Fast structure-based assignment of 15N HSQC spectra of selectively 15N-labeled paramagnetic proteins

Authors

Guido Pintacuda, Australian National University
Max A Keniry, Australian National University
Thomas Huber, University Of Queensland
Ah-Young Park, Australian National University
Nicholas E. Dixon, University of WollongongFollow
Gottfried Otting, Australian National University

RIS ID

16958

Publication Details

Pintacuda, G., Keniry, M., Huber, T., Park, A., Dixon, N. E. & Otting, G. (2004). Fast structure-based assignment of 15N HSQC spectra of selectively 15N-labeled paramagnetic proteins. Journal of the American Chemical Society, 126 (9), 2963-2970.

Abstract

A novel strategy for fast NMR resonance assignment of ¹⁵N HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively ¹⁵N-labeled samples. Comparison of sensitive undecoupled ¹⁵N HSQC spectra recorded of paramagnetic and diamagnetic samples yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole−dipole relaxation, and residual dipolar coupling. Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center. The method is demonstrated with the 30 kDa complex between the N-terminal domain of the ε subunit and the θ subunit of Escherichia coli DNA polymerase III. The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy.