of <100 nm and is expected to bring a tremendous benefit to society by revolutionizing the basic technology underpinning healthcare, agriculture, biotechnology, computing, communications, security, environmental technology and more; the annual value of nanotechnology-related products is expected to exceed US$1 trillion by 2015 [1]. Reflecting this future impact, the current total global investment in nanotechnology research and development is ∼€5 billion (∼US$6.7 billion) [2].

The nanotechnology revolution will have several aspects, but two are most relevant here. First, there will be a considerable focus on organic and inorganic nanomaterials. Many nanomaterials, i.e. materials with grain size <100 nm, exhibit novel and enhanced chemical and physical properties compared with their bulk forms, largely as a result of quantum effects and a substantial increase in the surface area/volume ratio. They will be the basis of a new age of high-performance materials. Secondly, new nanoscale manufacturing processes and instrumentation will be introduced that have tremendous engineering advantages over their micro- and macro-sized counterparts. In particular, construction from the ‘bottom up’, by direct atomic manipulation via nanotools or through self-assembly, will be used to design and build new nanostructures.

It is intriguing to consider that numerous examples of high-performance nanomaterials and exquisitely precise nanoengineering can be found in biology. Indeed, one of the most exciting prospects in nanoscience is to take inspiration from, or use directly, the tools and processes that have been developed by Nature over millions of years. This new discipline has become known as nanobiotechnology. These investigations may be at the level of the whole cell (the living embodiment of functional organization at the nanoscale) or in using different components of the cell. For example, nucleic acids (especially as DNA and RNA) are of interest because of their capacity to store information and to self-assemble into robust, complex two- and three-dimensional structures. Proteins are also of considerable interest, because they present unique nanoscale structural and functional capabilities. Nanobiotechnology is inherently an interdisciplinary field, drawing researchers from disciplines including protein biochemistry, molecular cell biology, organic and inorganic chemistry, materials science and others.

One area of current interest is the application of integral membrane proteins. These proteins, classified by their location in the lipid membrane of the cell, serve a number of critical biological functions. They are now being explored for use as nanoscale pores, switches, in new approaches to energy generation, as components of optoelectronic devices and more. The present review describes some of the recent applications of membrane proteins in nanobiotechnology. It is not intended to be comprehensive, but instead represents a broad survey of this rapidly growing and exciting field; the reader is directed towards more extensive reviews of specific areas where appropriate. I have deliberately confined this review to describing the potential applications of integral membrane proteins and have thus excluded the study of membrane protein structure and function by new enabling nanotools such as AFM (atomic force microscopy) [3], nanofluidics [4], solubilization in nanodiscs [5], single-molecule studies [6] and the use of DNA nanotubes to align proteins for structural work [7], although these are fascinating areas of research in their own right. I also do not consider the use of a lipid membrane alone (again, an area of intense current interest in nanobiotechnology), but rather the use of integral membrane proteins themselves.

Integral membrane proteins

Genomic sequence analysis suggests that 25–30% of all of the proteins in Nature will be integral membrane proteins [8,9]. This class of protein is defined by having some proportion of their amino acid sequence buried within a lipid bilayer, typically with at least one transmembrane domain, and they make extensive non-polar interactions with the hydrophobic core of the bilayer. Membrane proteins perform a wide variety of important biological functions, including the specific and non-specific transport of solutes and ions (pumps, transporters and channels), energy generation (ATP synthases), signal transduction (receptors), osmotic regulation (porins), membrane-associated biochemistry (kinases, proteases) and more. Many of these functions involve the transfer of material and/or information across the impermeable barrier presented by the lipid bilayer. This unique role as the cellular ‘gatekeepers’ is one of the aspects of membrane protein biochemistry that is particularly interesting in nanobiotechnology, because it suggests that membrane proteins can be used to communicate between two distinct environments separated by a lipid bilayer.

The sequence and structure of integral membrane proteins

The lipid bilayer is a complex heterogeneous environment. This complexity is reflected in the primary amino acid sequence and secondary and tertiary structure of integral membrane proteins. Lipid bilayers consist of two solvent-facing interface regions that comprise polar lipid headgroups, into which water can penetrate, and a hydrophobic core consisting of aliphatic lipid chains. Very little water, if any, is present within this apolar bilayer core. Thus membrane proteins that pass through the bilayer multiple times must have sequence and structural elements that can successfully interact with (i) the bulk solvent (ii) the interface region, and (iii) the hydrophobic bilayer centre.

This gives rise to some unusual amino acid bias when membrane proteins are compared with their ‘water-soluble’ counterparts that function in aqueous solutions such as the cytosol. Transmembrane segments are strongly biased towards amino acids that have hydrophobic side chains (with charged and polar groups disfavoured), and there are other trends in the amino acids found at the interface and solvent-exposed regions [10]. The absence of water in the membrane core makes it critical for main-chain hydrogen-bonding to be completely satisfied, and this requirement gives membrane proteins a relatively limited palette of secondary-structure motifs. Two types of structure have been found for the transmembrane domains of integral membrane proteins: β-barrels and α-helical bundles, with the α-helix type being predominant (see Figure 1). The transmembrane domains of multipass β-barrel and α-helical proteins are connected by external loops of various sizes and structures. In some proteins, these loops seem to be little more than structureless linkers, but in others, they are highly structured and play a central role in folding, assembly and function. There are currently no examples of mixed α/β structures within the bilayer, and unstructured regions are not normally accommodated within the bilayer core.

Examples of α-helical bundle and β-barrel structure in membrane proteins involved in signal transduction, photosynthesis and the formation of pores and channels

Figure 1
Examples of α-helical bundle and β-barrel structure in membrane proteins involved in signal transduction, photosynthesis and the formation of pores and channels

In all cases, α-helix is shown in red, β-sheet in gold and random coil in green. (a) The light-activated proton pump bacteriorhodopsin from the archaeon H. salinarum, with retinal cofactor shown in magenta (PDB code 1C3W); (b) the photosynthetic reaction centre from the purple bacterium R. sphaeroides, with various cofactors shown in blue (PDB code 1PCR); (c) the β-barrel pore α-haemolysin from S. aureus (PDB code 7AHL), with a view into the pore from below the membrane; (d) the mechanosensitive channel from Mycobacterium tuberculosis, MscL, in the closed state, with a view into the channel from above the membrane (PDB code 2OAR). Figures were generated using PyMOL (DeLano Scientific; http://www.pymol.org). The approximate position of the lipid bilayer is indicated by the black lines.

Figure 1
Examples of α-helical bundle and β-barrel structure in membrane proteins involved in signal transduction, photosynthesis and the formation of pores and channels

In all cases, α-helix is shown in red, β-sheet in gold and random coil in green. (a) The light-activated proton pump bacteriorhodopsin from the archaeon H. salinarum, with retinal cofactor shown in magenta (PDB code 1C3W); (b) the photosynthetic reaction centre from the purple bacterium R. sphaeroides, with various cofactors shown in blue (PDB code 1PCR); (c) the β-barrel pore α-haemolysin from S. aureus (PDB code 7AHL), with a view into the pore from below the membrane; (d) the mechanosensitive channel from Mycobacterium tuberculosis, MscL, in the closed state, with a view into the channel from above the membrane (PDB code 2OAR). Figures were generated using PyMOL (DeLano Scientific; http://www.pymol.org). The approximate position of the lipid bilayer is indicated by the black lines.

Integral membrane proteins in vitro

The technological potential of many membrane proteins can only be explored in vitro. This requires high protein concentrations, often obtained by recombinant expression (for example, using plasmid-based overexpression in Escherichia coli) and subsequent purification (for example, by introducing a hexahistidine tag and using nickel-affinity chromatography). However, membrane proteins require special treatment; the high overall hydrophobicity of integral membrane proteins must be compensated for when they are removed from the membrane if non-specific aggregation is to be avoided. Most commonly, attempts at recombinant expression often result in the formation of insoluble aggregates (inclusion bodies) in the cytoplasm, with only a fraction of the protein of interest being correctly sorted to the cell membrane. These difficulties in expression typically lead to low overall protein yields.

Solubilizing detergents must be used to extract membrane proteins from the native or heterologous bilayer. These detergents normally have a hydrophobic tail and a polar headgroup region, so that detergent micelles mimic the interactions that the protein has with the membrane. There are several detergents commonly in use, with favourites including DDM (n-dodecyl-β-D-maltopyranoside), LDAO (N,N-dimethyldodecylamine-N-oxide), FC-12 (fos-choline 12) and OG (n-octyl-β-D-glucopyranoside). A determined and systematic screening for the correct detergent conditions must be carried out for each individual protein. Finally, the protein can be characterized in this detergent solution or reconstituted into synthetic lipid bilayers for reactions under closely defined bilayer conditions.

Recombinant expression, purification and solubilization of integral membrane proteins are not trivial. The challenges in obtaining significant amounts of folded stable protein in vitro remains one of the major barriers to developing membrane protein technologies. This means that much of the work described below is based on proteins that are experimentally tractable because they are readily extracted in high yields from native membranes (i.e. photosynthetic reaction centres, bacteriorhodopsin) or happen to be easier to handle (i.e. α-haemolysin, which is expressed as soluble monomers).

Using structure and function in nanobiotechnology

There are two main facets of membrane proteins that can generally be used for the purposes of nanotechnology. First, because these proteins are confined within a planar two-dimensional bilayer, membrane proteins offer a unique structural support. These ‘scaffolding’ applications are particularly interesting since it has been found that several membrane proteins can self-assemble into crystalline arrays within the bilayer. Secondly, the biological function of the protein can itself be exploited. Both aspects will be explored here.

Molecular motors and generation of biochemical energy

The Fo-F1 ATPase is a celebrated example of a membrane-embedded biological motor. This protein complex converts electrochemical energy (a proton gradient, generated by photosynthesis or oxidative phosphorylation) into mechanical energy (rotation of the membrane-embedded Fo ring and stalk) to generate biochemical energy by synthesizing ATP. This process is reversible, so that ATP hydrolysis can drive rotation of the motor in the reverse direction and the enzyme can function as a proton pump. Hence the Fo-F1 ATPase potentially has at least three potential nanotechnological applications: as a molecular motor, as a means to supply biochemical energy in the form of ATP, or as a mechanism for generating a proton gradient.

Considerable effort has been focused on making use of these nanoscale rotors by tethering the soluble F1 subunit alone to various surfaces (e.g. [11]); this falls outside the scope of the present review, because it is the Fo subunit that is embedded in the membrane. However, co-reconstituting the light-activated proton pump bacteriorhodopsin and the entire Fo-F1 ATPase into polymer vesicles can link their activities [12]. When these polymersomes are exposed to light, the proton gradient generated by bacteriorhodopsin can power the ATP synthase, leading to rotary movement and associated ATP synthesis. This should provide the basis for developing vesicle-based light-powered nanoscale devices that use the rotary movement of the stalk and/or the biochemical fuel provided by ATP. An additional development of this approach is that similar proteoliposomes containing bacteriorhodopsin and the ATP synthase can be captured in a silica matrix [13], generating a solid-state device and potentially increasing the stability of the proteins.

Membrane proteins as structural scaffolds

S-layers

Self-assembly is a highly desirable characteristic of nanoscale systems, because it will allow the ‘bottom-up’ construction of nanoscale structures without the need for direct mechanical manipulation or other intervention. A family of membrane-associated proteins has been identified that can spontaneously self-assemble into two-dimensional crystalline lattices. Called cell-surface layers, or S-layers, these proteins are found at the outer membrane of many prokaryotes. In the context of the present review, S-layer proteins are better described as membrane-associated or membrane-bound proteins rather than integral membrane proteins, because typically only part of the S-layer protein is attached to or embedded within the membrane with the majority being displayed from the cell surface. As reviewed by Sleytr and colleagues [14,15], S-layer proteins form regular lattices with pores between 2 and 8 nm in width that can occupy up to 70% of the surface area of the cell. They probably serve a number of functions in vivo, including protection, nutrient filtration and participating in cell–cell interactions. S-layer lattices can exhibit oblique, square or hexagonal symmetry and represent a prime example of the versatile biological self-assembly of a single molecular species into robust crystalline structures.

The potential applications of these S-layer proteins has been the focus of an intense body of work, and only a brief summary can be given here (other recent reviews provide a far more comprehensive account; see [16,17]). Briefly, self-assembly of S-layer proteins to a crystalline lattice in vitro can be accomplished in suspension, upon solid substrates such as silicon wafers and synthetic polymers, upon synthetic lipid bilayers and at other interfaces (i.e. air/water).

The tightly controlled nanoscale ordering of S-layers has made them an attractive target for the defined periodic display of small and large biological moieties. This includes other proteins and antibodies, which can be non-covalently attached to a functionalized S-layer using affinity tags [18] or introduced as transcriptional fusions without disrupting the structure [19]. Displaying enzymes on the S-layer lattice generates dense ordered catalytic arrays, and attachment to S-layers may significantly improve enzyme stability [19,20]. Since S-layers can be formed upon a solid substrate, there is considerable potential for their use in ‘biochip’-based approaches, including biosensor and immunoassay applications [21,22]. Additionally, they could be used as a template for inorganic materials and nanomaterials [23] and for supported lipid bilayers [24].

Lattices

Other membrane proteins also exhibit ordered self-assembly into crystalline lattices. This notably includes bacteriorhodopsin, the light-driven proton pump ordered in a hexagonal lattice in the PM (purple membrane) of the archaeon Halobium salinarum. This structural aspect of the PM has now been used as a biological template for the synthesis and directed assembly of gold and silver nanoparticles [25] (Figure 2). Gold nanoparticles were coated with a maleimide linker that could react with thiol groups introduced into bacteriorhodopsin at specific locations, and this covalent interaction was used to assemble nanoparticle arrays on the PM lattice. Additionally, when nanoparticles were synthesized in the presence of these engineered membranes, the thiol-containing PM served as a template for the controlled synthesis of a two-dimensional nanoparticle array. It seems likely that this approach can be readily adapted for other inorganic materials.

Using membrane protein structure and function in nanotechnology: the example of bacteriorhodopsin

Figure 2
Using membrane protein structure and function in nanotechnology: the example of bacteriorhodopsin

Bacteriorhodopsin is organized into a hexagonal lattice in the native PM, and this structure can be used as a template to assemble a nanoparticle array (transmission electron microscopic image, right), where black dots are gold nanoparticles. Bacteriorhodopsin is a light-activated proton pump, and this functionality can be used to generate a photovoltage when it is integrated into a solid-state device by sandwiching layers of PM between layers of silica (left; scale bar, 1 mm). Images of photovoltaic device and response graph reproduced with permission from K.M. Bromley, A.J. Patil, A.M. Seddon, P.J. Booth and S. Mann, Bio-functional mesolamellar nanocomposites based on inorganic/polymer intercalation in purple membrane (bacteriorhodopsin) films. Advanced Materials, 2007, vol. 19, pp. 2433–2438. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Images of PM lattice and array reproduced with permission from X. Mo, M.P. Krebs and S. Yu, Directed synthesis and assembly of nanoparticles using purple membrane, Small, 2006, vol. 2, pp. 526–529. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 2
Using membrane protein structure and function in nanotechnology: the example of bacteriorhodopsin

Bacteriorhodopsin is organized into a hexagonal lattice in the native PM, and this structure can be used as a template to assemble a nanoparticle array (transmission electron microscopic image, right), where black dots are gold nanoparticles. Bacteriorhodopsin is a light-activated proton pump, and this functionality can be used to generate a photovoltage when it is integrated into a solid-state device by sandwiching layers of PM between layers of silica (left; scale bar, 1 mm). Images of photovoltaic device and response graph reproduced with permission from K.M. Bromley, A.J. Patil, A.M. Seddon, P.J. Booth and S. Mann, Bio-functional mesolamellar nanocomposites based on inorganic/polymer intercalation in purple membrane (bacteriorhodopsin) films. Advanced Materials, 2007, vol. 19, pp. 2433–2438. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Images of PM lattice and array reproduced with permission from X. Mo, M.P. Krebs and S. Yu, Directed synthesis and assembly of nanoparticles using purple membrane, Small, 2006, vol. 2, pp. 526–529. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.

Cell-surface display

Integral membrane proteins can also be used to provide a simple structural platform on to which other biological moieties can be mounted. This idea is the basis of cell-surface-display technology, where membrane proteins function as scaffolds that present other peptide and protein sequences of interest at the surface of a cell. The heterologous peptide sequences are normally inserted into a specific region of the membrane protein by transcriptional fusion, and it is possible to construct large and diverse libraries of short peptide inserts. These can then be used in ‘biopanning’ applications where cells displaying the peptide library are screened to search for binding partners. Of particular interest here is that cell-surface display can be used to screen for short peptide sequences that interact with inorganic compounds and can influence nanomaterial synthesis. The use of short peptide sequences to enable or control nanomaterials synthesis is well-established (for example, see [26]) and the use of membrane proteins in their identification is an exciting development (for a review, see [27]).

Peelle et al. [28] used a yeast surface-display system where a single-chain antibody library was fused to the C-terminus of a cell-adhesion receptor from the yeast outer membrane [29]. This generic method could be used to pan a library of 109 single-chain antibodies for sequences that bind to crystalline CdS (cadmium sulfide) and other inorganic compounds. Soluble peptides based on these sequences could then be used to promote and control the synthesis of fluorescent CdS nanoparticles.

This yeast-based system complements other approaches that use E. coli outer membrane proteins to screen for organic/inorganic interactions for nanotechnology. For example, a polypeptide library inserted into the porin LamB was displayed at the E. coli cell surface and screened for peptides binding to gold and chromium [30]; a commercial system based upon membrane-embedded flagella (called FliTrx) could be used to identify polypeptides that bind to ZnO and Cu2O [31]; and proteins that constitute the E. coli fimbriae could be used as scaffolds to host a peptide library that could be screened for interactions with ZnO [32]. Additionally, biomineralization proteins could be inserted into one of the structureless loops of the porin OmpA and used for the deposition of inorganic products at the E. coli cell surface [33].

Optical and electronic devices

Bacteriorhodopsin

Bacteriorhodopsin is a light-activated proton pump that is probably the best-studied integral membrane protein, largely because it can be easily extracted from the native PM and because it is unusually stable and robust. From the perspective of nanotechnology, bacteriorhodopsin is attractive because of the nanoscale ordering of the PM (discussed above) and because bacteriorhodopsin can transport protons across a lipid bilayer under illumination so can generate a light-induced pH gradient and protonmotive force. Additionally, bacteriorhodopsin contains a retinal chromophore which gives it a strong purple colour. This intrinsic absorbance signal reports directly upon bacteriorhodopsin structure and function, and bacteriorhodopsin exhibits detectable variations in colour during the photocycle. Bacteriorhodopsin is extremely stable in the PM form and it is the PM that is applied for many technologies.

The potential device applications of bacteriorhodopsin have been explored thoroughly and the extensive literature has been described in detail previously (e.g. [34,35]). Essentially, efforts have focused on using the photoelectrical and photochromic properties of bacteriorhodopsin in optoelectronics, data storage and optical devices. Many of these applications involve integrating the PM into solid-state devices, and various means of fabricating such a hybrid device have been proposed. These include standard chemical methods for the deposition of thin films, which can vary in the degree to which protein orientation is controlled (for a review, see [36]). Other approaches include binding PM to an antibody array [37], embedding bacteriorhodopsin in a polymer matrix [38], allowing the protein to adsorb to solid supports by coating them in suitable lipids [39] or by taking advantage of vesicle fusion [40]. Recently, mesolamellar composites have been created by sandwiching sheets of PM between thin layers of silica, organoclay or polymer materials [41]. This latter study showed that when PM was sandwiched between sheets of amorphous silica, the resulting organic–inorganic hybrid exhibits the standard photoelectrical and photochromic responses associated with native bacteriorhodopsin (Figure 2).

It has also been demonstrated that bacteriorhodopsin can be used to convert light energy into mechanical energy [42]. When the tip of a microcantilever was coated with PM using the biotin–streptavidin interaction, light-induced proton transport from the buffer to the protein–cantilever interface caused the reversible deflection of the cantilever arm.

Photosynthetic proteins

Membrane-bound photosynthetic complexes consist of integral membrane proteins, chemical pigments and other cofactors. These complexes are highly efficient at transforming light energy into chemical redox energy. This process involves the excitation of cholorophyll pigments and subsequent electron transfer to form ion pairs that are stabilized by a rapid charge separation. This is extremely efficient (quantum yield ∼1) and rapid (most steps of the reaction taking picoseconds to nanoseconds). It has been appreciated for several years that immobilizing photosynthetic proteins on conductive inorganic substrates should produce hybrid organic–inorganic photovoltaic nanodevices that can be used as light sensors, as sources of solar power and in optical applications. The prototypical reaction centres from purple bacteria are particularly well understood, with plenty of information available on structure and function. They are also relatively robust and easily purified from the native membrane at high yields and so are currently the focus of much effort in this area. Although the reaction centre core is surrounded by antenna complexes in vivo that help to harvest and channel light to increase photosynthetic efficiency, the antennae are not essential and can be substituted by chlorophyll pigments in vitro. Reaction centres can be extracted intact from the membrane in gentle detergents ready for in vitro use.

The critical step in integrating ‘soft’ photosynthetic proteins with ‘hard’ substrates (which, in fact, is one of the key challenges facing nanobiotechnology in general) is optimizing the interface between the protein and the inorganic surface. There are several considerations. First, the normal structure and function of the protein should be retained; ideally, the efficiency of electron transfer should be identical with that in the membrane, and the protein should remain sensitive to several wavelengths of light. A second goal is to enhance protein stability, so that the device is long-lived and reusable. Thirdly, because electron transport is directional, it is important to persuade the majority of the proteins to adopt the same orientation within the device. Fourthly, for high-efficiency devices, it is desirable to minimize the distance between the protein and the surface. Finally, high protein densities are needed for highest efficiency, but discrete patterning is desirable so that light sensitive regions can be clearly defined.

This has been achieved through a number of different approaches. For example, Yasuda et al. [43] found that Rhodopseudomonas viridis reaction centres solubilized in LDAO could be reconstituted into oriented monolayers in a Langmuir–Blodgett thin film, and that these films could be deposited upon solid supports and generate a photocurrent when coated with gold. However, most success has been enjoyed using variations of bifunctional linkers, chemical groups that have separate affinities for the surface substrate and for the protein of interest. Katz [44] describes the immobilization of orientated monolayers of Rhodobacter sphaeroides reaction centres at the surface of a carbon electrode using thiol chemistry between condensed aromatic compounds, which adsorb to the electrode surface via π–π interactions, and an available cysteine group within the protein.

Trammell et al. [45] used a similar approach to attach R. sphaeroides reaction centres to carbon electrodes by exploiting the non-covalent interaction between a polyhistidine affinity tag on the protein and a Ni-NTA (Ni2+-nitrilotriacetate)-functionalized linker at the electrode surface. The major advance of this study is that using these affinity tags (pioneered by Nakamura et al. [46]) allows the protein to be oriented with the M-subunit facing the electrode, rather than the H-subunit. This orientation presents the primary electron-donor site, or P-site, to the electrode surface and has the quinone-acceptor sites facing away from the electrode. This results in more efficient electron transfer between the protein and the substrate, generating higher photocurrents. A similar approach, again using bifunctional linkers, has also been used to introduce reaction centres to the interior of carbon nanotubes, and electrodes made of arrays of these protein–nanotube hybrids exhibit improvements in performance compared with standard graphite electrodes [47].

Proteins can also be immobilized at a surface by using linkers that are covalently attached to the substrate. When gold monolayers were modified with chemical groups that present Ni-NTA, the polyhistidine–Ni-NTA interaction could again be used to immobilize R. sphaeroides reaction centres with the P-site facing the electrode [48]. Das et al. [49] also took advantage of this immobilization technique to generate oriented monolayers (stabilized by the introduction of two peptide surfactants) of both the R. sphaeroides reaction centre and the larger, and more complex, Photosystem I from spinach chloroplasts on gold. These monolayers were then integrated into solid-state devices by coating them with an organic semiconductor.

Although the use of various linkers to attach these proteins to surfaces is generally essential to maintain activity, there are now examples where a direct protein–substrate interaction can be used successfully. For example, cysteine mutants of a cyanobacterial Photosystem I complex can bind covalently to a gold substrate to form well-oriented functional monolayers [50]. A similar approach using carbodi-imide chemistry can be used to covalently link these cysteine mutants to carbon nanotubes [51].

It is also interesting to consider some of the recent advances in generating patterned surfaces. AFM-based dip-pen nanolithography [52] and the chemical technique of nanoimprint lithography [53] can both be used to generate discrete amino-terminated domains on otherwise protected substrates, and photosynthetic complexes adsorb selectively to those amino domains through charge interactions. This gives rise to discrete photoactive regions on the substrate. The precise control of protein patterning offered by methods such as these will be critical for the manufacture of devices based on photosynthetic proteins.

Biological nanopores

Several different types of membrane protein are involved in the passage of materials through the membrane. These include channel proteins, which tend to be helical bundles and highly selective for small molecules or ions (for example, the potassium channel KcsA), and pore proteins, which tend to be β-barrels with a more open structure that makes them somewhat less selective (for example, OmpF or OmpA from the outer membrane of E. coli and the heptameric α-haemolysin from Staphylococcus aureus). The diameter of the hole formed by both channels and pore proteins in the membrane is <10 nm, and both are thus considered to be examples of naturally occurring nanoscale pores. There has been a particular focus on using α-haemolysin in vitro, because it is extremely stable and can be readily overexpressed as soluble monomers that self-assemble into the pore structure when introduced into lipid bilayers.

Nanopores in vitro: sequencing and sensing

Single-channel measurements in synthetic bilayers have been used to show that single strands of RNA and DNA can pass through α-haemolysin pores when they are kept continuously in the ‘open’ state by an applied transmembrane voltage [54]. The voltage drives the charged oligonucleotide into the pore, which then becomes blocked, leading to measurable changes in ion conductivity. The length of time that the channel is blocked by the presence of the oligonucleotide is related directly to the length of that oligonucleotide (i.e. the number of bases) and it is possible to discern different sequences (i.e. the type of base) [55]. Other pores with different channel dimensions, such as OmpF, have also been investigated for DNA-translocation activity and this approach has the potential to become a novel method for DNA sequencing and analysis [56,57].

Additionally nanopores may be useful as sensors. Again, α-haemolysin has been the poster child for the development of this technology and single-channel measurements under applied voltage in planar bilayers are used. These represent ‘stochastic sensors’ [58] where individual binding interactions are detected under single-channel conditions. The frequency of binding events represents the analyte concentration, and individual analytes can be discriminated by their characteristic binding signature that typically includes the extent of current block and the average length of each blocking event. An interesting recent example is the use of α-haemolysin to detect toxic mustard compounds [59] by exploiting the reactivity of these compounds with cysteine thiols. By using mutagenesis to introduce cysteine residues into the centre of the pore, the irreversible covalent binding of several nitrogen mustards was observed by monitoring the associated decrease in current. Additionally, a molecular adapter (a thiol-containing cyclodextrin group that binds non-covalently to the pore lumen) could be used to observe multiple reversible binding events.

Similar approaches using either chemical modifications of α-haemolysin, functionalized cyclodextrin adapters or genetically engineered pores have now been used to detect and discriminate between metal ions [58], organic compounds [60] (and remarkably, to discriminate between different enantiomers [61]) and small molecules important in biological signalling [62]. By tethering ligands to the pore, it is also possible to exploit protein–ligand interactions to detect proteins [63] and even to obtain a full description of ligand-binding kinetics [64,65].

This ‘stochastic sensor’ approach has now been extended to other proteins. Chen et al. [66] have been able to rationally design and engineer a ‘quiet’ version of the pore OmpG, where background noise from spontaneous pore gating was considerably reduced compared with wild-type protein. Once again, a cyclodextrin adapter was employed in single-molecule studies in planar bilayers to turn this modified pore into a sensor for ADP.

The same α-haemolysin system can also be adapted to serve as a nanoreactor for polymer synthesis [67]. A single cysteine group was introduced into the α-haemolysin lumen and repeating cycles of simple disulfide chemistry were used to grow a polymer attached to that site. The polymerization was followed by monitoring changes in pore current at each monomer addition step, so that reaction progress kinetics and the nature of the reaction products could be characterized simultaneously.

Cell permeabilization

One interesting application of nanopores may be the selective introduction of small molecules and/or ions into mammalian cells. A variant of the α-haemolysin pore, H5, is particularly useful in that the 2-nm-diameter pore can be reversibly closed and opened by introducing Zn2+ ions and chelating agents respectively. This method was used to introduce small molecules such as sugars and fluorescent dyes into fibroblast cells [68]. The potential to adapt this technology for the selective and non-selective introduction of therapeutics or imaging agents into nanopore-permeabilized cells has been recognized. Additionally, Panchal et al. [69] report an α-haemolysin construct that is able to specifically lyse cancer cells by permeabilization once a protective region is cleaved off by a protease secreted by certain tumours.

Ligand-activated nanopores

To achieve a ligand-responsive nanopore, Moreau et al. [70] constructed a transcriptional fusion between the GPCR (G-protein-coupled receptor) M2 and the K+ channel Kir6.2 and expressed this fusion in Xenopus oocytes. Ligand binding by the GPCR induced a conformational change that was transmitted to the channel, causing it to open. This direct mechanical coupling between receptor and channel results in a ligand-induced electrical signal as K+ ions flow across the oocyte cell membrane. The principle behind this technique appears to be applicable to other GPCRs, suggesting that the fusion between a receptor and a nanopore could be used to screen for new GPCR-binding drugs or as a diagnostic tool.

The examples above indicate that biological nanopores will be one of the cornerstones of nanobiotechnology. With this in mind, it is especially interesting to note recent progress in successfully immobilizing functional nanopores in a chip format [71].

Light-activated membrane proteins

There has been considerable interest in using non-invasive and precise triggers such as light to activate membrane proteins in vivo and in vitro, and several different strategies have now been adopted to generate optically activated nanoscale valves and switches (for a review, see [72]).

One of the most popular approaches has been the use of photochemistry, where membrane proteins are activated or inactivated by the light-induced changes in the structure or physicochemistry of a chemical group. For example, photoinduced changes in the structure and charge state of a covalently bound spiropyran group can direct the reversible opening and closing of the mechanosensitive channel MscL in a synthetic bilayer at different wavelengths of light [73]. This reversibly introduces a non-selective pore of ∼3 nm diameter into synthetic lipid bilayers. Another approach used an azobenzene group to control the proximity of an agonist to the allosteric regulatory site of the ligand-gated ion channel iGluR [74]. The receptor agonist, glutamate, was covalently coupled to the receptor via the azobenzene linker. When whole cells containing this derivatized iGluR are illuminated at longer wavelengths of light (500 nm), azobenzene is in the trans form and the agonist is kept away from the allosteric binding site. Upon illumination with shorter wavelengths (380 nm), reversible photoisomerization of the azobenzene to the cis form presents the ligand to the binding site and activates the channel. This is evident from photocurrents that can be detected by whole-cell patch–clamp recordings.

pH-responsive membrane proteins

Cellular pH is a key indicator of cellular health. Conditions including cancer, inflammation and stroke result in localized tissue acidification and so there is considerable interest in developing pH-responsive nanotechnologies that can specifically deliver a biological activity, such as a therapeutic small molecule, to acidified tissue.

One approach is to use the native pH response of the porin OmpF. When this nanopore is reconstituted into ∼100-nm-diameter vesicles made from an amphiphilic copolymer [75], the pore is found to be in the closed state at pH 7.5, but open at pH 5. Thus, at moderate pH, the vesicle is a sealed container, but, at low pH, the pore opens and small-molecule substrates can enter or exit the vesicle interior. In this particular example, the vesicles were loaded with the enzyme acid phosphatase and the entry of a fluorigenic phosphatase substrate was observed when the pore was open at low pH. These vesicles were made of a stable biocompatible polymer so that they could potentially be used in therapeutic applications such as drug delivery. If the polymer vesicles were loaded with a bioactive molecule, this would be sequestered within the vesicle chamber at normal physiological pH, but released specifically in regions of acidified tissue.

An exciting recent development is the demonstration that pH can also be used to control the insertion of transmembrane domains across a lipid bilayer. A short peptide, originally derived from the bacteriorhodopsin transmembrane helix C, has been found to adopt a soluble disordered structure in solution at neutral pH, but to insert across a bilayer as a classical transmembrane α-helix under acidic conditions [76]. The reversible insertion of this so-called ‘pHLIP’ peptide into lipid bilayers at low pH may prove extremely useful in targeted drug delivery and in cell imaging applications [77].

Conclusions

Nanobiotechnology is an exciting new area of research. This genuinely interdisciplinary enterprise should exploit the enormous nanotechnological potential of the natural world and the structure and function of integral membrane proteins can provide the basis for new nanoscale sensors, optoelectronics and much more. It will be fascinating to watch these technologies mature and to see new ideas emerge from the fertile ground of biological nanotechnology.

Early Career Research Award Delivered at Robinson College, Cambridge, on 8 January 2009 Paul Curnow

Early Career Research Award Delivered at Robinson College, Cambridge, on 8 January 2009 Paul Curnow
Early Career Research Award Delivered at Robinson College, Cambridge, on 8 January 2009 Paul Curnow

Early Career Research Award:

Abbreviations

     
  • AFM

    atomic force microscopy

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • LDAO

    N,N-dimethyldodecylamine-N-oxide

  •  
  • Ni-NTA

    Ni2+-nitrilotriacetate

  •  
  • PM

    purple membrane

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