Biology subjects

Decarreau, J.

Publications and source records attributed to Decarreau, J..

12 recordsLinked to original sources

bioRxiv2026

Discovering and designing multistate protein assemblies

Evolved protein assemblies exploit multiple weak interfaces to package cargo, facilitate metabolism, and direct cell movement, often through architectures that depart from strict geometric symmetry. Designed protein assemblies can also break symmetry through conformational flexibility or strain-based defects, but have generally lacked the capacity for large-scale structural rearrangements characteristic of evolved systems. Here, we characterize the structures and biophysical properties of emergent protein assemblies and establish a strategy for designing quasisymmetric materials that switch between assembly states. Rather than relying on intrinsic flexibility, our approach programs multiple self-assembling interfaces onto rigid oligomers. Using multistate design and ensemble predictions, we generate assemblies in which protomers can adopt alternative designed interface modes, enabling identical amino acid sequences to form distinct configurations depending on assembly pathway and environmental conditions. As a proof of principle, we construct quasisymmetric capsids that transition into micron-scale 2D arrays in response to temperature. This work establishes a strategy for encoding alternative assembly geometries within a single protein sequence and provides a foundation for engineering materials with environmentally tunable properties.

biochemistry↗

bioRxiv2026

Programmable De Novo Design of Mesoporous Protein Crystal Frameworks

Three-dimensional protein crystals are ordered, porous macroscopic materials with potential applications in catalysis, biosensing, and biomedicine. However, most protein crystals are obtained by empirical screening, providing limited control over the lattice architecture, pore geometry or component composition that determine material function. Here, we present a modular strategy for the programmable design of highly porous, framework-like protein crystals using predefined protein-protein interactions. This strategy yielded over 30 distinct protein crystals, including single-component and multicomponent P213 and I213 lattices that grow to over 100 micrometers in size. Small-angle X-ray scattering and electron microscopy showed close agreement between experimental lattices and computational models. RFdiffusion-guided design generated isomorphous variants with matched lattice parameters, enabling coherent protein crystal alloys, epitaxial core-shell growth and reversible shell assembly. The designed crystals exhibit tunable mesoporous architectures, with limiting apertures of 2-18 nm, and support genetically encoded incorporation of fluorescent protein guests. These results establish a general route to programmable lattice engineering of protein crystals and position them as genetically encoded, compositionally tunable mesoporous materials.

synthetic biology↗

bioRxiv2025

De-novo design of a random protein walker

Molecular machines hold great potential. Design of static monomeric and oligomeric protein structures has advanced tremendously, but few dynamic protein systems have been designed. Here we present the design and characterization of a random protein walker that diffuses along a designed protein track. For the track, we designed micro-meter long fibres and developed a method to rigidly decorate them with arbitrary proteins. The walkers consist of homo-oligomers that reversibly bind the track using heterodimeric feet; we tested multiple heterodimer interfaces for reversibility and designed six walkers with different numbers of feet. Cryo-EM experiments confirmed the structure of the track and walkers. We performed detailed single molecule tracking and kinetics characterisation and found that the walkers with more feet diffuse along the track faster. The system represents a tuneable starting point for future powered protein molecular robots.

synthetic biology↗

bioRxiv2025

Design of Orthogonal Far-Red, Orange and Green Fluorophore-binding Proteins for Multiplex Imaging

Fluorescent proteins and small molecule dyes have complementary strengths for biological imaging: the former are genetically manipulatable enabling tagging of specific proteins and detection of protein interactions, while the latter have greater photostability and brightness but are difficult to target. To combine these strengths, we used de novo protein design to generate binders to three bright, stable, cell-permeable dyes spanning the visible spectrum: JF657 (far red), JF596 (orange-red) and JF494 (green). For each dye, we obtain nanomolar binders with weak or no binding to the other two dyes; the accuracy of the design approach is confirmed by a crystal structure of one binder which is very close to the design model. Fusion of the JF567, JF596 and JF494 binders to three different targets followed by staining with the three dyes simultaneously enables multiplex imaging. We further expand functionality by incorporating an active site carrying out nucleophilic aromatic substitution to form a covalent linkage with the dye, and developing split versions which reconstitute fluorescence at subcellular locations where both halves are present, enabling both protein-protein interaction detection and chemically induced dimerization with fluorescence reporting. Our designs combine the advantages of fluorescent proteins and small molecule dyes and should be broadly useful for cellular imaging.

biochemistry↗

bioRxiv2025

De novo design of inducibly assembling multi-component filaments

The design of multi-component nanomaterials is an outstanding challenge. Here, we describe the computational design of protein filaments with two or three distinct structural components that assemble into micron-scale, well-ordered fibers when mixed. CryoEM structure determination of four fiber designs was close to the computational design models. Filament assembly can be initiated by mixing the components, and modulated by addition and/or phosphorylation of designed regulatory subunits. This work demonstrates that regulatable multi-component protein filament systems can now be designed, opening the door to a wide range of engineered materials.

bioengineering↗

bioRxiv2024

Computational design of bifaceted protein nanomaterials with tailorable properties

Recent advances in computational methods have led to considerable progress in the design of self-assembling protein nanoparticles. However, nearly all nanoparticles designed to date exhibit strict point group symmetry, with each subunit occupying an identical, symmetrically related environment. This limits the structural diversity that can be achieved and precludes anisotropic functionalization. Here, we describe a general computational strategy for designing multi-component bifaceted protein nanomaterials with two distinctly addressable sides. The method centers on docking pseudosymmetric heterooligomeric building blocks in architectures with dihedral symmetry and designing an asymmetric protein-protein interface between them. We used this approach to obtain an initial 30-subunit assembly with pseudo-D5 symmetry, and then generated an additional 15 variants in which we controllably altered the size and morphology of the bifaceted nanoparticles by designing de novo extensions to one of the subunits. Functionalization of the two distinct faces of the nanoparticles with de novo protein minibinders enabled specific colocalization of two populations of polystyrene microparticles coated with target protein receptors. The ability to accurately design anisotropic protein nanomaterials with precisely tunable structures and functions could be broadly useful in applications that require colocalizing two or more distinct target moieties.

bioengineering↗

bioRxiv2024

Diffusing protein binders to intrinsically disordered proteins

Proteins which bind intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) with high affinity and specificity could have considerable utility for therapeutic and diagnostic applications. However, a general methodology for targeting IDPs/IDRs has yet to be developed. Here, we show that starting only from the target sequence of the input, and freely sampling both target and binding protein conformation, RFdiffusion can generate binders to IDPs and IDRs in a wide range of conformations. We use this approach to generate binders to the IDPs Amylin, C-peptide and VP48 in a range of conformations with Kds in the 3 -100nM range. The Amylin binder inhibits amyloid fibril formation and dissociates existing fibers, and enables enrichment of amylin for mass spectrometry-based detection. For the IDRs G3bp1, common gamma chain (IL2RG) and prion, we diffused binders to beta strand conformations of the targets, obtaining 10 to 100 nM affinity. The IL2RG binder colocalizes with the receptor in cells, enabling new approaches to modulating IL2 signaling. Our approach should be widely useful for creating binders to flexible IDPs/IDRs spanning a wide range of intrinsic conformational preferences.

biochemistry↗

bioRxiv2024

The kinesin motor Kif9 regulates centriolar satellite positioning and mitotic progression.

Centrosomes are the principal microtubule-organizing centers of the cell and play an essential role in mitotic spindle function. Centrosome biogenesis is achieved by strict control of protein acquisition and phosphorylation prior to mitosis. Defects in this process promote fragmentation of pericentriolar material culminating in multipolar spindles and chromosome missegregation. Centriolar satellites, membrane-less aggrupations of proteins involved in the trafficking of proteins toward and away from the centrosome, are thought to contribute to centrosome biogenesis. Here we show that the microtubule plus-end directed kinesin motor Kif9 localizes to centriolar satellites and regulates their pericentrosomal localization during interphase. Lack of Kif9 leads to aggregation of satellites closer to the centrosome and increased centrosomal protein degradation that disrupts centrosome maturation and results in chromosome congression and segregation defects during mitosis. Our data reveal roles for Kif9 and centriolar satellites in the regulation of cellular proteostasis and mitosis.

cell biology↗

bioRxiv2023

De novo design of modular protein hydrogels with programmable intra- and extracellular viscoelasticity

Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment and molecular dynamics (MD) simulation, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in non-Newtonian biomaterials exhibiting fluid-like properties under rest and low shear, but shear-stiffening solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly, in correlation with matching formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology. SignificanceProtein-based hydrogels have many applications in cellular engineering and medicine. Most genetically encodable protein hydrogels are made from naturally harvested proteins or protein-polymer hybrid constructs. Here we describe de novo protein hydrogels and systematically investigate the impact of microscopic properties of the building blocks (e.g., supramolecular interaction, valencies, geometries, flexibility) on the resultant macroscopic gel mechanics, both intra-and extracellularly. These de novo supramolecular protein assemblies, whose properties can be tuned from solid gels to non-Newtonian fluids, provide expanded opportunities for applications in synthetic biology and medicine.

bioengineering↗

bioRxiv2023

Computational design of non-porous, pH-responsive antibody nanoparticles

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: a de novo designed tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

bioengineering↗

bioRxiv2023

Design of amyloidogenic peptide traps

Segments of proteins with {beta}-strand propensity can self associate to form amyloid fibrils associated with many diseases. These regions often adopt alternative structures in their folded states, or are intrinsically disordered in solution, making it difficult to generate binders or inhibitors with existing strategies. Here we describe a general approach to bind such segments in {beta}-strand and {beta}-hairpin conformations using de novo designed scaffolds that contain deep peptide binding clefts flanked by {beta}-strands that form hydrogen bonds to the peptide upon binding. The designs bind their cognate peptides in vitro with nanomolar affinities and in mammalian cells. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide binding cleft is protected in the apo state. We use the approach to design binders to segments of the amyloid forming proteins Transthyretin, Tau, Serum amyloid A1 and A{beta}42. The A{beta} binders block assembly of A{beta} fibrils as effectively as the most potent of the clinically tested antibodies to date.

synthetic biology↗

bioRxiv2020

Design of Biologically Active Binary Protein 2D Materials

Proteins that assemble into ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers,6,7 but with the exception of a single lattice formed through non-rigid template streptavidin linkers,8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages.9-12 Here we describe a computational method to generate de-novo binary 2D non-covalent co-assemblies by designing rigid asymmetric interfaces between two distinct protein dihedral building-blocks. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly-crystalline micrometer-scale p6m arrays nearly identical to the computational design model in vitro and in cells without the need of a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces to drive extensive receptor clustering, downstream protein recruitment, and signaling. Using quantitative microscopy we show that arrays assembled on living cells have component stoichiometry and likely structure similar to arrays formed in vitro, suggesting that our material can impose order onto fundamentally disordered substrates like cell membranes. We find further that in sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems. One Sentence SummaryCo-assembling binary 2D protein crystals enables robust formation of complex large scale ordered biologically active materials

synthetic biology↗