NMR detection of protein 15N-spins near paramagnetic lanthanide ions

Authors

Michael John, Australian National University
Ah-Young Park, Australian National University
Nicholas E. Dixon, University of WollongongFollow
Gottfried Otting, Australian National University

RIS ID

16852

Publication Details

John, M., Park, A., Dixon, N. E. & Otting, G. (2007). NMR detection of protein 15N-spins near paramagnetic lanthanide ions. Journal of the American Chemical Society, 129 (3), 462-463.

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 N_z-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.