A modular protein engineering toolkit

Natural proteins perform an amazing repertoire of functions. A key goal of synthetic biology technology development is to design new functional proteins that are not derived from natural sequences. Protein design is a young field, beginning with DNA and peptide synthesis technologies. The first designed proteins were four-helix bundles, surprisingly stable, but not functional. Protein design is not yet routine, and has several limitations. Most successful design methods use a potential energy optimisation approach in an all-atom forcefield, which is computationally expensive and therefore limited to smaller domains. In contrast, the highly regular and predictable structure of DNA allows large "origami" objects to be predictably designed and constructed.

Here, we propose a modular protein construction kit that uses regular and extendable backbone scaffolds, but can have smaller computationally designed functional domains inserted with atomic precision. Such a system would have some of the composability and designability of highly regular DNA origami, with all of the advantages of computational protein design. The project begins with synthetic beta-solenoid proteins in the RFR family. This family has a 5 residue repeat, consensus ADLSG. Four of these repeats form a near-square and these squares stack to form the solenoid. The A and L residues of the repeat form the hydrophobic core, and there is a great deal of freedom for the other residues which form the surface. The solenoid core is about the same width as DNA, but much more chemically versatile.

We have made synthetic solenoid proteins of variable lengths with repeat sequences drawn from a residue frequency table. Into these solenoids we have inserted computationally designed loops, using a novel hierarchical fragment-free technique, and entire protein domains (MacDonald et al. 2016). This work defined an extendable protein scaffold part, which we now wish to extend into a multi-part modular protein design toolkit. We will extend the scaffold with functional loops, and design interfaces to enable the modular building of higher order structures. The project will involve computational protein design, protein characterization and structural biology.