Exploring the Design Space of 3 10 -helical Bundles using Non-canonical Amino Acids

Abstract

Steric constraints and favored hydrogen-bonding patterns result in peptides and proteins based on α-amino acids being largely confined to α-helical and β-strand secondary structures. Although stand-alone 310 helices can be stabilized by incorporating α,α-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.