An ability to mimic the boundaries of biological compartments would improve our understanding of ... more 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.
Protein−carbohydrate interactions play pivotal roles in health and disease. However, defining and... more Protein−carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C−H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C−H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C−H bonds engage more often in CH−π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate−aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C−H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein−carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein−carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites.
The design of bioinspired nanostructures and materials of defined size and shape is challenging a... more 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.
The design of bioinspired nanostructures and materials of defined size and shape is challenging a... more 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.
The role of amino acid sequence in conformational switching observed in prions and proteins assoc... more The role of amino acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study ␣ to  conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an ␣؊helical leucine zipper and a ؊hairpin. The parent peptide, Template؊␣, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce ؊structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template؊␣T. Unlike the parent peptide, Template؊␣T underwent a heat-inducible switch to ؊structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilization of the native states facilitate transitions to amyloid, destabilization of the leucine-zipper form of Template؊␣T did not promote a transformation. Cross-linking the termini of the peptides compatible with the alternative ؊hairpin design, however, did promote the change. Furthermore, despite screening various conditions, only the internally cross-linked form of the parent, Tem-plate؊␣, peptide formed amyloid-like fibrils. These findings demonstrate that, in addition to general properties of the polypeptide backbone, specific residue placements that favor ؊structure promote amyloid formation.
Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medici... more Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs, and as supports for cell growth and tissue engineering 1 . Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials 2-4 . Here, we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks of fibrils melt on heating, whereas those formed through hydrophobic fibril-fibril interactions strengthen when warmed. The hSAFs are dual-peptide systems that gel only on mixing, which gives tight control over assembly 5 . These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.
The role of amino acid sequence in conformational switching observed in prions and proteins assoc... more The role of amino acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study alpha to beta conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an alpha-helical leucine zipper and a beta-hairpin. The parent peptide, Template-alpha, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce beta-structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template-alphaT. Unlike the parent peptide, Template-alphaT underwent a heat-inducible switch to beta-structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilization of the native states facilitate transitions ...
An ability to mimic the boundaries of biological compartments would improve our understanding of ... more 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.
Protein−carbohydrate interactions play pivotal roles in health and disease. However, defining and... more Protein−carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C−H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C−H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C−H bonds engage more often in CH−π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate−aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C−H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein−carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein−carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites.
The design of bioinspired nanostructures and materials of defined size and shape is challenging a... more 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.
The design of bioinspired nanostructures and materials of defined size and shape is challenging a... more 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.
The role of amino acid sequence in conformational switching observed in prions and proteins assoc... more The role of amino acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study ␣ to  conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an ␣؊helical leucine zipper and a ؊hairpin. The parent peptide, Template؊␣, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce ؊structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template؊␣T. Unlike the parent peptide, Template؊␣T underwent a heat-inducible switch to ؊structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilization of the native states facilitate transitions to amyloid, destabilization of the leucine-zipper form of Template؊␣T did not promote a transformation. Cross-linking the termini of the peptides compatible with the alternative ؊hairpin design, however, did promote the change. Furthermore, despite screening various conditions, only the internally cross-linked form of the parent, Tem-plate؊␣, peptide formed amyloid-like fibrils. These findings demonstrate that, in addition to general properties of the polypeptide backbone, specific residue placements that favor ؊structure promote amyloid formation.
Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medici... more Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs, and as supports for cell growth and tissue engineering 1 . Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials 2-4 . Here, we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks of fibrils melt on heating, whereas those formed through hydrophobic fibril-fibril interactions strengthen when warmed. The hSAFs are dual-peptide systems that gel only on mixing, which gives tight control over assembly 5 . These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.
The role of amino acid sequence in conformational switching observed in prions and proteins assoc... more The role of amino acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study alpha to beta conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an alpha-helical leucine zipper and a beta-hairpin. The parent peptide, Template-alpha, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce beta-structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template-alphaT. Unlike the parent peptide, Template-alphaT underwent a heat-inducible switch to beta-structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilization of the native states facilitate transitions ...
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