Complex biological machines arise from self-assembly on the basis of structural features programmed into sequence-specific macromolecules (i.e. polypeptides and polynucleotides) at the molecular level. As a consequence of the near-absolute control of macromolecular architecture that results from such sequence specificity, biological structural platforms may have advantages for the creation of functional supramolecular assemblies in comparison with synthetic polymers. Thus biological structural motifs present an attractive target for the synthesis of artificial nanoscale systems on the basis of relationships between sequence and supramolecular structure that have been established for native biological assemblies. In the present review, we describe an approach to the creation of structurally defined supramolecular assemblies derived from synthetic α-helical coiled-coil structural motifs. Two distinct challenges are encountered in this approach to materials design: the ability to recode the canonical sequences of native coiled-coil structural motifs to accommodate the formation of structurally defined supramolecular assemblies (e.g. synthetic helical fibrils) and the development of methods to control supramolecular self-assembly of these peptide-based materials under defined conditions that would be amenable to conventional processing methods. In the present review, we focus on the development of mechanisms based on guest–host recognition to control fibril assembly/disassembly. This strategy utilizes the latent structural specificity encoded within sequence-defined peptides to couple a conformational transition within the coiled-coil motifs to incremental changes in environmental conditions. The example of a selective metal-ion-induced conformational switch will be employed to validate the design principles.
The oriented axial assembly of protein motifs represents the primary structural feature that underlies the formation of native protein-based fibrils such as keratin, collagen, tubulin, and F-actin (filamentous actin) . Self-assembly occurs via linear propagation of the subunits that is mediated through specific molecular recognition interactions at structurally complementary interfaces. The structural features that guide self-assembly are encoded within the sequences of the protein subunits and include complementary packing of side chains at the interface, as well as electrostatic, hydrophobic and hydrogen-bonding interactions. Given the richness of the molecular-scale information programmed within the sequences of polypeptides, it should not be surprising that synthetic materials have yet to recapitulate the self-assembly behaviour of native protein-based assemblies. Thus elucidating design principles that underlie the self-assembly of fibrillogenic proteins into structurally defined supramolecular structures is of interest not only in generating synthetic polypeptides that mimic native structural proteins , but also for the design of new materials with biological, chemical and mechanical properties that exceed those of currently available synthetic polymers.
The simplest structural units that comprise protein-based fibrils are based on fundamental secondary-structure elements such as α-helices and β-strands. Although self-assembling β-sheet peptides based on amyloid and related de novo amino acid sequences are being actively scrutinized as functional nanoscale materials [3–13], the design of self-assembled fibrils derived from α-helical peptides [14,15] has not advanced to a similar extent. Helical scaffolds differ in critical structural features from β-sheet assemblies, including the structural periodicity of the fibre repeat , the orientation of protomers relative to the long axis of the fibril  and the physical interactions that guide self-association. These considerations suggest that helical protomers may be effectively considered as complementary blocks to β-strands, and that the resulting coiled-coil fibrils may have significantly different physical properties from those of β-sheet fibrils . Moreover, in contrast with amyloidogenic peptides, the principles that govern the association of helical protomers into discrete coiled-coil assemblies  have been elucidated in detail on the basis of structural studies of model peptides such as leucine zippers [19–24]. However, until recently, the potential of these helical peptides for the construction of synthetic self-assembled materials remained largely untapped, despite the fact that fibrils derived from α-helical coiled-coil motifs occur widely in native biological systems . Thus α-helical proteins based on coiled-coil structural motifs are an attractive target for de novo engineering of structurally defined fibrils from self-assembly of synthetic helical protomers .
Coiled-coil structural motifs occupy multiple roles in biological systems, including protein–protein recognition, locomotion, signal transduction and sensing/actuation, many of which would be desirable to emulate in synthetic nanoscale systems . Two distinct challenges are encountered in this approach to materials design: the ability to re-code the canonical sequences of native coiled-coil structural motifs to accommodate the formation of structurally defined supramolecular assemblies (e.g. synthetic helical fibrils), and the development of methods to control supramolecular self-assembly of these peptide-based materials under defined conditions that would be amenable to conventional fibre-processing methods. Previous research in our laboratory [27–29], as well as others [30–36], has provided insight into the critical structural parameters that govern the formation of extended helical assemblies. Although significant structural characterization remains to be performed on these systems, in particular, to establish the structural relationship between peptides within the assembly in the context of the design rules, the main focus of the present review is the development of methods to control the self-assembly process. We envisage a strategy based on the rational design of selective binding sites for guest species (metal ions, complex ions and small molecules) within de novo-designed coiled-coil peptides as a mechanism to control the reversible assembly/disassembly of helical fibrils based on these structural motifs.
Guest–host interactions within coiled-coil structural motifs
Native biomacromolecular assemblies are characterized not only by well-defined structures, but also by unique functions that arise as a consequence of those structures. One important function is the ability to sense and respond to incremental changes in environmental conditions. A critical challenge in the creation of synthetic biomolecular assemblies is the rational design of responsive mechanisms that can be coupled to supramolecular structure to recreate the self-assembly behaviour that is characteristic of native biological systems. Coiled-coil motifs are the best understood and most extensively characterized structures among natively folded protein families, and therefore represent an attractive structural platform for de novo protein design studies directed towards the introduction of function into synthetic peptide systems. These studies have focused on attempts to engineer selective recognition sites for guest species into internal positions with the coiled-coil structure, affording model systems for understanding the binding specificity associated with ligand recognition and enzymatic catalysis.
The potential for engineering guest binding sites into coiled-coil structures can be assessed initially from consideration of crystallographic structural determinations performed on discrete helical bundles [19–24]. Cross-sectional analyses of these coiled-coil structures indicate that, for oligomerization states greater than two, the core residues define internal cavities within the helical bundle that can accommodate small guest species (usually water molecules or counterions) [37,38]. The cross-sectional shape of the cavities reflects that of the underlying symmetry of the helical bundle, whereas the cavity dimensions correlate approximately with the oligomerization state of the coiled-coil structure and the size and orientation of the side chains of amino acid residues at the positions that define the cavities. In certain circumstances, coiled-coils with higher oligomerization states can form continuous channels that span the length of the structure, and which can serve as hosts for adventitious guest species such as PEG [poly(ethylene glycol)] oligomers or hexanediol molecules [23,24]. These prototypical coiled-coil structures have been employed as models to guide the introduction of amino acid substitutions at appropriate positions within the heptad sequences to generate binding sites for specific groups of guest species within a defined structural context. Our initial studies have focused primarily on the design of selective metal-ion-binding sites of defined co-ordination geometry (see below). The engineering of metal-binding sites into discrete helical bundles of defined oligomerization state provides support for this approach. Numerous antecedents have been reported in which the selective binding of metal ions can trigger a conformational transition within this structural context. These results suggest that the binding of metal ions at engineered sites within coiled-coil assemblies may be employed as a potential mechanism to control fibril assembly/disassembly.
Metal-ion-binding sites in peptides and proteins
Metal ions in proteins frequently play critical functional roles that include stabilization of protein structure, induction of conformational transitions, and facilitation of electron transfer, small-molecule transport and enzymatic catalysis . Numerous peptide models have been examined to define and potentially replicate the native functional roles of metal ions within defined polypeptide structural contexts [40–45]. One of the most striking observations to emerge from these studies is the ability of metal ions to induce conformational transitions from the unfolded to folded state [46–52] or between different folded states [53–55] through interaction with the side chains of appropriately placed amino acid ligands within the polypeptide backbone. Moreover, metal ions can influence the supramolecular assembly of disease-related protein fibrils [56,57], either enhancing or inhibiting the process, depending on the stereoelectronic properties of the specific metal. Preliminary studies in our laboratory  (see below) indicate that an appropriately chosen metal ion can trigger the self-assembly of a de novo-designed peptide into a structurally defined nanoscale material. This process involves selective recognition and binding of the metal ion at a complementary site within the peptide sequence, which induces a conformational transition that results in a specific mode of self-association consistent with fibril self-assembly. These results suggest that the rational design of selective binding sites for co-ordination of metals and other complexes within de novo-designed peptides may represent a promising approach for the controlled fabrication of nanoscale self-assembled materials.
The trimeric coiled-coil structural template
Although a number of alternative coiled-coil sequence motifs may be considered as candidates for the design of self-assembled materials, we focused on the trimeric coiled-coil motif derived from an isoleucine zipper (Figure 1) based on the following considerations. First, numerous model studies within this structural context have established sequence–structure correlations that would serve as useful principles to guide the sequence design. Secondly, the oligomerization state of the trimeric coiled-coil meets the minimum requirements for creation of functional cavities within the hydrophobic core of the self-assembled complex (see above). Finally, structural prototypes for metal ion and small-molecule switches based on discrete trimeric coiled-coil assemblies have been described previously, which provides confidence with regard to the feasibility of this approach. The design of the first-generation peptide, TZ1 , comprised six heptad repeats of a coiled-coil structural motif that was derived from the amino acid sequence of the isoleucine zipper peptide GCN4-pII . The latter peptide had been demonstrated on the basis of crystallographic data to form a three-stranded helical bundle. Consequently, the incorporation of isoleucine residues at the (a/d)-core positions of TZ1 should favour the formation of a trimeric assembly. Lateral registration between adjacent helical protomers in the structure of TZ1 was specified through manipulation of the coulombic interactions between charged residues at the (e)- and (g)-positions of the heptad repeats [58,59]. The sequence of the TZ1 peptide was designed such that the electrostatic interactions between the (e/g)-residues on structurally adjacent protomers would be completely charge-complementary only in a staggered alignment in which the peptides self-assemble into a helical fibril corresponding to a three-stranded rope as depicted in Figure 1. Structural characterization of peptide TZ1 was consistent with the formation of high-aspect-ratio helical fibrils based on the adoption of an α-helical conformation. This structural prototype has been successfully employed as the basis for the creation of sequence variants in which selected isoleucine residues within core positions have been replaced with alternative amino acid residues to create guest-binding sites that can trigger environmentally responsive self-assembly (see below).
Design of peptide TZ1: a structural template for fibril self-assembly derived from a trimeric coiled-coil motif
Engineering guest-binding sites in coiled-coil motifs
Using the isoleucine zipper sequence of TZ1 as a structural template, we envisaged that mutations could be introduced into the peptide sequence at structurally critical core positions, so that the self-assembly of fibrils could be controlled through incremental changes in environmental stimuli (pH, metal ion or small molecule) within a sharply defined range under physiologically relevant conditions. Peptide TZ1 had been demonstrated previously to self-assemble into high-aspect-ratio fibrils based on an α-helical coiled-coil conformation; however, the stability of the hydrophobic isoleucine core was such that it was difficult to completely denature the helical structure even in the presence of strong chemical denaturants and thermolysis. We hypothesized that the substitution of appropriately functionalized amino acid residues into the TZ1 peptide would permit us to control the self-assembly of these variants through manipulation of the relative stability of the α-helical conformation vis-à-vis the random coil conformation under appropriately defined experimental conditions. Thus the self-assembly of these variant peptides could be coupled to a random-coil-to-α-helix conformational transition that could be induced due to the effect of changes in environmental conditions on the chemical state of the responsive residues.
Design of responsive peptide sequences
We envisaged that a wide range of recognition processes might be employed to trigger a coil-to-helix conformational transition and subsequent fibril assembly within the TZ1 structural template. At least three different types of guest molecule can potentially act as triggers for this conformational transition: transition metal ions, complex ions and hydrophobic small molecules. Sets of ligands corresponding to the side chains of natural or synthetic amino acids may be postulated for the creation of binding sites for specific guest species within the coiled-coil structure of TZ1; however, the sequence of the template must be appropriately modified to accommodate selective binding of the guest species. The necessity to maintain the critical structural features that stabilize the fibril formation places restrictions on the number and position of the binding sites that can be introduced into the peptide sequence of TZ1. The 3-fold symmetry of the trimeric coiled-coil structural template and precisely staggered alignment of peptides within the fibril (Figure 1) requires that three amino acid substitutions are introduced at core (a/d)-positions that are separated by two heptad repeats within the sequence (Figure 2). This arrangement ensures that the critical charge-complementary interactions between (e/g)-residues are preserved and that the variant residues align in the self-assembled state to create the guest-binding cavities. These mutations within the sequence will define three guest-binding sites along the contour length of each peptide. The identity of the variant residues will determine the selectivity for binding of the guest and the ability of the guest to trigger a conformational transition and the associated fibril assembly. Given the different orientations of amino acid side chains that occupy the (a)- and (d)-positions of a trimeric coiled-coil structure , two different versions of the TZ1 template can be constructed, TZ1Xa (I9X/I23X/I37X) and TZ1Xd (I5X/I19X/I33X), in which the substitutions are introduced at the (a)-positions and (d)-positions respectively (Figure 2). In the case of discrete coiled-coil host species, differences have been observed in the thermodynamics of guest binding between substitutions at the (a)- and (d)-positions.
Cross-sectional representations (A) and amino acid sequence patterns (B) of the variant structural templates TZ1Xa and TZ1Xd within the trimeric assembly
A metal-ion-induced switch for self-assembly
A structural variant of the parent peptide, TZ1Hd, was created in which histidine residues were introduced into the core (d)-positions of alternate heptads to afford a triple mutant (I5H/I19H/I33H) of TZ1 [28–29]. In the suppositious fibril structure of Figure 1, the core histidine residues should reside at adjacent positions across the helical interface. The histidine residues of TZ1Hd can be reversibly protonated at the imine nitrogen of the imidazole side chain to interconvert between charged and uncharged states, which induces a reversible transition between the random coil and α-helical conformations respectively. We have demonstrated that peptide TZ1Hd self-assembles into high-aspect-ratio helical fibrils in buffered aqueous solutions in which the pH value exceeds the pKa of the histidine side chains, which coincides with the random-coil-to-α-helix conformational transition .
The successful pH-triggered formation of the self-assembled helical fibrils validated the structural principles underlying the design of TZ1Hd ; however, we also noted that the layers of three proximal histidine residues within the suppositious fibril structure provided a potential metal-ion-binding site and therefore a mechanism for coupling a metal-ion-induced conformational transition to peptide self-assembly . The trigonal planar geometry within the hypothetical metal-ion-binding site, although encountered infrequently, has precedence for some electron-rich late-transition metal ions. In particular, the silver(I) ion can adopt trigonal planar co-ordination in the presence of soft-donor nitrogen ligands, such as substituted imidazoles  with minimal deviations from the idealized geometry in sterically unconstrained systems . We anticipated that peptide TZ1Hd could sterically accommodate the silver(I) ion within the trimeric binding site, as larger ionic species have been observed as guests within cavities created between similarly sized residues in trimeric coiled-coil structures .
Silver(I) ion binding was assessed by both CD spectropolarimetry and ITC (isothermal titration calorimetry) under acidic conditions (pH 5.6), which are not conducive to pH-triggered self-assembly. Both techniques confirmed a binding event, which, in the case of CD spectropolarimetry, could be demonstrated to coincide with a transition in peptide secondary structure from coil to helix (Figure 3). The ITC data indicated approximately one binding site per peptide. Each peptide contains three histidine residues that could contribute one-third of a trigonal binding site for a silver(I) ion, thus yielding a theoretical value of one binding site per peptide. Transmission electron microscopy indicated that the silver(I) ion-binding event and coil-to-helix structural transition coincided with formation of high-aspect-ratio fibrils. STEM (scanning transmission electron microscopy) imaging of the fibrils was accomplished in bright-field phase-contrast mode and backscatter mode. The intensity of the latter signal depends on electron density, and the observed intensity along the contour length of the fibril was consistent with the presence of an electron-dense element such as silver. The presence of silver was confirmed through energy-dispersive X-ray spectroscopic analysis. Surprisingly, silver(I) ions bound very selectively to peptide TZ1Hd, being removed only under conditions of treatment with a strongly co-ordinating ligand such as a thiosulfate anion. Several other transition metals were assessed for their ability to bind to peptide TZ1Hd and trigger self-assembly. Notably, CD titration experiments indicated that the isoelectronic (d10) zinc(II) ion, the (d9) copper(II) ion or the (d8) nickel(II) ion could not effectively induce a comparable conformational transition within TZ1Hd even in the presence of excess metal ion. Since zinc(II), copper(II) and nickel(II) prefer alternative co-ordination geometries, these metal ions may not be accommodated easily within the trigonal planar co-ordination sites of TZ1Hd. However, these results suggested that, if helical assemblies could be designed that presented an appropriate co-ordination environment, then selective metal ion switches might be selected based on co-ordination preferences.
A silver(I) ion-selective switch for peptide self-assembly
We have proposed a general design for the synthesis of responsive peptide systems that can self-assemble into structurally defined high-aspect-ratio helical fibrils upon binding of an appropriately chosen guest species. This approach has been validated in the context of a peptide, TZ1Hd, that self-assembles selectively in response to the presence of a silver(I) ion guest. These results suggest that the rational design of selective guest-binding sites within de novo-designed peptides represents a promising approach to the controlled fabrication of nanoscale self-assembled materials that exploits the latent structural specificity encoded within these sequence-defined molecules.
Bionanotechnology II: from Biomolecular Assembly to Applications: Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 7–9 January 2009. Organized and Edited by Tony Cass (Imperial College London, U.K.) and Dek Woolfson (Bristol, U.K.).
Supported by National Science Federation [grant number CHE-0414434] and Department of Energy [grant number ER-15377] grants.