Aimee L. Boyle

Self-assembling cages from coiled-coil peptide modules

From Coils to Cages Self-assembly strategies that mimic protein assembly, such as the formation of viral coats, often begin with simpler peptide assemblies. Fletcher et al. (p. 595, published online 11 April; see the Perspective by Ardejani and Orner) designed two coiled-coil peptide motifs, a heterodimer, and a homotrimer. Both peptides contained cysteine residues and could link through disulfide bonds, so that the trimer could form the vertices of a hexagonal network and the dimer its edges. However, these components are flexible and, rather than form extended sheets, they closed to form particles ∼100 nanometers in diameter. Hexagonal networks form from heterodimeric and homotrimeric coiled coils and create ~100-nanometer-diameter cages. [Also see Perspective by Ardejani and Orner] An ability to mimic the boundaries of biological compartments would improve our understanding of self-assembly and provide routes to new materials for the delivery of drugs and biologicals and the development of protocells. We show that short designed peptides can be combined to form unilamellar spheres approximately 100 nanometers in diameter. The design comprises two, noncovalent, heterodimeric and homotrimeric coiled-coil bundles. These are joined back to back to render two complementary hubs, which when mixed form hexagonal networks that close to form cages. This design strategy offers control over chemistry, self-assembly, reversibility, and size of such particles.

A de novo peptide hexamer with a mutable channel

The design of new proteins that expand the repertoire of natural protein structures represents a formidable challenge. Success in this area would increase understanding of protein structure, and present new scaffolds that could be exploited in biotechnology and synthetic biology. Here we describe the design, characterisation and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, 6-helix bundle with a channel running through it. Although lined exclusively by hydrophobic leucine and isoleucine side chains, the 6 Å channel is permeable to water. One layer of leucine residues within the channel is mutable accepting polar aspartic acid (Asp) and histidine (His) side chains, and leading to subdivision and organization of solvent within the lumen. Moreover, these mutants can be combined to form a stable and unique (Asp-His)3 heterohexamer. These new structures provide a basis for engineering de novo proteins with new functions.

De novo designed peptides for biological applications

In recent years our ability to design and assemble peptide-based materials and objects de novo (i.e. from first principles) has improved considerably. This brings us to a point where the resulting assemblies are quite sophisticated and amenable to engineering in new functions. Whilst such systems could be used in a variety of ways, biological applications are of particular interest because of the demand for biocompatible, readily produced systems with potential as drug-delivery agents, components of biosensors and scaffolds for 3D cell and tissue culture. This tutorial review describes the building blocks (or tectons) that are being used in peptide assembly, highlights a range of materials and objects that have been produced, notably hydrogels and virus-like particles, and introduces a number of potential applications for the designs.

A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology

Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.

A Set of de Novo Designed Parallel Heterodimeric Coiled Coils with Quantified Dissociation Constants in the Micromolar to Sub-nanomolar Regime

The availability of peptide and protein components that fold to defined structures with tailored stabilities would facilitate rational protein engineering and synthetic biology. We have begun to generate a toolkit of such components based on de novo designed coiled-coil peptides that mediate protein-protein interactions. Here, we present a set of coiled-coil heterodimers to add to the toolkit. The lengths of the coiled-coil regions are 21, 24, or 28 residues, which deliver dissociation constants in the micromolar to sub-nanomolar range. In addition, comparison of two related series of peptides highlights the need for including polar residues within the hydrophobic interfaces, both to specify the dimer state over alternatives and to fine-tune the dissociation constants.

‘Pincer’ pyridine dicarbene complexes of nickel and their derivatives. Unusual ring opening of a coordinated imidazol-2-ylidene

The reaction of NiBr2(DME), DME = 1,2-dimethoxyethane, with the pincer pyridine dicarbene ligands (C-N-C) ( 2) and (C-NMe-C) ( 2Me), (C-N-C = 2,6-bis-[(DiPP)imidazol-2-ylidene]pyridine, C-NMe-C = 2,6-bis-[(DiPP)imidazol-2-ylidene]-3,5-dimethylpyridine, DiPP = 2,6-diisopropylphenyl) gave the square planar complexes [Ni(C-N(Me)-C)Br]Br, 3.( Br)- and 3Me.( Br)- respectively. Transmetallation from [(C-NMe-C)2Ag2](Ag6I8), 6Me.( Ag6 I8)2- to NiBr2(DME) gave [Ni(C-NMe-C)Br](AgI2), 3Me.( AgI2)-. Reaction of 3.( Br)- with KPF6 resulted only in exchange of the ionic bromide, however the reaction of 3.( Br)- with AgBF4 in MeCN or AgOTf in THF resulted in the exchange of both coordinated and ionic bromides, giving rise to the square planar 4.( BF4)-2 and octahedral 5, respectively. In contrast, the reaction of 3Me.( AgI2)-, with excess AgOTf resulted in an unusual reverse transmetallation leading to 6Me.( OTf)-. The substitution of tmeda in Ni(CH3)2(tmeda), tmeda = N,N,N,N-tetramethylethylenediamine, by 2 produced the complex 7, in which ring opening of the heterocyclic imidazole ring of one of the NHC functional groups has taken place.

Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.

The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo.

A Helix Swapping Study of Two Protein Cages

Protein cages have been the focus of studies across multiple scientific disciplines. They have been used to deliver drugs, as templates for nanostructured materials, as substrates in the development of bio-orthogonal chemistry, and to restrict diffusion to study spatially confined reactions. Although their monomers fold into four-helix bundle structures, two cage proteins, DPS and BFR, self-assemble to form a 12-mer with tetrahedral symmetry and an octahedrally symmetric 24-mer, respectively. These monomers share strong similarities of both sequence and tertiary structure. However, they differ in the presence of a short additional helix. In BFR, the fifth helix is at the C-terminus and is positioned along the 4-fold symmetry axis, whereas with DPS, an extra helix helps to define the 2-fold axis in the cage and is located between the second and third helices in the monomer bundle. In an attempt to investigate if these short helices govern protein assembly, mutants were designed and produced that delete and swap these minidomains. All mutants form highly helical structures that unfold cooperatively as evidenced by thermal melting followed by circular dichroism. Dynamic light scattering, size exclusion chromatography, and sedimentation equilibrium experiments demonstrated that although many of the BFR mutants do not self-assemble and form lower-order complexes, many DPS mutants could form cages despite their unnatural design. Taken together, our data indicate that the BC helix is less important than the E helix for overall cage self-assembly, suggesting that dimerization may not play a role in nanostructure formation that is as key as previously assumed. Additionally, we found that fusing the minidomain from BFR onto DPS results in a mutant that assembles into a homogeneous population of a novel protein oligomer. This assembled cage while still formed from 12 subunits is larger in overall shape than that of the native protein.

Rational design of peptide-based building blocks for nanoscience and synthetic biology

The rational design of peptides that fold to form discrete nanoscale objects, and/ or self-assemble into nanostructured materials is an exciting challenge. Such efforts test and extend our understanding of sequence-to-structure relationships in proteins, and potentially provide materials for applications in bionanotechnology. Over the past decade or so, rules for the folding and assembly of one particular protein-structure motif--the alpha-helical coiled coil have advanced sufficiently to allow the confident design of novel peptides that fold to prescribed structures. Coiled coils are based on interacting alpha-helices, and guide and cement many protein-protein interactions in nature. As such, they present excellent starting points for building complex objects and materials that span the nano-to-micron scales from the bottom up. Along with others, we have translated and extended our understanding of coiled-coil folding and assembly to develop novel peptide-based biomaterials. Herein, we outline briefly the rules for the folding and assembly of coiled-coil motifs, and describe how we have used them in de novo design of discrete nanoscale objects and soft synthetic biomaterials. Moreover, we describe how the approach can be extended to other small, independently folded protein motifs--such as zinc fingers and EF-hands--that could be incorporated into more complex, multi-component synthetic systems and new hybrid and responsive biomaterials.

Designed coiled coils promote folding of a recombinant bacterial collagen.

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.