Fast Structure-Based Assignment of15N HSQC Spectra of Selectively15N-Labeled Paramagnetic Proteins
- 13 February 2004
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 126 (9) , 2963-2970
- https://doi.org/10.1021/ja039339m
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.Keywords
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