Improving the accuracy of NMR structures of large proteins using pseudocontact shifts as long-range restraints.
We demonstrate improved accuracy in protein structure determination for large (>/=30 kDa), deuterated proteins (e.g. STAT4(NT)) via the combination of pseudocontact shifts for amide and methyl protons with the available NOEs in methyl-protonated proteins. The improved accuracy is cross validated by Q-factors determined from residual dipolar couplings measured as a result of magnetic susceptibility alignment. The paramagnet is introduced via binding to thiol-reactive EDTA, and multiple sites can be serially engineered to obtain data from alternative orientations of the paramagnetic anisotropic susceptibility tensor. The technique is advantageous for systems where the target protein has strong interactions with known alignment media.
Gaponenko
Vadim
V
Structural Biophysics Laboratory, National Cancer Institute, P.O. Box B, Frederick, MD 21702-1201, USA.
Sarma
Siddhartha P
SP
Altieri
Amanda S
AS
Horita
David A
DA
Li
Jess
J
Byrd
R Andrew
RA
D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D050797:STAT4 Transcription Factor; D015534:Trans-Activators / Q000737:chemistry
D004268:DNA-Binding Proteins; D010455:Peptides; D011506:Proteins; D050797:STAT4 Transcription Factor; D015534:Trans-Activators