Programmable self assembling functional Origami from designed protein – ProteOrigami

The objectives of the ProteOrigami project are to design, produce and characterize self-assembling supramolecular Protein Origami and to use this new platform to precisely organize optical nanomaterials in space. This experimental project gathers four partners with complementary expertise in protein engineering (I2BC, Orsay) , biophysical characterization (IPR, Rennes), electron microscopy and optical nanomaterials design and characterization of the hybrid protein-inorganic architectures (CEMES and CBI, Toulouse). Naturals proteins spontaneously form sophisticated architectures with essential biological functions. However designing self-assembling artificial proteins remains highly challenging and predominantly aimed at biological and medical applications.
ProteOrigami will use extremely stable artificial alphaRep proteins, created by I2BC, as elementary units. These proteins have a modular structure that facilitates new designs and are efficiently mass-produced by standard molecular biology methods. We propose a new concept of protein "brick & staple" whereby stable and soluble alphaRep brick proteins are assembled and ordered in a predefined 3D geometry by specific alphaRep staple proteins. The precise molecular recognition between brick and staple is engineered by directed evolution and drives the 3D supramolecular origami assembly. Preliminary results obtained by the four partners clearly demonstrate that this original and potentially general approach works efficiently.
A first work package is devoted to the design of a range of new origami building blocks in order to control the origami structure and morphology. Bricks with different sizes or altered geometries will be used to tune the origami shape and its helical periodicity. Stop modules will be designed to control the origami size. Orthogonal bricks and staples pairs will be generated to induce a sequentially ordered protein organization. Finally, multimeric proteins will be built as branching nodes to established 2D/3D origami networks. All origami architectures will be characterized by complementary structural methods (SAXS, TEM, cryoEM).
The second work package translates the precise spatial organization of proteins in origamis into the spatial ordering of functional optically active moieties ('cargo') and studies their structural and optical properties . Since the different elements (brick, staple, stop modules) are separately produced, they can be labelled by single and selective coupling reactions with a component to be placed in a predefined position. The regioselective origami decoration will be used to position a single or a spatially ordered ensemble of cargos such as fluorophores, emitting or plasmonic nanoparticles. We anticipate these hybrid nanoconstruction to exhibit synergistic properties when regularly ordered at the nanoscale (superradiance, lasing, coupled plasmon modes,…) or when brought into the near-field (i.e. within 1-10 nm distance) of each other (Purcell effect, FRET, plasmon-mediated energy transfer). Fluorescence, scattering and spectroscopic properties will be explored on bulk and individual objects.
A last work package exploits a new approach of alphaRep-templated growth of (111)-faceted plasmonic gold nanocrystals recently established by two ProteOrigami partners. Adding this interacting functionality to the origami bricks is possible as the self-assembly leaves their main affinity surface available. The objective is to grow the plasmonic structure inside or on one side of the origami while programming the position(s) of the emitter(s) at a predefined distance from the gold surface that maximizes the coupling between the emitting dipole and the plasmonic near-field modal landscape. In such a configuration, the protein origami will be the first reported strategy to allow a programmable relative placement of plasmonic and emitting nanostructures on a scalable template. The structural and nanooptical properties will be studied.