De novo design of structural and functional extensions of the SynRFR beta-solenoid scaffold

Protein design is a rapidly developing field, with potentially substantial future benefits for humanity. De novo protein design provides an opportunity to test and improve our understanding of fundamental protein biophysics and biochemistry, such as protein folding and the sequence-structure-function relationship, whilst generating useful catalysts, vaccines and nanomaterials not found in nature. Both novel structures and functions must be computationally explored to expand the man-made proteome. The routine production of novel designs has been made possible by the improved performance of software packages, such as Rosetta, and an increase in computing power, enabling more researchers to join the protein design effort. The beta-solenoid scaffold SynRFR, designed in the Murray group (MacDonald et al, 2016) is based on the pentapeptide repeat family of proteins. SynRFR can support both loop and whole-domain insertions, enabling easier design of functional components, whilst the repeating structure theoretically allows the incorporation of multiple such components on a single scaffold. The length of the SynRFR scaffold can also be altered, providing control over the number of functional modules that can be accommodated.
The project will consist of 3 parts:

1. De novo design of structural insertions to generate a miniprotein core. Miniproteins are defined as polypeptide chains smaller than 40 amino acids but with ordered and stable 3D structures. A beta-hairpin extension of the SynRFR scaffold has already been successfully designed (MacDonald et al, 2016), raising the possibility of assembling miniprotein-like extensions using two or more such insertions. For example, the beta hairpin could theoretically be expanded into a minimal beta sandwich through the design of an adjacent beta-hairpin, and then into a triple-hairpin beta-sandwich, not observed in nature. A further extension of interest is a helical hairpin.
2. Rational design of modified SynRFR scaffolds. Functionalisable SynRFR scaffolds will be designed through introduction of elements of the SpyCatcher/SpyTag system (Zakeri et al, 2012). In addition, the SynRFR24.1 variant will be split into two self-assembling components. Finally, if successful, the two designs will be combined into functionalisable self-assembling building blocks.
3. Bioinformatic analysis of beta-helical structures to inform an exploration of the 'limits' of the beta-solenoid. Comparative analysis of beta-helical structures from the PDB could provide general design principles for introducing deviations from ideality into the structure of the SynRFR scaffold such as a bend or the replacement of parts of the beta-solenoid with different secondary structure elements.