Biology subjects

Boyle, A. L.

Publications and source records attributed to Boyle, A. L..

3 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↗

bioRxiv2023

Novel histones and histone variant families in prokaryotes

Histones are important chromatin-organizing proteins in eukaryotes and archaea. They form superhelical structures around which DNA is wrapped. Recent studies have shown that some archaea and bacteria contain alternative histones that exhibit different DNA binding properties, in addition to highly divergent sequences. However, the vast majority of these new histones are identified in metagenomes and thus are difficult to study in vivo. The recent revolutionary breakthroughs in computational protein structure prediction by AlphaFold2 and RoseTTAfold allow for unprecedented insights into the potential function and structure of previously uncharacterized proteins. Here, we categorize the prokaryotic histone space into 17 distinct groups based on AlphaFold2 predictions. We identify a new superfamily of histones, termed 3 histones, which are common in archaea and present in several bacteria. Importantly, we establish the existence of a large family of histones throughout archaea and in some bacteriophages that, instead of wrapping DNA, bridge DNA, thereby diverging from conventional nucleosomal histones.

bioinformatics↗

bioRxiv2021

Novel anti-repression mechanism of H-NS proteins by a phage "early protein".

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the early protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 modulates MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an "electrostatic zipper" between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their "half-open" conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

biochemistry↗