Biology subjects

Kibler, R. D.

Publications and source records attributed to Kibler, R. D..

12 recordsLinked to original sources

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↗

bioRxiv2026

De novo design of selective kinase modulators

Protein kinases are critical regulators of cellular signaling, but precise modulation of their activity remains challenging due to their high structural conservation. Here, we present de novo designed genetically encoded miniproteins capable of activating or inhibiting focal adhesion kinase (FAK) by directly targeting the kinase domain itself. Among 96 binders designed to stabilize distinct conformational states of FAK, 33 modulated kinase activity. Biochemical characterization of the four most potent modulators revealed that two designs inhibit FAK with low-nanomolar IC50 values while the remaining two potentiated FAK activity by more than two-fold. When expressed in cells, the modulators preserved the same inhibitory and activating effects observed in vitro, establishing that designed conformational binders can directly tune FAK signaling in living cells. Taking advantage of the high similarity between kinases, we redesigned the FAK inhibitors to inhibit Src kinase. Our approach establishes a versatile platform for selective and genetically encoded kinase control as a way to rewire cell signaling and as a starting point for the discovery of novel modulatory sites of kinases.

biochemistry↗

bioRxiv2026

Generative design of programmable asymmetric β-barrel nanopores

Protein nanopores are powerful tools for molecular sensing, sequencing, and separation, but designing pores with programmable function remains challenging. Native homo-oligomeric transmembrane {beta} barrels (TMBs) are used for these applications, but their uniform lumens limit spatial resolution and analyte discrimination. Although monomeric TMBs can be designed using energy-based methods, these approaches remain highly manual and limited to structural design rather than function. Here, we present a generative AI framework for TMB design, with backbone and sequence design models trained on a curated distillation set. We characterized 48 designs spanning 0.7-1.5 nm in diameter, diverse lumen chemistries, and hydrophobic thicknesses. Crystal structures closely match the design models. We demonstrate that our method produces customizable nanopores for ion sensing, DNA translocation, and transport across polymer membranes.

biochemistry↗

bioRxiv2026

Templating and confining calcium phosphate mineralization within designed protein assemblies

Bone formation involves the deposition of ordered hydroxyapatite (HAp) on collagen fibrils, but the underlying molecular mechanisms remain largely unresolved, limiting the design of protein-apatite hybrid materials. Here we show that computationally designed de novo proteins can template and confine HAp mineralization. We design C3-symmetric oligomers with inner surfaces chemically complementary to the HAp {010} facet, and assemble these through additional interfaces into D3 oligomers, one-dimensional nanotubes, and two-dimensional arrays. Electron microscopy revealed templated mineralization and confinement at each hierarchical level, with mineral shape guided by the underlying protein architecture. Conversion of an initially formed amorphous phase to HAp is driven by the designed protein-mineral interface. Our results establish a framework for programmable protein-guided mineralization, providing a foundation for next-generation hybrid materials.

biochemistry↗

bioRxiv2025

Accelerating protein design by scaling experimental characterization

Recent advances in de novo protein design have greatly outpaced standard protein biochemistry workflows, and experimental testing has been a bottleneck in the validation of new designs and methodologies. Here, we describe experimental and computational workflows to address the issues of scale, speed and reproducibility of common in vitro protein testing methods, enabling at least an order of magnitude increase in throughput while reducing wetlab time. Semi-Automated Protein Production (SAPP) is a rapid, modular, scalable and cost-effective protocol, enabling up to milligram-scale protein production, and standardized characterization including yield, dispersity, and oligomeric state of hundreds of designs per day, at the cost-equivalent of a few DNA oligos per construct. End-to-end protocol execution takes 48 hours, with about 6 hours spent benchside using mostly standard laboratory equipment. This protocol has become the standard at our institute, providing critical experimental validation for dozens of projects spanning tens of thousands of designs. We showcase the power of the platform by using it to rapidly characterize de novo designed inhibitors of respiratory syncytial virus. Since at least 80% of SAPPs total cost comes from synthetic DNA, we also developed a scalable demultiplexing protocol (DMX) to leverage oligo pools as input DNA, providing a further 5-fold reduction in costs, enabling >1000 designs to be purified and characterized in arrayed, clonal format at a cost of $5 per construct. By reframing standard molecular biology practices and orchestrating wetlab workflows with partial automation instead of complex end-to-end robotics, these protocols should be widely adoptable, accelerating protein design.

biochemistry↗

bioRxiv2024

Bottom-up design of calcium channels from defined selectivity filter geometry

Native ion channels play key roles in biological systems, and engineered versions are widely used as chemogenetic tools and in sensing devices1,2. Protein design has been harnessed to generate pore-containing transmembrane proteins, but the capability to design ion selectivity based on the interactions between ions and selectivity filter residues, a crucial feature of native ion channels3, has been constrained by the lack of methods to place the metal-coordinating residues with atomic-level precision. Here we describe a bottom-up RFdiffusion-based approach to construct Ca2+ channels from defined selectivity filter residue geometries, and use this approach to design symmetric oligomeric channels with Ca2+ selectivity filters having different coordination numbers and different geometries at the entrance of a wide pore buttressed by multiple transmembrane helices. The designed channel proteins assemble into homogenous pore-containing particles, and for both tetrameric and hexameric ion-coordinating configurations, patch-clamp experiments show that the designed channels have higher conductances for Ca2+ than for Na+ and other divalent ions (Sr2+ and Mg2+). Cryo-electron microscopy indicates that the design method has high accuracy: the structure of the hexameric Ca2+ channel is nearly identical to the design model. Our bottom-up design approach now enables the testing of hypotheses relating filter geometry to ion selectivity by direct construction, and provides a roadmap for creating selective ion channels for a wide range of applications.

biochemistry↗

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

Bond-centric modular design of protein assemblies

We describe a modular bond-centric approach to protein nanomaterial design inspired by the rich diversity of chemical structures that can be generated from the small number of atomic valencies and bonding interactions. We design protein building blocks with regular coordination geometries and bonding interactions that enable the assembly of a wide variety of closed and opened nanomaterials using simple geometrical principles. Experimental characterization confirms successful formation of more than twenty multi-component polyhedral protein cages, 2D arrays, and 3D protein lattices, with a high (10-50 %) success rate and electron microscopy data closely matching the corresponding design models. Because of the modularity, individual building blocks can assemble with different partners to generate distinct regular assemblies, resulting in an economy of parts and enabling the construction of reconfigurable systems.

biochemistry↗

bioRxiv2023

Design of four component T=4 tetrahedral, octahedral, and icosahedral protein nanocages through programmed symmetry breaking

Four, eight or twenty C3 symmetric protein trimers can be arranged with tetrahedral (T-sym), octahedral (O-sym) or icosahedral (I-sym) point group symmetry to generate closed cage-like structures1,2. Generating more complex closed structures requires breaking perfect point group symmetry. Viruses do this in the icosahedral case using quasi-symmetry or pseudo-symmetry to access higher triangulation number architectures3-9, but nature appears not to have explored higher triangulation number tetrahedral or octahedral symmetries. Here, we describe a general design strategy for building T = 4 architectures starting from simpler T = 1 structures through pseudo-symmetrization of trimeric building blocks. Electron microscopy confirms the structures of T = 4 cages with 48 (T-sym), 96 (O-sym), and 240 (I-sym) subunits, each with four distinct chains and six different protein-protein interfaces, and diameters of 33nm, 43nm, and 75nm, respectively. Higher triangulation number viruses possess very sophisticated functionalities; our general route to higher triangulation number nanocages should similarly enable a next generation of multiple antigen displaying vaccine candidates10,11 and targeted delivery vehicles12,13.

biochemistry↗

bioRxiv2023

Blueprinting expandable nanomaterials with standardized protein building blocks

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures1. Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight "train track" assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.

biochemistry↗

bioRxiv2023

Stepwise design of pseudosymmetric protein hetero-oligomers

Pseudosymmetric hetero-oligomers with three or more unique subunits with overall structural (but not sequence) symmetry play key roles in biology, and systematic approaches for generating such proteins de novo would provide new routes to controlling cell signaling and designing complex protein materials. However, the de novo design of protein hetero-oligomers with three or more distinct chains with nearly identical structures is a challenging problem because it requires the accurate design of multiple protein-protein interfaces simultaneously. Here, we describe a divide-and-conquer approach that breaks the multiple-interface design challenge into a set of more tractable symmetric single-interface redesign problems, followed by structural recombination of the validated homo-oligomers into pseudosymmetric hetero-oligomers. Starting from de novo designed circular homo-oligomers composed of 9 or 24 tandemly repeated units, we redesigned the inter-subunit interfaces to generate 15 new homo-oligomers and recombined them to make 17 new hetero-oligomers, including ABC heterotrimers, A2B2 heterotetramers, and A3B3 and A2B2C2 heterohexamers which assemble with high structural specificity. The symmetric homo-oligomers and pseudosymmetric hetero-oligomers generated for each system share a common backbone, and hence are ideal building blocks for generating and functionalizing larger symmetric assemblies. Significance StatementProtein oligomers composed of multiple unique subunits are versatile building blocks for creating functional materials and controlling biological processes. However, designing robust hetero-oligomers with distinct subunits and precise structural symmetry remains a major challenge. Here, we present a general strategy for designing such complexes by breaking down the problem into simpler steps by first symmetrically re-designing the interfaces of homo-oligomeric proteins, and then recombining validated variants to form pseudosymmetric hetero-oligomers. Using this method, we generated 17 hetero-oligomers with up to three unique subunits that assemble with high specificity. Our approach can be extended to create a wide range of pseudosymmetric assemblies for manipulating cellular signaling and as building blocks for advanced protein materials. These pseudosymmeteric heterotrimers have already enabled the construction of a set of massive nanocages, including a T=4 icosahedral nanocage with a 70 nm diameter and 240 subunits.1

biochemistry↗

bioRxiv2022

Hallucinating protein assemblies

Deep learning generative approaches provide an opportunity to broadly explore protein structure space beyond the sequences and structures of natural proteins. Here we use deep network hallucination to generate a wide range of symmetric protein homo-oligomers given only a specification of the number of protomers and the protomer length. Crystal structures of 7 designs are very close to the computational models (median RMSD: 0.6 [A]), as are 3 cryoEM structures of giant rings with up to 1550 residues, C33 symmetry, and 10 nanometer in diameter; all differ considerably from previously solved structures. Our results highlight the rich diversity of new protein structures that can be created using deep learning, and pave the way for the design of increasingly complex nanomachines and biomaterials. One-sentence summaryDeep network-based protein design enables the generation of cyclic homo-oligomers across the nanoscopic scale.

biophysics↗