Four-bond deuterium isotope effects on the chemical shifts of amide nitrogens in proteins

An approach towards precision NMR measurements of four-bond deuterium isotope effects on the chemical shifts of backbone amide nitrogen nuclei in proteins is described. Three types of four-bond N-15 deuterium isotope effects are distinguished depending on the site of proton-to-deuterium substitution: N-4(Ni-1D), N-4(Ni+1D) and N-4(C,i-1D). All the three types of isotope shifts are quantified in the (partially) deuterated protein ubiquitin. The N-4(Ni+1D) and N-4(C,i-1D) effects are by far the largest in magnitude and vary between 16 and 75ppb and -18 and 46ppb, respectively. A semi-quantitative correlation between experimental N-4(Ni+1D) and N-4(C,i-1D) values and the distances between nitrogen nuclei and the sites of H-1-to-D substitution is noted. The largest isotope shifts in both cases correspond to the shortest inter-nuclear distances. Copyright (c) 2013 John Wiley & Sons, Ltd.