Accurate measurements of the effects of deuteration at backbone amide positions on the chemical shifts of 15N, 13Cα, 13Cβ, 13CO and 1Hα nuclei in proteins

An approach towards accurate NMR measurements of deuterium isotope effects on the chemical shifts of all backbone nuclei in proteins (15N, 13Cα, 13CO, 1Hα) and 13Cβ nuclei arising from 1H-to-D substitutions at amide nitrogen positions is described. Isolation of molecular species with a defined protonation/deuteration pattern at successive backbone nitrogen positions in the polypeptide chain allows quantifying all deuterium isotope shifts of these nuclei from the first to the fourth order. Some of the deuterium isotope shifts measured in the proteins ubiquitin and GB1 can be interpreted in terms of backbone geometry via empirical relationships describing their dependence on (φ; ψ) backbone dihedral angles. Because of their relatively large variability and notable dependence on the protein secondary structure, the two- and three-bond 13Cα isotope shifts, 2ΔCα(NiD) and 3ΔCα(Ni+1D), and three-bond 13Cβ isotope shifts, 3ΔCβ(NiD), are useful reporters of the local geometry of the protein backbone.