NMR detection of protein 15 N spins near paramagnetic lanthanide ions

Abstract

Pronounced paramagnetic relaxation enhancement (PRE) due to paramagnetic metal ions prevents the observation of NMR signals from 1H spins near the metal. While 15N spins are less prone to PRE, the intrinsic sensitivity of 15N NMR spectroscopy is low. This Communication presents a 1H detected out-and-back Nz-exchange experiment which allows the measurement of pseudocontact shifts of 15N spins located as close as 6 Å from a Dy3+ ion in a 30 kDa protein complex. The experiment relies on the chemical exchange between paramagnetic and diamagnetic metal ions during two mixing times during which the 15N magnetization is stored as PRE-insensitive longitudinal magnetization. It is demonstrated with the complex between the subunit θ and the N-terminal domain of the subunit ε of E. coli DNA polymerase III, prepared with a mixture of Dy3+ and La3+. Pseudocontact shifts were measured for 61 15N spins which were not observable in a conventional 15N-HSQC spectrum.