A NATIVE-LIKE 3-ALPHA-HELIX BUNDLE PROTEIN FROM STRUCTURE-BASED REDESIGN - A NOVEL MAQUETTE SCAFFOLD

A uniquely structured 65 amino acid helix-loop-helix'-loop-helix '' th ree-alpha-helix bundle, alpha(3)-1, was designed and chemically synthe sized, using the crystallographically characterized three stranded coi led coil ''Coil-Ser'', as a starting point. The circular dichroism spe ctrum of alpha(3)-1 has a typical alpha-helical signature, with a [the ta](222) = -22 600 deg.cm(2).dmol(-1), indicating a 80.5% alpha-helica l content. Sedimentation equilibrium analytical ultracentrifugation re vealed that alpha(3)-1 is monomeric in solution. Consistent with the d esign parameters, the fluorescence emission maximum of the unique hydr ophobic core tryptophan residue occurs at 324 nm. The evaluated Delta G(H2O) 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 H-1-NMR spectrum of alpha(3)-1 illustrates excellent chemical shift dispersion and reso lution. All 35 expected methyl correlations are accounted for in the C -13-HSQC spectrum, providing stringent evidence for the existence of a native-like hydrophobic core. The monomeric nature of alpha(3)-1 shou ld facilitate NMR structural studies and kinetic protein folding analy sis of the current design, and on future variants with engineered bind ing sites. The utility of this single-chain three-a-helix bundle frame work for expanding the range of biochemical cofactors bound in maquett es is being explored.