[show abstract][hide abstract] ABSTRACT: Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address difficulties in studying and understanding natural systems, and to provide routes to new biomaterials with potential applications in nanotechnology and medicine. We have designed a self-assembling fiber system, the SAFs, in which two small α-helical peptides are programmed to form a dimeric coiled coil and assemble in a controlled manner. The resulting fibers are tens of nm wide and tens of μm long, and, therefore, comprise millions of peptides to give gigadalton supramolecular structures. Here, we describe the structure of the SAFs determined to approximately 8 Å resolution using cryotransmission electron microscopy. Individual micrographs show clear ultrastructure that allowed direct interpretation of the packing of individual α-helices within the fibers, and the construction of a 3D electron density map. Furthermore, a model was derived using the cryotransmission electron microscopy data and side chains taken from a 2.3 Å X-ray crystal structure of a peptide building block incapable of forming fibers. This was validated using single-particle analysis techniques, and was stable in prolonged molecular-dynamics simulation, confirming its structural viability. The level of self-assembly and self-organization in the SAFs is unprecedented for a designed peptide-based material, particularly for a system of considerably reduced complexity compared with natural proteins. This structural insight is a unique high-resolution description of how α-helical fibrils pack into larger protein fibers, and provides a basis for the design and engineering of future biomaterials.
Proceedings of the National Academy of Sciences 07/2012; 109(33):13266-71. · 9.74 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: α-Helical coiled coils are ubiquitous protein-protein-interaction domains. They share a relatively straightforward sequence repeat, which directs the folding and assembly of amphipathic α-helices. The helices can combine in a number of oligomerisation states and topologies to direct a wide variety of protein assemblies. Although in nature parallel dimers, trimers and tetramers dominate, the potential to form larger oligomers and more-complex assemblies has long been recognised. In particular, complexes above pentamer are interesting because they are barrel-like, having central channels or pores with well-defined dimensions and chemistry. Recent empirical and rational design experiments are beginning to chart this potential new territory in coiled-coil space, leading to intriguing new structures, and possibilities for functionalisation and applications.
Current Opinion in Structural Biology 03/2012; 22(4):432-41. · 8.74 Impact Factor
[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. · 12.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ability to modify peptide- and protein-based biomaterials selectively under mild conditions and in aqueous buffers is essential to the development of certain areas of bionanotechnology, tissue engineering and synthetic biology. Here we show that Self-Assembling peptide Fibers (SAFs) can incorporate multiple modified peptides non-covalently, stoichiometrically and without disrupting their structure or stability. The modified peptides contain groups suitable for post-assembly click reactions in water, namely azides and alkenes. Labeling of these groups is achieved using the orthogonal Cu(I)-catalyzed azide-alkyne and photoinitiated thiol-ene reactions, respectively. Functionalization is demonstrated through the conjugation of biotin followed by streptavidin-nanogold particles, or rhodamine, and visualized by electron and light microscopy, respectively. This has been shown for fibers harboring either or both of the modified peptides. Furthermore, the amounts of each modified peptide in the fibers can be varied with concomitant changes in decoration. This approach allows the design and assembly of fibers with multiple functional components, paving the way for the development of multi-component functionalized systems.
[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.
[show abstract][hide abstract] ABSTRACT: One possible route to develop new synthetic-biological systems is to assemble discrete nanoscale objects from programmed peptide-based building blocks. We describe an algorithm to design such blocks based on the coiled-coil protein-folding motif. The success of the algorithm is demonstrated by the production of six peptides that form three target parallel, blunted-ended heterodimers in preference to any of the other promiscuous pairings and alternate configurations, for example, homodimers, sticky-ended assemblies, and antiparallel arrangements. The peptides were linked to promote the assembly of larger, defined nanoscale rods, thus demonstrating that targeted peptide-peptide interactions can be specified in complex mixtures.
Journal of the American Chemical Society 01/2009; 131(3):928-30. · 10.68 Impact Factor