Fast structure-based assignment of 15N HSQC spectra of selectively 15N-labeled paramagnetic proteins.
A novel strategy for fast NMR resonance assignment of (15)N HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively (15)N-labeled samples. Comparison of sensitive undecoupled (15)N 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 epsilon subunit and the theta 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.
Pintacuda
Guido
G
Australian National University, Research School of Chemistry, Canberra, ACT 0200, Australia.
Keniry
Max A
MA
Huber
Thomas
T
Park
Ah Young
AY
Dixon
Nicholas E
NE
Otting
Gottfried
G
D000465:Algorithms; D016880:Anisotropy; D004258:DNA Polymerase III / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D007553:Isotope Labeling; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D021122:Protein Subunits; D012984:Software
D009587:Nitrogen Isotopes; D021122:Protein Subunits; D004258:DNA Polymerase III