Quantification of H/D Isotope Effects on Protein Hydrogen-bonds by ^h3J_NC′ and ¹J_NC′ Couplings and Peptide Group ¹⁵N and ¹³C′ Chemical Shifts

Abstract

The effect of hydrogen/deuterium exchange on proteinhydrogen bond coupling constants ^h3J_NC′ has been investigated in the small globular protein ubiquitin. The couplings across deuterated or protonated hydrogen bonds were measured by a long-range quantitative HA(CACO)NCO experiment. The analysis is combined with a determination of the H^N/D^N isotope effect on the amide group ¹J_NC′ couplings and the ¹⁵N and ¹³C′ chemical shifts. On average, H-bond deuteration exchange weakens ^h3J_NC′ and strengthens ¹J_NC′ couplings. A correlation is found between the size of the ¹⁵N isotope shift, the ¹⁵N chemical shift, and the ^h3J_NC′ coupling constants. The data are consistent with a reduction of donor-acceptor overlap as expected from the classical Ubbelohde effect and the common understanding that H^N/D^N exchange leads to a shortening of the N-hydron bond length.

Abbreviations: H-bond – hydrogen bond.