![circular statistics in biology. circular statistics in biology.](http://i.ytimg.com/vi/KCNiHsZVFXM/maxresdefault.jpg)
While these cTRP constructs have many favorable properties, we have previously noted that their ability to self-assemble from smaller subunits is compromised by relatively limited contacts and small surface areas that are involved in packing between repeats and subunits. cTRPs that display multiple binding domains can display significant avidity effects (due to multimeric display of those domains around the molecular surface) and have been used to create novel formulations of immunological stimulatory and signaling molecules that can be used for biotechnology applications such as therapeutic T-cell manufacture. The largest of these constructs was used to create symmetric protein nanoparticles that can incorporate a variety of functional protein via covalent attachment around their periphery 5. We have previously described the creation of an array of circular tandem repeat proteins (‘cTRPs’) that are constructed from repeated two-helix bundles and that display a wide range of sizes and symmetries 5, 13. Engineered TRPs display many of the same properties as naturally evolved repeat proteins, including relatively few constraints on their length and size, significant flexibility that allows them to sample a wide variety of curvature and shapes, and a propensity to exhibit high thermostability and solubility. Multiple types of computationally designed tandem repeat proteins (using either naturally existing or computationally designed motifs and topologies) have been described that form either extended linear shapes (in which the N- and C-termini are at opposite ends of the protein chain) 12 or closed circular shapes (in which the N- and C-termini contact with one another and thereby complete a closed protein ring) 5, 13, 14.
![circular statistics in biology. circular statistics in biology.](https://d3i71xaburhd42.cloudfront.net/29046c9c3480570dd4abc7f23aca756a3f0b4ddc/5-Figure2-1.png)
They are particularly amenable to de novo design via purely computational approaches, due to their highly modular architectures 8, 9. Tandem repeat proteins (‘TRPs’) contain modular units of repeated protein sequence and structure, that can be composed of a variety of structural motifs including α-helical bundles, β-sheets, or mixed topologies 8, 9, 10, 11. Whether they are created via de novo computational design or laboratory selections (or a combination of those two approaches), engineered proteins are now being used for a wide variety of biotechnology applications, including those that can benefit from new ligand binding proteins 2, 3, 4, novel immunological regulators 5 or targeted protein therapeutics 6, 7.
![circular statistics in biology. circular statistics in biology.](https://media.springernature.com/w500h319/springer-static/image/art%3A10.1038%2Fnrn3475/MediaObjects/41583_2013_Article_BFnrn3475_Fig1_HTML.jpg)
The discipline of protein engineering has been greatly facilitated by at least three significant recent advances: the development of accurate computational algorithms for de novo structural design, an enhanced ability to construct and screen large protein libraries, and increasingly efficient and affordable deep sequencing approaches. Protein engineering has enabled the creation of novel protein folds and assemblages, as well as the modification of existing protein molecules 1. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. Circular tandem repeat proteins (‘cTRPs’) are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures.