[Show abstract][Hide abstract] ABSTRACT: 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.
Chemical Science 02/2014; 5(2):507-514. DOI:10.1039/C3SC52019F · 9.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 μM - sub-nM 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.
Journal of the American Chemical Society 03/2013; 135(13). DOI:10.1021/ja312310g · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The type three secretion system (T3SS) is a large and complex protein nano-machine that many gram-negative pathogens employ to infect host cells. A key structure of this machine is a proteinaceous pore that inserts into the target membrane and forms a channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly formed from two large membrane proteins called translocators. Importantly, effective secretion and thus pore formation of the translocators depends on their binding to and being transported by small specialized chaperones after synthesis in the bacterial cytosol. Recent crystal structures have shown these chaperones are formed from modular tetratricopeptide repeats. However, each crystal structure produced different homodimeric structures, suggesting flexibility in their topology that may be of importance to function. Given the crucial role of the translocator chaperones, we investigated the conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis coupled with analytical centrifugation and equilibrium denaturations showed that LcrH is a weak and thermodynamically unstable dimer (K(D) ≈ 15 µM, ΔG = 7.4 kcalmol(-1)). The modular TPR structure of the dimer allows it to readily unfold in a non-cooperative manner to a one-third unfolded dimeric intermediate (ΔG = 1.7 kcalmol(-1)), before cooperatively unfolding to a monomeric denatured state (ΔG = 5.7 kcalmol(-1)). Thus under physiological conditions the chaperone is able to populate C-terminally unravelled partially folded states, whilst being held together by its dimeric interface. Such ability suggests a fly-casting mechanism as a route to binding their far larger translocator cargo.
[Show abstract][Hide abstract] ABSTRACT: The design of bioinspired nanostructures and materials of defined size and shape is challenging as it pushes our understanding of biomolecular assembly to its limits. In such endeavors, DNA is the current building block of choice because of its predictable and programmable self-assembly. The use of peptide- and protein-based systems, however, has potential advantages due to their more-varied chemistries, structures and functions, and the prospects for recombinant production through gene synthesis and expression. Here, we present the design and characterization of two complementary peptides programmed to form a parallel heterodimeric coiled coil, which we use as the building blocks for larger, supramolecular assemblies. To achieve the latter, the two peptides are joined via peptidic linkers of variable lengths to produce a range of assemblies, from flexible fibers of indefinite length, through large colloidal-scale assemblies, down to closed and discrete nanoscale objects of defined stoichiometry. We posit that the different modes of assembly reflect the interplay between steric constraints imposed by short linkers and the bulk of the helices, and entropic factors that favor the formation of many smaller objects as the linker length is increased. This approach, and the resulting linear and proteinogenic polypeptides, represents a new route for constructing complex peptide-based assemblies and biomaterials.
Journal of the American Chemical Society 08/2012; 134(37):15457-67. DOI:10.1021/ja3053943 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The de novo design of systems comprising peptides and proteins as building blocks presents exciting new challenges in the areas of self-assembly and supramolecular chemistry and biochemistry. A key issue is that the rules relating protein sequence-to-structure are not fully elucidated. However, through the use of rational, “bottom-up” approaches, systems can be constructed by the careful design and programming of peptidic tectons that use existing sequence-to-structure relationships and test new ones. Indeed, there has been considerable success in the design and development of effectively infinite, fibrous systems constructed from α-helical, β-structured or collagen-based tectons. Some designs are now well enough developed that they can be readily functionalized, leading to potential applications in bionanotechnology, synthetic biology, and tissue engineering.The design of discrete assemblies using similar approaches, however, is proving more challenging, with precise control of assembly being particularly difficult. As a result, there are fewer examples of such systems being reduced to practice.This chapter describes design rules and approaches exemplified with fibrous and discrete systems, as well as discussing routes to the functionalization of such assemblies and their potential applications.Keywords:peptide;protein;self-assembly;nanostructure;supramolecular;discrete;de novo design
[Show abstract][Hide abstract] ABSTRACT: 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, characterization and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, six-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 and histidine side chains, which leads 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.
Nature Chemical Biology 12/2011; 7(12):935-41. DOI:10.1038/nchembio.692 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Chemical Society Reviews 03/2011; 40(8):4295-306. DOI:10.1039/c0cs00152j · 33.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Solution-phase spectroscopy and mass spectrometry are used to probe interactions between divalent metal ions and a synthetic Cys(2)His(2) zinc-finger peptide (vCP1). Both methods provide the same order of binding affinity, zinc ≥ cobalt ≫ copper ≫ calcium. Collision-cross-section measurements show that both apo and holo forms are compact. This is corroborated by molecular-dynamics simulations.
Chemical Communications 01/2011; 47(1):412-4. DOI:10.1039/c0cc02445g · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.