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 pKa of the ionizable carboxyl group when the amides of the compounds are either protonated or deuterated. From the pKa shift, we obtain a quantitative scale factor: SF = ∂(ΔGHB)/∂(RT In Φ), where ΔGHB 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 "Cm 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.
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