Fluorous Effect in Proteins:  De Novo Design and Characterization of a Four-α-Helix Bundle Protein Containing Hexafluoroleucine

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (ΔΔGunfold) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with l-5,5,5,5‘,5‘,5‘-hexafluoroleucine (hFLeu). We find that ΔΔGunfold is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in ΔΔGunfold of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally fr...