Design, synthesis, and characterization of protein origami based on self-assembly of a brick and staple artificial protein pair

Abstract

A versatile strategy to create an inducible protein assembly with predefined geometry is demonstrated. The assembly is triggered by a binding protein that staples two identical protein bricks together in a predictable spatial conformation. The brick and staple proteins are designed for mutual directional affinity and engineered by directed evolution from a synthetic modular repeat protein library. As a proof of concept, this article reports on the spontaneous, extremely fast and quantitative self-assembly of two designed alpha-repeat (αRep) brick and staple proteins into macroscopic tubular superhelices at room temperature. Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM with staining agent and cryoTEM) elucidate the resulting superhelical arrangement that precisely matches the a priori intended 3D assembly. The highly ordered, macroscopic biomolecular construction sustains temperatures as high as 75 °C thanks to the robust αRep building blocks. Since the α-helices of the brick and staple proteins are highly programmable, their design allows encoding the geometry and chemical surfaces of the final supramolecular protein architecture. This work opens routes toward the design and fabrication of multiscale protein origami with arbitrarily programmed shapes and chemical functions.

Engineering of brick and staple components for ordered assembly of synthetic repeat proteins

“…The nanotubular structures appear aligned into highly ordered crystals similar to the fully indexed crystals obtained with Brick B (Moreaud et al, 2023).…”

“…The original Brick B design, described in (Moreaud et al, 2023), has been improved by introducing new H-Bonds and salt bridges at the Brick/Brick interface between the last repeat of one Brick protein and the first repeat of the next one in the assembly. This was done by substituting positions 22, 23, 26 and 30 of the first and last repeat of the cleaved brick protein.…”

“…We have recently described a new type of self-assembling protein structure based on ɑReps (Moreaud et al, 2023). ɑReps are a family of artificial proteins based on HEAT-like repeat that fold as curved solenoids (Urvoas et al, 2010).…”

Precisely patterned nanofibres made from extendable protein multiplexes

“…The top and bottom surfaces of the CHR monomers are primarily hydrophilic residues in the bounded multiplexes presented in previous sections. We attempted to replace these with the hydrophobic residues found on the core repeats, but most of these ‘uncapped’ designs did not express in E. coli , likely due to the increased hydrophobicity and lack of specificity in forming the intended fibre geometry 21 .…”

Designing Multifunctional Biomaterials via Protein Self‐Assembly

“…Minard's group [52] showed a universal strategy for creating an inducible protein assembly with a predefined geometry. This work paves the way for the design and manufacture of multi‐scale protein origami with arbitrarily programmed shapes and chemical functions.…”