Quantification of H/D isotope effects on protein hydrogen-bonds by (h3)J(NC ') and (1)J(NC ') couplings and peptide group N-15 and C-13 ' chemical shifts

摘要

The effect of hydrogen/deuterium exchange on protein hydrogen bond coupling constants (h3)J(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 (1)J(NC') couplings and the N-15 and C-13' chemical shifts. On average, H-bond deuteration exchange weakens (h3)J(NC') and strengthens (1)J(NC') couplings. A correlation is found between the size of the N-15 isotope shift, the N-15 chemical shift, and the (h3)J(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.