Observation of Through-Hydrogen-Bond JHC′ in a Perdeuterated Protein

It is demonstrated that J connectivity between amide protons and hydrogen-bond-accepting carbonyl carbons can be observed in perdeuterated human ubiquitin. A selective pulse scheme is used to detect these small ^2hJ_HC′ interactions in the presence of the much larger through-covalent-bond ²J_HC′ and ³J_HC′ couplings. The ratio of the observed through-H-bond correlation intensity and the ²J_HC′ connectivity observed in a reference spectrum indicates ^2hJ_HC′ values of ca. 0.4–0.6 Hz, which are only slightly smaller than the corresponding ^3hJ_NC′ values. However, for technical reasons, ^2hJ_HC′ couplings are more difficult to measure than ^3hJ_NC′.