Rapid measurement of pseudocontact shifts in metalloproteins by proton-detected solid-state NMR spectroscopy.
Pseudocontact shifts (PCSs) arise in paramagnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the paramagnetic center and the position relative to the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of (1)H-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved (1)H PCSs, in a large metalloprotein, Co(2+)-substituted superoxide dismutase (Co(2+)-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with (1)H-(1)H distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.
Centre de RMN à Très Hauts Champs, UMR 5280 CNRS/ENS Lyon/UCB Lyon 1, Université de Lyon, 5 rue de la Doua, 69100 Villeurbanne, France.
Michael J
MJ
Knight
Isabella C
IC
Felli
Roberta
R
Pierattelli
Ivano
I
Bertini
Lyndon
L
Emsley
Torsten
T
Herrmann
Guido
G
Pintacuda
D003035:Cobalt / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000592:standards*; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D011522:Protons; D012015:Reference Standards; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism
D008667:Metalloproteins; D011522:Protons; D003035:Cobalt; D013482:Superoxide Dismutase