NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands.
Selective isotopic labeling of larger proteins greatly simplifies protein NMR spectra and reduces signal overlap, but selectively labeled proteins cannot be easily assigned since the sequential assignment method is not applicable. Here we describe a strategy for resonance assignment in selectively labeled proteins. Our approach involves a spin-labeled analog of a ligand of which the three-dimensional structure in complex with the target protein is known. Other methods for introduction of the spin label are possible. The paramagnetic center causes faster relaxation of all neighboring nuclei in a distance-dependent manner. Measurement of this effect allows to deduce distances between isotopically labeled residues and the paramagnetic center which can be used for resonance assignment. The method is demonstrated for the catalytic domain of Abl kinase in complex with the inhibitor, STI571.
Cutting
Brian
B
Novartis Institutes of Biomedical Research, P.O. Box CH 4002, Basel, Switzerland.
Strauss
André
A
Fendrich
Gabriele
G
Manley
Paul W
PW
Jahnke
Wolfgang
W
D001549:Benzamides; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000068877:Imatinib Mesylate; D008024:Ligands; D008280:Magnetics; D008958:Models, Molecular; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010879:Piperazines; D017434:Protein Structure, Tertiary; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry* / Q000378:metabolism*; D011743:Pyrimidines / Q000737:chemistry* / Q000378:metabolism*; D013113:Spin Labels
D001549:Benzamides; D008024:Ligands; D010879:Piperazines; D011743:Pyrimidines; D013113:Spin Labels; D000068877:Imatinib Mesylate; D016315:Proto-Oncogene Proteins c-abl