Biology subjects

Clayden, J.

Publications and source records attributed to Clayden, J..

5 recordsLinked to original sources

bioRxiv2026

Exploring the Design Space of 310-helical Bundles using Non-canonical Amino Acids

Steric constraints and favored hydrogen-bonding patterns result in peptides and proteins based on -amino acids being largely confined to -helical and {beta}-strand secondary structures. Although stand-alone 310 helices can be stabilized by incorporating ,-disubstituted amino acids such as amino-isobutyric acid (Aib, U), assemblies based solely on this structure are rare. Previously, we reported a de novo designed peptide, (ELUULK)4, that forms stable and defined 310-helical bundles. The Leu residues create a consolidated hydrophobic core, the Aib residues flank this core and promote 310-helix formation, and the Glu/Lys residues define the water-soluble surface of the bundles. Here, we examine the mutability of these sites and their contributions to maintaining this unusual fold. Surface substitutions of Glu and Lys with Asp, Arg, or the cyclic non-canonical amino acid 4-aminopiperidine-4-carboxylic acid (Api) are well tolerated, and all variants form stable 310-helical oligomers. In contrast, mutation of Aib at the flanking positions reveals strong constraints: only the cyclopropane analogue, 1-aminocyclopropane-1-carboxylic acid (Ac3c), preserves a 310-helical bundle, albeit with reduced thermal stability. Core substitutions prove even more restrictive. Of the proteinogenic hydrophobic residues, Leu uniquely supports bundle formation. Cyclic analogues largely disrupt assembly, although 1-aminocyclopentane-1-carboxylic acid (Ac5c) is partially tolerated. However, extending the design to seven hexads, (ELUULK)7, enables mutation of the two central core positions to Ala or Gly and retention of stable, hexameric 310-helical assemblies. These variants create hydrophobic cavities that bind environment-sensitive dyes. Together, these studies define sequence constraints for de novo 310-helical bundles and establish routes to their functionalization.

synthetic biology↗

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

De novo designed 3-helix bundle peptides and proteins with controlled topology and stability

Computational protein design is advancing rapidly. However, approaches and methods are needed to increase success rates and to elaborate designs. Here we describe the combination of rational and computational design to deliver three-helix bundle (3HB) peptide assemblies and single-chain proteins with control over topology and thermal stability. First, we garner sequence-to-structure relationships from antiparallel 3HBs in the Protein Data Bank. This gives core-packing rules, including layers of hydrogen-bonded polar residues, which are combined with surface-charge patterning to design complementary sequences for acidic (A), basic (B), and neutral (N) helices. By altering the design of the N helix, two sets of synthetic peptides are generated for clockwise and anticlockwise arrangements of the three-helix assemblies. Solution-phase characterization shows that both ABN peptide mixtures form stable, heterotrimeric assemblies consistent with the targeted up-down-up topologies. Next, AlphaFold2 models for both designs are used to seed computational designs of single-chain proteins where the helices are connected by loop building. Synthetic genes for these express in E. coli to yield soluble, monomeric, and thermally stable proteins. By systematically introducing additional polar layers within the core, the thermal stability of these proteins is varied without compromising the specificity of the helix-helix interactions. Chemical and thermal denaturation reveals comparable thermodynamic parameters to those of highly stable natural proteins. Four X-ray crystal structures confirm that the design models and AlphaFold2 predictions match to sub-[A] accuracy.

biophysics↗

bioRxiv2021

Constructing synthetic-protein assemblies from de novo designed 310 helices

Compared with the iconic helix, 310 helices occur much less frequently in protein structures. The different 310-helical parameters lead to energetically less favourable internal energies, and a reduced tendency to pack into defined higher-order structures. Consequently, in natural proteins, 310 helices rarely extend past 6 residues, and do not form regular supersecondary, tertiary, or quaternary interactions. Here, we show that despite their absence in nature, synthetic protein-like assemblies can be built from 310 helices. We report the rational design, solution-phase characterisation, and an X-ray crystal structure for water-soluble bundles of 310 helices with consolidated hydrophobic cores. The design uses 6-residue repeats informed by analysing natural 310 helices, and incorporates aminoisobutyric acid residues. Design iterations reveal a tipping point between -helical and 310-helical folding, and identify features required for stabilising assemblies in this unexplored region of protein-structure space.

synthetic biology↗

bioRxiv2018

On the interpretation of tractography-based parcellations

Connectivity-based parcellation of subcortical structures using diffusion tractography is now a common paradigm in neuroscience. These analyses often imply voxel-level specificity of connectivity, and the formation of compact, spatially coherent clusters is often taken as strong imaging-based evidence for anatomically distinct subnuclei in an individual. In this study, we demonstrate that internal structure in diffusion anisotropy is not necessary for a plausible parcellation to be obtained, by spatially permuting diffusion parameters within the thalami and repeating the parcellation. Moreover, we show that, in a winner-takes-all paradigm, most voxels receive the same label before and after this shuffling process--a finding that is stable across image acquisitions and tractography algorithms. We therefore suggest that such parcellations should be interpreted with caution.

neuroscience↗