Assignment of paramagnetic 15N-HSQC spectra by heteronuclear exchange spectroscopy

Authors

Michael John, Australian National University
Madeleine J. Headlam, Australian National University
Nicholas E. Dixon, University of WollongongFollow
Gottfried Otting, Australian National University

RIS ID

16851

Publication Details

John, M., Headlam, M. J., Dixon, N. E. & Otting, G. (2007). Assignment of paramagnetic 15N-HSQC spectra by heteronuclear exchange spectroscopy. Journal of Biomolecular N M R, 37 (1), 43-51.

Abstract

Paramagnetic metal ions in proteins provide a rich source of structural information, but the resonance assignments required to extract the information can be challenging. Here we demonstrate that paramagnetically shifted ¹⁵N-HSQC cross-peaks can be assigned using N_Z-exchange spectroscopy under conditions in which the paramagnetic form of the protein is in dynamic equilibrium with its diamagnetic form. Even slow exchange of specifically bound metal ions may be detected within the long lifetime of ¹⁵N longitudinal magnetization of large proteins at high magnetic fields. Alternatively, the exchange can be accelerated using an excess of metal ions. In the resulting exchange spectra, paramagnetic ¹⁵N resonances become visible for residues that are not directly observed in a conventional ¹⁵N-HSQC spectrum due to paramagnetic ¹H^N broadening. The experiments are illustrated by the 30 kDa lanthanide-binding ɛ186/θ complex of DNA polymerase III in the presence of sub-stoichiometric amounts of Dy³⁺ or a mixture of Dy³⁺ and La³⁺.