Contribution of hydrogen bonding to protein stability estimated from isotope effects

An unresolved issue in structural biology concerns the relative contribution of H bonds to protein stability. We use the small molecules 4-acetamidobenzoic acid and N-acetylanthranilic acid as model compounds to relate the energetic contribution from hydrogen bonds (H bonds) to the deuterium/hydrogen amide isotope effect. N-Acetylanthranilic acid models carbonyl - amide H bonds formed during protein folding; 4-acetamidobenzoic acid models the unfolded state in which the amide H bonds to water. NMR is used to measure shifts in the pK_a of the ionizable carboxyl group when the amides of the compounds are either protonated or deuterated. From the pK_a shift, we obtain a quantitative scale factor: SF = ∂(ΔG^HB)/∂(RT In Φ), where ΔG^HB is the change in free energy of an H bond upon isotope substitution and Φ is the fractionation factor. Isotope effect data also are reported for a small globular protein, λ repressor, using the "C_m experiment". The protein's isotope effect, which reports on the shape of the energy well, is converted to H-bonding free energy by applying the scale factor. We estimate that amide-related H bonds (amide - carbonyl and amide - water) contribute favorably to protein stability by ∼30-50 kcal/mol in λ repressor, GCN4 coiled coil, and cytochrome c but unfavorably by ∼6 kcal/mol in ubiquitin. The results indicate that H-bond strength varies from one protein to another and presumably at different sites within the same protein.