Biology subjects

McManus, J. J.

Publications and source records attributed to McManus, J. J..

5 recordsLinked to original sources

bioRxiv2026

Expanding all-α-helical protein space through rational computational design

De novo protein design is advancing rapidly1,2. This is being driven by AI to generate protein backbones, sequences, and structural models3-7. As a result, de novo designed proteins are becoming larger and more complex8-10, and increasingly explore new protein structures11,12. By contrast, natural proteins have evolved structural and functional complexity by modular combination of recurring protein domains13. Approximately 25% of these natural domains are mostly -helical structures14. Here we show how these can be expanded using rational computational design. Following the domain classification scheme CATH15, we build complex all- de novo proteins hierarchically using sequence-to-structure relationships for helix-helix interactions, systematic rules to connect helices, computational tools to design loops, and in silico evaluation. The pipeline starts with a target architecture of free-standing helices. These are connected into a topology by considering local arrangements of helical bundles using understood sequence-to-structure relationships for helix packing. Single-chain sequences are completed using template- and AI-based methods. Finally, AlphaFold models are assessed to give small numbers of designs for experimental validation. We test 31 designs for 14 different architectures and 25 topologies. 75% of these express as stable, monomeric, water-soluble proteins; and >30% yield X-ray crystal structures matching the designs to atomic accuracy and with new-to-nature structures. Finally, several of the scaffolds are functionalised through one-shot designs to deliver ion, small-molecule and protein binders.

synthetic biology↗

bioRxiv2025

Biomolecular condensation using de novo designed globular proteins

De novo protein design is advancing rapidly, but many targets remain inaccessible to current AI-based tools. Here we describe de novo designed globular domains that drive biomolecular condensation. Starting from a water-soluble, monomeric protein, we make variants with the same amino-acid composition but different surface-charge distributions: one with large patches of surface charge, and another with a more-homogeneous charge distribution. The individual domains form stable and discrete structures in solution, with the large-patch variant exhibiting more-attractive interprotein interactions. Next, two copies of each variant are joined with disordered linkers to generate dumbbell-like proteins. When expressed in eukaryotic cells, the large-patch variant forms intracellular puncta, whereas that with small patches does not. The assemblies are dynamic, liquid condensates in vitro and in cells. The structured domains facilitate functionalisation: we introduce fluorophore-binding sites to visualise fluorescent condensates directly in cells without a GFP reporter; and we manipulate the condensates using motor proteins.

synthetic biology↗

bioRxiv2025

Rapid Assessment of Size, Shape, and Chemical Complementarity of Ligands for Computational Protein Design

Driven by deep-learning approaches, computational protein design is advancing rapidly, and it is now possible to generate many de novo protein structures quickly and robustly. This sets new frontiers for the field, including designing proteins that bind small molecules tightly and specifically, and understanding the non-covalent interactions that underpin such designs to make binding predictable and tunable. Here we address these challenges with a rapid physics-based computational method to generate isosteric and chemically complementary binding pockets for small-molecule targets in de novo designed proteins. We test this experimentally by constructing and characterizing binding proteins for several synthetic and natural chromophores. By evaluating only single-digit numbers of designs, the pipeline delivers stable proteins with pre-organized binding sites confirmed by X-ray crystallography, which bind the targets selectively with micromolar affinities or better. To illustrate the scope and applications of this approach, we incorporate distinct and coupled chromophore-binding sites in a two-domain de novo protein enabling controlled energy transfer between the two sites, and we develop a small de novo binding protein that can be used in live mammalian cells to visualize sub-cellular structures.

biophysics↗

bioRxiv2024

Maturation and conformational switching of a de novo designed phase-separating polypeptide

Cellular compartments formed by biomolecular condensation are a widespread feature of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels, through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arise in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using the droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely {beta} conformations, analogous to models of pathological phase separation.

synthetic biology↗

bioRxiv2023

Assembling membraneless organelles from de novo designed proteins

Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological process and augment natural systems. The bottom-up design of self-assembled objects like microcompartments and membraneless organelles is one such challenge, which also presents opportunities for chemical and synthetic biology. Here, we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli (E. coli). To do this, we combine de novo -helical sequences, intrinsically disordered linkers, and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from diffusion-limited assemblies to dynamic condensates. The designs are characterised in cells and in vitro using biophysical and soft-matter physics methods. Finally, we use the designed polypeptide to co-compartmentalise a functional enzyme pair in E. coli.

synthetic biology↗