Biomaterials Made from Coiled-Coil Peptides
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The development of biomaterials designed for specific applications is an important objective in personalized medicine. While the breadth and prominence of biomaterials have increased exponentially over the past decades, critical challenges remain to be addressed, particularly in the development of biomaterials that exhibit highly specific functions. These functional properties are often encoded within the molecular structure of the component molecules. Proteins, as a consequence of their structural specificity, represent useful substrates for the construction of functional biomaterials through rational design. This chapter provides an in-depth survey of biomaterials constructed from coiled-coils, one of the best-understood protein structural motifs. We discuss the utility of this structurally diverse and functionally tunable class of proteins for the creation of novel biomaterials. This discussion illustrates the progress that has been made in the development of coiled-coil biomaterials by showcasing studies that bridge the gap between the academic science and potential technological impact.
KeywordsCoiled-coils Biomaterials Hydrogels Nanotubes Nanomedicine
Enhanced permeability and retention
cartilage oligomeric matrix protein
polyethylene glycol diacrylate
hydrogelating self-assembling fibres
analytical ultra centrifugation
Severe Acute Respiratory Syndrome
The N-hydroxysuccinimide ester of 4-(N-maleimidomethyl) cyclohexanecarboxylic acid
17.1 Introduction: The Emerging Role of Biomaterials
Biomaterials derived from designed peptide and protein sequences have become the focus of significant scientific research effort over the last two decades. Rapid developments in genome sequencing, bioinformatic analysis, atomic resolution structural determination, and computational protein design, in combination with extant synthetic and analytical methods, present an unprecedented opportunity to create peptide- and protein-based biomaterials of defined structure and controllable function. Progress toward the goal of creating tailorable biomaterials for applications in nanotechnology and human health has been impressive, although the practical promise has yet to be realized on a substantial scale. One strategy has involved the identification of stable structural motifs that can serve as flexible scaffolds for the introduction of function. This approach requires the identification of protein folds that can accommodate substantial sequence modification without compromising structure or function. Ideally, these protein motifs would be highly designable and amenable to computational structural prediction. The α-helical coiled-coil motif is the most studied protein fold and fulfills many of the criteria that one would envision as a scaffold for biomaterials design.
Well-understood design rules (Lupas and Gruber 2005)
Accessibility of a variety of oligomeric states (Harbury et al. 1993)
Versatility in sequence and structure
As discussed in previous chapters, coiled-coils can be designed de novo using a handful of simple rules that can be implemented computationally to select for oligomerization state with a great degree of reliability (Thompson et al. 2014; Huang et al. 2005; Wood et al. 2014).
As evidenced by their ubiquity in the proteomes of sequenced organisms, coiled-coil proteins comprise varied sequences, adopt a variety of native structures, and display a range of functions (Testa et al. 2009). The scope of coiled-coil sequences and structures has been further expanded through the carefully applied use of rational design. Non-canonical amino acids can be incorporated at specified sites in the sequences using either solid-phase peptide synthesis methods or supplementation of growth media used in in vitro or in vivo protein expression. The introduction of non-native chemical functionality can further expand the material properties of this flexible scaffold. Indeed, coiled-coils are a versatile and robust scaffold for generating a diverse range of functional biomaterials that can be tailored for specific applications through modification of the sequence at structurally permissive sites.
17.2 Coiled-Coil Therapeutics
A major problem facing modern medicine is the dearth of strategies for treating diseases that present differently in each patient, e.g. cancer. The need for easily customizable treatments has led to the development of a broad field known as nano-theranostics (a portmanteau derived from therapeutics and diagnostics), which seeks to use nano-scale materials to image and/or treat diseases. A dominant strategy in nano-theranostics is to specifically target diseased cells for the delivery of small doses of extremely cytotoxic low molecular weight drugs. A clear challenge in this field is thus developing site-specific drug delivery to reduce side effects from apoptosis of healthy cells. Coiled-coil motifs have recently been explored as potential nano-theranostic agents, due to their lack of immunogenicity, exquisite sensitivity to biologically relevant environmental changes (such as pH), and well-known engineering rules. This inherent designability makes coiled-coils an attractive scaffold from which to develop materials for nano-scale treatments.
Coiled-coil-based therapeutics can be broken up into two main categories: coiled-coil drug carriers, and drug-free coiled-coil systems. The first of these categories is more familiar to those studying nano-medicine. Essentially, a low molecular weight drug is encapsulated by a self-assembling coiled-coil, which dissociates upon some environmental change (typically pH, which can vary in a diseased state) (Yu 2002). The low molecular weight drug is generally highly cytotoxic and, as such, the design focus for this approach is towards target specificity (Pola et al. 2013). These systems also offer the potential for multi-valent drug binding, which improves the loading efficiency by minimizing the entropic penalty for binding. Assuming one chain in a heterodimeric coiled-coil system contains a single binding site, its sequence may be doubled to introduce a second binding site (Assal et al. 2015). Recently, Klok and co-workers investigated a heterodimeric coiled-coil carrier comprising two peptides (K3 and E3), which self-assemble at pH 7.0, but dissociate at pH 5.0 (endosomal pH), thereby releasing their cargo (Apostolovic et al. 2010, 2011). These peptides were bound to a poly (N-(2-hydroxypropyl)-methacrylamide (PHPMA) polymer backbone and designed to carry an anti-cancer drug, methotrexate (MTX) in one case, and a fluorescent dye in another. By combining these two technologies, it follows that one could monitor cellular uptake of the drug carrier while simultaneously treating the patient. Though uptake of the drug into the endosomes was shown, the distribution of the drug throughout the organelles has not yet been obtained.
17.3 Coiled-Coil Hydrogels
Hydrogels are formed from covalent or non-covalent cross-linking of individual polymer chains into a three-dimensional water-swollen network. Hydrogel materials offer a wealth of potential applications (Elisseeff 2008), in that the biological, chemical, and mechanical properties may be tailored for particular applications through modification of the structure of the polymer and cross-linker. They have innate capacity to absorb and retain large volumes of water relative to the mass of the hydrogel itself (Elisseeff 2008; Xu and Kopeček 2008; Yao et al. 2014). Moreover, hydrogels are often highly biocompatible due to their capacity for water absorption, physical elasticity, and relative softness (Kopeček 2007, Yao et al. 2014). The aforementioned qualities make hydrogels a convincing substitute for native extracellular matrix (Slaughter et al. 2009; Yao et al. 2014), as well as suitable candidates for cell culture applications (Frampton et al. 2011; Jung et al. 2011), biomedical uses such as wound treatment (Wong et al. 2011; Lin et al. 2011), and drug delivery (Garbern et al. 2011; Guziewicz et al. 2011). Unsurprisingly, hydrogels were the first biomaterials developed for medical uses in humans (Kopeček 2007; Kopeček and Yang 2012). As biomedical materials, coiled-coil-based hydrogels offer an alternative to the more commonly employed β-sheet hydrogels. One potential concern with the latter materials is that the β-sheet morphology is analogous to amyloid structures, such as those observed in prion and amyloid diseases. These toxic assemblies often develop via a misfolding of endogenous proteins and can be auto-catalytically propagated in the presence of a β-sheet template (Fletcher et al. 2011). Due to the risk of initiating protein misfolding disease states, implementation of β-sheet-based hydrogels must be carefully scrutinized for the manifestation of deleterious physiological effects (Fletcher et al. 2011). Biomaterials based on coiled-coil structures do not display similar behaviour, as self-assembly in most cases is restricted to the formation of closed oligomers. This section will cover the past decade of advances within coiled-coiled-based hydrogels.
Hydrogels based on coiled-coil self-association can offer several notable advantages (Kopeček 2007). The coiled-coil is an exceptionally well understood structural motif, when compared to other protein folding domains (vide supra) (Lupas and Gruber 2005). Due to the wealth of information regarding the coiled-coil’s structure and properties (in comparison to other super-secondary and tertiary structures), the coiled-coil has the potential to serve as an ideal ‘tuning knob’ for hydrogel properties. Though the coiled-coil is certainly not the only factor that governs hydrogel properties, it represents the most facile mechanistic handle. Tuning coiled-coil properties can involve a procedure as simple as altering the sequence of amino acids within the constituent alpha helices, allowing the researcher to alter hydrogel properties with relative ease and efficiency. Coiled-coils offer versatility in terms of oligomeric state as well (Harbury et al. 1993). Moreover, due to the widespread prevalence of coiled-coils within native proteins, coiled-coil-based biorecognition domains are also pervasive throughout the natural world. Co-opting such domains may yield significant advantages in the engineering and design of hydrogels, as doing so may offer a conduit through which artificially engineered hydrogels may interact with natural systems with high specificity (Kopeček 2007).
The self-assembly of these ABA block copolymers is driven by two interconnected forces: hydrophobic collapse of A ends and polar association of water with the B interior region. One advantage of hydrogel formation from these ABA block copolymers is their reversibility, wherein virtual cross-links resultant from the coiled-coil interactions can be reversibly denatured, yielding a gel to sol transition. However, the A blocks retain the capacity to re-form the coiled-coil structure upon removal of the denaturing conditions. Moreover, the interaction between helices is highly tunable; minute changes in sequence, especially to the alpha helical regions can produce discernible effects on the biophysical properties of the resultant morphologies (Kopeček 2007). Xu and Kopeček noted that the physical characteristics of their ABA triblock and AB diblock hydrogels depended on the relative lengths of the A block and B block, the sequence composition, and the introduction of specific electrostatic interactions. Tirrell and co-workers programmed further specificity into these systems through diversification of the polypeptide sequence to create an ABC triblock system. In this architecture, the A and C domains comprise different coiled-coil sequences that exhibit little tendency to associate heteromerically, that is, the A and C blocks are structurally orthogonal (Shen et al. 2006). The presence of these orthogonal coiled-coil sequences inhibits the formation of intramolecular loops, which were prevalent within hydrogels derived from the ABA triblock systems, and, consequently, lowers the rate of erosion of free polymer from the surface of the resultant hydrogel.
An additional advantage of the coiled-coil motif lies in its ability to accommodate mutations at specific positions while retaining its structural integrity (Harbury et al. 1993, 1994). As a consequence, sequences that encode biological functions can be introduced into the scaffold of the coiled-coil structural units. This structural robustness enabled Huang et al. to implant the RGD domain between two hexa-heptad sequences (A2) derived from a previously published leucine zipper (LZ) coiled-coil peptide. The RGD domain was separated from each A2 sequence by a nine amino acid spacer (C1). A C-terminal tail (C2), containing cysteine, was also added, completing an RGD-containing system constructed as follows: A2-C1-RGDS-C1-A2-C2. The insertion of RGDS imbued cell adhesion to the hydrogel (Huang et al. 2014). Specifically, Huang et al. found that RGD sequences introduced into LZ hydrogels promoted the attachment (as determined via MTS cell proliferation assay kit, a colorimetric analysis which measures the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, or MTS) and proliferation of metabolically active cells at quantitatively higher levels than the control system: A2-C1-C1-A2-C2, which lacked the RGD domain. These results were corroborated in vivo using human marrow stem cells in a murine host system, which demonstrated cell attachment, growth and even neovascularity, thus proving that a mutable, tunable, coiled-coil-based hydrogel has potential for tissue engineering applications (Huang et al. 2014). This “customization” of a hydrogel is possible primarily due to the structural stability encoded within the coiled-coil LZ domain, and its ability to incorporate mutations near it without loss of its structure/function relationship.
Hybrid hydrogels are derived from self-assembly of synthetic polymers to which coiled-coil sequences have been covalently attached, usually at the termini, to afford ABA-type protein-polymer conjugates (Kopeček 2007). These hybrid materials combine the specificity of coiled-coil interactions with the well-known mechanical properties and biocompatibility of synthetic biomaterials. Kopecek and Yang (2012) linked N-(2-Hydroxypropyl) methacrylamide (HPMA) to (N’,N’-dicarboxymethylaminopropyl) methacrylamide (DAMA) to create a copolymer that served as the main polymer component of the hydrogel. The HPMA polymer, a hydrophilic molecule, promotes the water absorption and swelling of the hydrogel. DAMA, a metal-chelating agent, provided a mechanism to couple the polymer chain to the peptide moiety of the hydrogel via Ni2+ chelation between DAMA and a His-tag domain within the peptide. The HPMA-DAMA copolymer was conjugated to two differing coiled-coil-forming peptide sequences with differing lengths and amino acid compositions: CC1 and CC2. CC1 was derived from a portion of the stalk domain of kinesin, adopting a tetrameric coiled-coil structure. In contrast, CC2 was designed by the Kopeček group as a tetrameric coiled-coil, but comprised a shorter sequence than CC1. Importantly, it was observed that CC2 exhibited greater thermostability than CC1. These investigators hypothesized that differences in amino acid sequence between the two coiled-coil motifs (CC1 and CC2) would produce measurable effects in subsequent downstream biophysical properties. The difference in sequence between CC1 and CC2 influences the thermodynamic stability of the resulting coiled-coils, which, in turn, would affect the properties of the resultant hydrogel. For example, the Tm of a coiled-coil could be raised or lowered through sequence control, which enabled the formation of hydrogels of varying thermostability. When subjected to a temperature increase from 25° to 70 °C, the CC1-based hydrogel volume decreased tenfold (Wang et al. 1999). The mid-point temperature of the gel-sol transition coincided with the Tm of the constituent coiled-coil that was determined from its CD melting transition. Conversely, CC2 did not exhibit a hydrogel volume transition within the same temperature range (Wang et al. 1999). Kopeček asserted that this data indicated that “properties of a well-defined coiled-coil protein motif can be imposed onto a hybrid hydrogel” (Kopeček 2007).
In the pursuit of enhanced stability and a dynamic mechanical response, Yao et al. created a hybrid hydrogel that incorporated reversible physical cross-links and photo-polymerizable groups (Yao et al. 2014). A reactive cysteine residue was introduced into the sequence of a coiled-coil derived from the five-helix bundle of cartilage oligomeric matrix protein (COMP). The COMP peptide was conjugated to a macromer of polyethylene glycol diacrylate (PEGDA) through Michael addition to one of the terminal acrylate groups. The resultant pentameric assembly contained PEG chains that were terminally functionalized with the remaining acrylate group. Photo-induced polymerization of the terminal acrylates afforded a network in which the pentameric coiled-coil acted as a physical cross-link between polymer chains. This process has the advantage of retaining the dynamic physical cross-links associated with the coiled-coil motif, while permitting rapid in situ fabrication of the hydrogel using photo-induced polymerization (Yao et al. 2014). Fibroblast cells could be encapsulated within a 3D hydrogel matrix, which was generated in situ via photo-polymerization of an RDG-modified peptide-macromer. The reversibility of the coiled-coil self-association enabled self-healing behaviour within the hydrogel, which promoted cell spreading and migration in 3D culture (Yao et al. 2014). These hydrogel systems demonstrate a key advantage in the use of coiled-coils as structural units within self-assembling biomaterials, namely the potential for customization of the selectivity and stability of the interaction.
In the cases described above, the coiled-coil was employed primarily as a bio-recognition motif in order to create the virtual cross-links that defined the hydrogel. The hydrophilic central block provided the flexibility to accommodate the swelling solvent. However, Woolfson and co-workers demonstrated hydrogels could be created solely from α-helical peptide sequences, which they termed hydrogelating self-assembling fibres (hSAFs) (Banwell et al. 2009). The hSAF system is based on previous work performed by the Woolfson group, wherein they engineered self-assembling fibres (SAFs) via two α-helices co-assembling such that the resultant structure was a dimer of the two helices. Importantly, this dimer featured sticky ends, and resultant end-to-end assembly produced coiled-coil fibrils, which in turn aggregated to form large fibres. It was observed that these fibres demonstrated a high level of order, leading to their precipitation from solution (Banwell et al. 2009). Using the SAF as a starting scaffold, the researchers inserted alanine residues (to encourage hydrophobic interactions between fibrils) and glutamine residues (to promote hydrogen bonding). These mutations were performed only at the b, c, and f positions of the heptad repeat, as these lie on the exposed surface of the helix, while other residue positions are more critical in driving dimerization of the two constituent helices to form a coiled-coil. Differing levels of alanine and glutamine were introduced at the aforementioned positions to form hydrogels driven largely by hydrophobic interactions between fibrils or driven by hydrogen bonding. It was observed that the hydrophobically-driven hydrogels (those featuring proportionally large amounts of alanine at the variable positions) showed an increase in gel strength when heated. Conversely, the hydrogel variants driven primarily by hydrogen bonding (those featuring proportionally large levels of glutamine at the variable positions) melted and lost their gel strength upon heating. A distinct advantage of this system is that each constituent helix has its own sequence identity, and hydrogelation only occurs upon mixing. Lastly, Woolfson and co-workers showed that these hSAF systems are compatible with, and support both cell growth and cell differentiation. A recurring theme regarding coiled-coil biomaterials is the flexibility of this protein fold as a structural platform. Mehrban et al. demonstrated that these fibrillar coiled-coils could be decorated with the RGDS cell-adhesive sequence. The resultant hydrogels could promote the growth, directional migration, and differentiation of murine embryonic neural stem cells in culture (Banwell et al. 2009; Mehrban et al. 2015).
17.4 Immunogenic Coiled-Coil Assemblies
An important factor to consider in the development of biomaterials for in vivo applications is immunogenic response. For tissue engineering applications, minimizing the immune response to the coiled-coil construct facilitates integration into the body with minimal risk of rejection. In contrast, immunotherapies rely on a strong immune response to the coiled-coil material (Boato et al. 2007). It follows that it is of great interest to not only measure the immunogenic potential of coiled-coil assemblies under physiologically relevant conditions for in vivo applications, but also to design systems in which immunological response can be controlled.
For a biomaterial to provoke an immune response, it must interact with the immune system either by being recognized as a foreign body or by breaking down the native B-cell tolerance (if it is sufficiently homologous to a protein in the host organism). In both cases, aggregated (Maas et al. 2007) or oligomerized (Rosenberg 2008) proteins have been shown to be more immunogenic than their monomeric counterparts. Biomaterials that do not provoke an immune response are invisible to the host cell and function freely within the cell. Because coiled-coil systems can access a variety of oligomeric states, they are ideal for tuning an immune response.
Recently the Collier group explored the effect of PEGylation on the immunogenicity of coiled-coil assemblies. PEGylation generally reduces immune response, while aggregation increases the immune response (Rudra et al. 2010a). The interesting question then, is how strong will the immune response be when a PEGylated peptide aggregates? To address this question, they investigated the murine immune response to three peptides: a domain from mouse fibrinogen (which disfavours coiled-coil formation due to poor charge pairings and buried polar residues), a mutated version of the same with a coiled-coil optimized sequence (γKEI), and a triblock peptide-polymer conjugate (γKEI-PEG-γKEI) (Rudra et al. 2010b). They found that the native peptide and its coiled-coil derivative elicited no immune response, while the triblock peptide copolymer caused a mild immune response. They attributed this change in immune response to the higher degree of oligomerization observed in the triblock copolymer, as determined from analysis using analytical ultracentrifugation (AUC). These results suggested that the immunogenicity of the peptide was not based on the folding, as the native random coil peptide and the coiled-coil forming mutant elicited similarly low immune responses.
17.5 Coiled-Coil Based Nanotubes, Fibrils, and Fibres
One-dimensional assemblies derived from proteins and peptides have been the focus of significant scientific research over the last two decades (Scanlon and Aggeli 2008). A significant motivation for this research stems from the presence of extended helical assemblies in native biological systems, in which they display a variety of functional roles that would be desirable to replicate within synthetic biomaterials. The knobs-into-holes packing typically associated with coiled-coils has been observed to play a significant role in stabilizing the structures of helical assemblies such as intermediate filaments (Chernyatina et al. 2015), type IV pili (Craig et al. 2006), bacterial flagella (Yonekura et al. 2003), the type III secretion system needle complex (Loquet et al. 2012), and filamentous phage capsids (Morag et al. 2015). Significant research effort has been directed toward the de novo design of helical assemblies based on coiled-coil structural motifs, which could be employed for the creation of synthetic scaffolds for biomedical applications (see above, for example). The primary approach of this research focused on design of sticky-ended coiled-coils that could oligomerize to form infinite one-dimensional polymers through non-covalent interactions. The staggered alignment of helices was promoted through introduction of electrostatic interaction between helical protomers that enforced an out-of-register alignment across the helical interface. However, most designs focused on coiled-coils of lower oligomerization state (usually dimers and trimers), primarily due to the difficulty in controlling alignment between adjacent helices for coiled-coils in larger helical bundles. In addition, at that juncture, limited structural information was available on coiled-coils of higher oligomerization state. One attractive feature of the latter systems is that coiled-coils of oligomerization state ≥5 helices display an interior channel in which the diameter depends on the bundle size. Self-assembly of coiled-coils of higher oligomerization state affords the potential to create nanotube structures in which the channel residues can be tailored for encapsulation of small molecules and the outer surface can be functionalized to enhance applications such as selective targeting for controlled release.
Numerous strategies have been described to drive the formation of tubular assemblies from peptides and proteins, though few of these approaches are applicable to coiled-coil systems (Burgess et al. 2015). The most promising approach to nanotube fabrication involves the end-to-end stacking of coiled-coils of defined oligomerization state (n ≥ 5) into extended structures through selective introduction of attractive non-covalent interactions at the interfaces between subunits. As coiled-coils are perhaps the best understood protein fold, with a wealth of available structural information, they represent a promising test bed to develop methods to reliably generate structurally-defined supramolecular assemblies.
Xu et al. (2013) described an approach to promote the end-to-end self-assembly of a seven-helix bundle into a helical nanotube. These researchers employed a structurally characterized 7-helix bundle, GCN4-pAA, as the starting point for the design. In this case, the 7-helix bundle has helical symmetry, in that successive peptides in the assembly are axially translated by an increment of one residue (1.43 Å rise per residue for the superhelix) between successive helices in the bundle. This translation results in a registry shift corresponding to a heptad repeat between the first and last helix in the bundle. The corresponding edges at the N- and C-termini are solvent-exposed and, upon interaction, could bury sufficient hydrophobic surface area to promote stacking of the 7-helix bundles. Xu et al. (2013) used the analogy of a helical lock washer to describe the structure of the coiled-coil subunits. The sequence of GCN4-pAA was redesigned to afford a fibrillogenic peptide sequence, 7HSAP1. This sequence preserved the residues that were critical for formation of the 7-helix bundle, but introduced features that would promote stacking of the subunits (Xu et al. 2013). Hydrophobically-driven association of complementary helix faces yielded a non-covalent assembly of helices with an inner lumen. In addition, the N-terminus and C-terminus of 7HSAP1 were populated with positively charged residues and negatively charged residues, respectively. This design promoted charge complementary association of helical heptamers through head-to-tail stacking of the heptameric subunits. The resultant nanotubes defined an internal cavity of approximately 7 Å in diameter, in analogy with the crystal structure of GCN4-pAA, which was capable of binding shape-appropriate small molecules such as the solvatochromic fluorophore PRODAN (Xu et al. 2013).
Hume et al. constructed a fibrillar assembly using the pentameric coiled-coil region of cartilage oligomeric matrix protein (COMPcc) as the basis for design. A variant of COMPcc was constructed in which residues that were non-essential for pentameric assembly were deleted (Hume et al. 2014). An N-terminal pocket, inherent to the wild type matrix protein and consisting of three leucines and a valine, was shifted with respect to the overall sequence, but preserved in identity and conserved with respect to its occurrence within the heptad repeat sequence. The presence of the N-terminal pocket was necessary to retain the preference for the pentameric assembly (over alternative oligomeric states or lack of assembly). Moreover, in the re-designed peptide, Q, charge density was introduced at positions on the outer (lateral) surface of the assembly in order to promote complementary interactions between oppositely charged regions on adjacent pentameric assemblies. Fibre formation would be promoted through head-to-tail stacking of helical bundles, which would be reinforced through lateral association between fibrils mediated through electrostatic complementarity. Peptide Q was found to form nano-fibres having diameters as large as half a micron (Hume et al. 2014). The pentameric assembly also defined a central pore of sufficient size to accommodate small molecules, such as the therapeutic agent curcumin. Curcumin was demonstrated not only to bind to the fibrillar assemblies, but also to promote further lateral association through a mechanism that has yet to be fully elucidated. Using this approach, Hume et al. succeeded in engineering the first documented experimentally-assembled protein-based microfibre, observing diameters of up to sixteen microns via aggregation of nano-scale moieties, while preserving a central cavity for encapsulation (Hume et al. 2014).
While larger coiled-coil bundles (>7 subunits) have been observed in native protein assemblies, the de novo design of larger diameter coiled-coils remains a challenge, which has thus far limited the fabrication of nanotubes with larger lumens. Walshaw and Woolfson (Walshaw and Woolfson 2001) described general principles for design of these types of assemblies based on an analysis of the 12-helix bundle observed in the structure of the tolC homotrimer (Koronakis et al. 2000). However, such large diameter tubular structures based on coiled-coils have yet to be realized. Egelman et al. (2015) investigated the self-assembly of a pair of de novo designed coiled-coils based on the structural principles proposed by Walshaw and Woolfson. The sequences of these two peptides were based on a canonical heptad repeat motif in which hydrophobic residues populate the a/d and c/f positions, producing hydrophobic seams along the helix offset by two residue spacing (Koronakis et al. 2000). In contrast to the α-helical cylinder of the tolC sequence (Calladine et al. 2001) hydrophobic residues at the outer a and f positions were substituted with alanines, which feature smaller sidechains than the residues of the native tolC. This substitution served to decrease steric interference between the flanking residues of the two hydrophobic pockets to encourage larger tube diameter (Egelman et al. 2015). Large sidechains at the outer positions would create steric interactions between neighbouring residues, and in the relaxation and mitigation of this steric stress, force the tube to constrict towards a smaller diameter. This was negated in the synthetic peptide via mutations to alanine at the aforementioned heptad positions a and f to encourage large diameter growth. Moreover, complementary pairs of charged residues populate the b and e positions in the synthetic peptide to promote antitypic helix-helix association (Egelman et al. 2015). The sole difference between the two sequences was the substitution of four arginine residues in the first peptide (Form I) with four lysine residues in the second peptide (Form II). Both peptides formed extended tubular assemblies in aqueous buffer, but differed significantly in diameter (6 nm for the Form I assemblies and 12 nm for the Form II assemblies).
Electron cryomicroscopy with direct electron detection was employed for the structural analysis of the Form I and Form II assemblies. Egelman et al. (2015) funnelled data through the iterative helical real space reconstruction algorithm (Egelman 2010) to produce near atomic resolution structural data for Forms I and II. Form I features a unilaminar helical motif, wherein helices are packed nearly perpendicular to the nanotube axis in which the cross-section displays near four-fold symmetry in the left-handed one-start helix. The Form II assemblies feature a bilaminar helical motif, wherein three bilayer stacks of helices are packed together and define a nearly circular cross-section in which the helices are slightly inclined with respect to the cross-sectional plane. In either structure, the nanotube lumen and outer surface are lined with functional groups from polar amino acid residues. One might envision that the inner and outer surfaces of the tubes could be selectively functionalized to promote applications in controlled encapsulation and release.
17.6 Coiled-Coil Composites
This review has largely focused largely on developments related to the potential of coiled-coils for applications in biomedicine, and, specifically, their interactions with other biological materials in vitro or in vivo. Recently, however, significant research has been focused on integrating inorganic materials with coiled-coil biomaterials. In particular, the use of coiled-coils has been proposed for applications involving the templating of inorganic materials. For example, numerous studies have used genetically engineered viral capsids for directing the deposition of inorganic materials, leading to the formation of highly ordered inorganic structures (Lee et al. 2002; Huang et al. 2005). These strategies have produced very efficient electron-capture devices (Dang et al. 2011), as well as highly functioning lithium batteries (Lee et al. 2009). While using a phage capsid is an elegant solution to the selection problem, a coiled-coil scaffold would be preferable due to its ease of production, robust structure, and well-known design rules. Several examples have been recently published in which coiled-coils, or assemblies derived from their self-association, have been employed to direct nanoparticle synthesis and assembly (Ryadnov 2006; Slocik et al. 2007; Wagner et al. 2009; Hume et al. 2015; Abram et al. 2016). These materials might ultimately find use as imaging agents or therapeutics in medical applications, especially as they could leverage the favourable electronic, optical, or magnetic properties of the inorganic nanoparticle and the structural specificity of the coiled-coil interaction (Seo et al. 2015).
Recent technological advances, such as structurally predictive computational algorithms and higher resolution characterization techniques, are expanding our ability to design and structurally characterize novel coiled-coil assemblies. The availability of this information has facilitated the development of novel biomaterials scaffolds based on coiled-coils. The natural abundance and structural diversity of coiled-coils, as well as their well-understood design rules, make them an obvious choice for biomaterials scaffolding. In addition, the versatility of the coiled-coil motif allows for the incorporation of artificial amino acids, which enhances the ability to do site-specific chemistry and generate more complex materials. As a result, coiled-coils have been used to develop a variety of structures, including hydrogels, nanotubes, nanosheets, and even composite materials with synthetic polymers or inorganic nanoparticles. Given the ever-increasing need for tailored materials, coiled-coils are emerging as a highly modular scaffold capable of a wide variety of functions.
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