A Native-Like Three-R-Helix Bundle Protein from Structure-Based Redesign: A Novel Maquette Scaffold

A uniquely structured 65 amino acid helix-loop-helix′-loop-helix′′ three-R-helix bundle, R3-1, was designed and chemically synthesized, using the crystallographically characterized three stranded coiled coil “Coil-Ser”, as a starting point. The circular dichroism spectrum of R3-1 has a typical R-helical signature, with a [θ]222 ) -22 600 deg‚cm2‚dmol-1, indicating a 80.5% R-helical content. Sedimentation equilibrium analytical ultracentrifugation revealed that R3-1 is monomeric in solution. Consistent with the design parameters, the fluorescence emission maximum of the unique hydrophobic core tryptophan residue occurs at 324 nm. The evaluated ∆GH2O based on reversible guanidine hydrochloride denaturation is -4.6 ( 0.3 kcal‚mol-1 (m ) 2.2 ( 0.2 kcal‚mol-1‚M-1) as measured by CD spectroscopy. The amide-aromatic region of the 1H-NMR spectrum of R3-1 illustrates excellent chemical shift dispersion and resolution. All 35 expected methyl correlations are accounted for in the 13C-HSQC spectrum, providing stringent evidence for the existence of a native-like hydrophobic core. The monomeric nature of R3-1 should facilitate NMR structural studies and kinetic protein folding analysis of the current design, and on future variants with engineered binding sites. The utility of this single-chain three-R-helix bundle framework for expanding the range of biochemical cofactors bound in maquettes is being explored.