Precision NMR measurements of deuterium isotope effects on the chemical shifts of backbone nuclei in proteins ((15)N, (13)CO, (13)C(α), and (1)HN) arising from (1)H-to-(2)H substitutions at aliphatic carbon sites. Isolation of molecular species with a defined protonation/deuteration pattern at carbon-α/β positions allows distinguishing and accurately quantifying different isotope effects within the protein backbone. The isotope shifts measured in the partially deuterated protein ubiquitin are interpreted in terms of backbone geometry via empirical relationships describing the dependence of isotope shifts on (φ; ψ) backbone dihedral angles. Because of their relatively large magnitude and clear dependence on the protein secondary structure, the two- and three-bond backbone amide (15)N isotope shifts, (2)ΔN(C(α,i)D) and (3)ΔN(C(α,i-1)D), can find utility for NMR structural refinement of small-to-medium size proteins.