Biology subjects

Eisenach, H.

Publications and source records attributed to Eisenach, H..

3 recordsLinked to original sources

bioRxiv2025

De novo Design of All-atom Biomolecular Interactions with RFdiffusion3

Deep learning has accelerated protein design, but most existing methods are restricted to generating protein backbone coordinates and often neglect interactions with other biomolecules. We present RFdiffusion3 (RFD3), a diffusion model that generates protein structures in the context of ligands, nucleic acids and other non-protein constellations of atoms. Because all polymer atoms are modeled explicitly, conditioning the model on complex sets of atom-level constraints for enzyme design and other challenges is both simpler and more effective than previous approaches. RFD3 achieves improved performance compared to prior approaches on a range of in silico benchmarks with one tenth the computational cost. Finally, we demonstrate the broad applicability of RFD3 by designing and experimentally characterizing DNA binding proteins and cysteine hydrolases. The ability to rapidly generate protein structures guided by complex sets of atom-level constraints in the context of arbitrary non-protein atoms should further expand the range of functions attainable through protein design.

biochemistry↗

bioRxiv2025

Massively parallel assessment of designed protein solution properties using mass spectrometry and peptide barcoding

Library screening and selection methods can determine the binding activities of individual members of large protein libraries given a physical link between protein and nucleotide sequence, which enables identification of functional molecules by DNA sequencing. However, the solution properties of individual protein molecules cannot be probed using such approaches because they are completely altered by DNA attachment. Mass spectrometry enables parallel evaluation of protein properties amenable to physical fractionation such as solubility and oligomeric state, but current approaches are limited to libraries of 1,000 or fewer proteins. Here, we improved mass spectrometry barcoding by co-synthesizing proteins with barcodes optimized to be highly multiplexable and minimally perturbative, scaling to libraries of >5,000 proteins. We use these barcodes together with mass spectrometry to assay the solution behavior of libraries of de novo-designed monomeric scaffolds, oligomers, binding proteins and nanocages, rapidly identifying design failure modes and successes.

biochemistry↗

bioRxiv2024

Design of a Soluble Multivalent Notch Agonist

Designed protein agonists can enhance the efficiency of endogenous signaling pathways, and provide a powerful means to control cellular functions and develop disease therapeutics. Designing a soluble cytokine-like agonist for Notch signaling, an evolutionarily conserved pathway that regulates cell fate in embryonic and adult development, is especially challenging because Notch receptor activation requires a mechanical force that is typically mediated by cell-associated transmembrane ligands at sites of cell-cell contact. Moreover, free soluble Notch ligand is signal inhibitory. Here, we exploit computationally designed protein oligomers with precise geometries and valencies to generate cytokine-like, protein only, multivalent soluble Notch agonists. These tools promote cell-cell contact, cluster Notch proteins in synapses at the cell surface, and activate Notch signaling in reporter cell lines and cells expressing endogenous receptors. We demonstrate the utility of these soluble Notch agonists in T cell differentiation from cord blood (CB) and human induced pluripotent stem cells (iPSCs), and in bioreactor production of T cells in liquid suspension. Soluble multivalent Notch agonists can be applied broadly to in vitro cellular differentiation methods to generate clinical cell products and to develop immunotherapies.

synthetic biology↗