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

Abstract

Discussion and ConclusionThe (ELUULK) 4 3 10 -helical bundle displays strongly position-dependent mutational tolerance.Surface residues can be readily substituted while preserving the 3 10 architecture, whereas the interfacial Aib positions and hydrophobic core impose much tighter geometric constraints.Computational analysis indicates that successful 3 10 -bundle formation requires both compatible backbone conformational preferences and precise side-chain geometry, explaining why small changes can redirect folding towards α-helical conformations or disrupt assembly entirely.Despite these constraints, extending the scaffold to seven hexad repeats enables controlled modification of the central hydrophobic core.Substitution of two Leu residues with Ala or Gly generates stable hexameric 3 10 -helical bundles containing hydrophobic cavities that bind small environmentsensitive dyes in the µM range.This demonstrates that sequence extension can overcome local packing constraints and provides a route to functionalising 3 10 -helical scaffolds through selective removal of methylene groups.Together, these results establish a sequence-structure framework for de novo 3 10 -helical bundle design and show that, although more constrained than canonical α-helical assemblies, 3 10 helices can serve as programmable scaffolds for tuning oligomerisation and creating defined molecular-recognition sites.Overview Peptides and proteins based on α-amino acids predominantly adopt α-helical and β-strand structures, while extended assemblies of the noncanonical 3 10 helix remain rare.We previously designed the de novo peptide (ELUULK) 4 , which forms a hexameric 3 10 -helical bundle in which Aib promotes 3 10 -helix formation, Leu forms the hydrophobic core, and Glu/Lys define the solvent-exposed surface. 1 Here, we investigate the sequence requirements underlying this noncanonical fold and explore routes to its functionalisation.Surface substitutions are well tolerated, whereas the Aib flanking positions show substantially greater constraints.Core positions are more restrictive, with Leu uniquely supporting bundle formation among proteinogenic hydrophobic residues.Extending to seven hexad repeats enables substitution of two central core Leu residues with Ala or Gly while retaining stable, hexameric 3 10 -helical assemblies.These substitutions generate hydrophobic cavities capable of binding environment-sensitive dyes.Together, these results define sequence constraints governing the stability of de novo 3 10 -helical bundles and demonstrate how controlled core modification can alter these otherwise highly constrained assemblies. K-D 8.0 E-K 6.2 E-