Amide-to-E-Olefin versus Amide-to-Ester Backbone H-Bond Perturbations: Evaluating the O−O Repulsion for Extracting H-Bond Energies

Abstract
Both amide-to-ester and amide-to-E-olefin backbone amide mutation methods were employed to perturb the same H-bond (formed by the NH of F23 and the CO of R14) in the Pin WW domain. Comparison of the thermodynamic folding energies of the ester mutant and the E-olefin mutant, accounting for the transfer free energy differences measured on relevant model compounds, yielded an estimated value of 0.3 kcal/mol for the O−O repulsion term (ΔGO-Orep) in a β-sheet context. The value of ΔGO-Orep enabled us to calculate the intrinsic F23−R14 H-bond free energy to be 1.3 kcal/mol.