Bioorthogonal dual functionalization of self-assembling peptide fibers.

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.

More than just bare scaffolds: towards multi-component and decorated fibrous biomaterials.

We are entering a new phase in biomaterials research in which rational design is being used to produce functionalised materials tailored to specific applications. As is evident from this Themed Issue, there are now a number of distinct types of designed, self-assembling, fibrous biomaterials. Many of these are ripe for development and application for example as scaffolds for 3D cell culture and tissue engineering, and in templating inorganic materials. Whilst a number of groups are making headway towards such applications, there is a general challenge to translate a wealth of excellent basic research into materials with a genuine future in real-life applications. Amongst other contemporary aspects of this evolving research area, a key issue is that of decorating or functionalising what are mostly bare scaffolds. There are a number of hurdles to overcome to achieve effective and controlled labelling of the scaffolds, for instance: maintaining biocompatibility, i.e., by minimising covalent chemistry, or using milder bioconjugation methods; attaining specified levels of decoration, and, in particular, high and stoichiometric labelling; introducing orthogonality, such that two or more functions can be appended to the same scaffold; and, in relevant cases, maintaining the possibility for recombinant peptide/protein production. In this critical review, we present an overview of the different approaches to tackling these challenges largely for self-assembled, peptide-based fibrous systems. We review the field as it stands by placing work within general routes to fibre functionalisation; give worked examples on our own specific system, the SAFs; and explore the potential for future developments in the area. Our feeling is that by tackling the challenges of designing multi-component and functional biomaterials, as a community we stand to learn a great deal about self-assembling biomolecular systems more broadly, as well as, hopefully, delivering new materials that will be truly useful in biotechnology and biomedical applications (107 references).

The non-covalent decoration of self-assembling protein fibers.

The design of self-assembling fibers presents challenges in basic science, and has potential for developing materials for applications in areas such as tissue engineering. A contemporary issue in the field is the construction of multi-component, functionalized systems. Previously, we have developed peptide-based fibers, the SAF system, that comprises two complementary peptides, which affords considerable control over assembly and morphology. Here we present a straightforward route to functionalizing the SAFs with small molecules and, subsequently, other moieties. This is achieved via non-covalent recruitment of charged peptide tags, which offers advantages such as further control, reversibility, and future prospects for developing recombinant tags. We demonstrate the concept by appending fluorescent labels and biotin (and thence gold nanoparticles) to the peptides, and visualising the resulting decorated SAFs by light and electron microscopy. The peptide tags bind in the nm-mum range, and show specificity compared with control peptides, and for the SAFs over similar alpha-helix-based peptide fibers.

Assembly pathway of a designed alpha-helical protein fiber.

Interest in the design of peptide-based fibrous materials is growing because it opens possibilities to explore fundamental aspects of peptide self-assembly and to exploit the resulting structures--for example, as scaffolds for tissue engineering. Here we investigate the assembly pathway of self-assembling fibers, a rationally designed alpha-helical coiled-coil system comprising two peptides that assemble on mixing. The dimensions spanned by the peptides and final structures (nanometers to micrometers), and the timescale over which folding and assembly occur (seconds to hours), necessitate a multi-technique approach employing spectroscopy, analytical ultracentrifugation, electron and light microscopy, and protein design to produce a physical model. We show that fibers form via a nucleation and growth mechanism. The two peptides combine rapidly (in less than seconds) to form sticky ended, partly helical heterodimers. A lag phase follows, on the order of tens of minutes, and is concentration-dependent. The critical nucleus comprises six to eight partially folded dimers. Growth is then linear in dimers, and subsequent fiber growth occurs in hours through both elongation and thickening. At later times (several hours), fibers grow predominantly through elongation. This kinetic, biomolecular description of the folding-and-assembly process allows the self-assembling fiber system to be manipulated and controlled, which we demonstrate through seeding experiments to obtain different distributions of fiber lengths. This study and the resulting mechanism we propose provide a potential route to achieving temporal control of functional fibers with future applications in biotechnology and nanoscale science and technology.

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.