Measurement of one-bond 15N-13C' dipolar couplings in medium sized proteins.
A simple and accurate method is described for measurement of 1J(C'N) splittings in isotopically enriched proteins. The method is of the quantitative J correlation type, and the 1J(C'N) splitting is derived from the relative intensity in two 3D TROSY-HNCO spectra with 1J(C'N) dephasing intervals of approximately 1/(2 1J(C'N)) (reference intensity) and approximately 1/1J(C'N) (residual intensity). If the two spectra are recorded under identical conditions and with the same number of scans, the random error in the 1J(C'N) value extracted in this manner is inversely related to the signal-to-noise (S/N) in the reference spectrum. A S/N of 30:1 in the reference spectrum yields random errors of less than 0.2 Hz in the extracted 1J(C'N) value. Dipolar couplings obtained from the difference in 1J(C'N) splitting in the isotropic and liquid crystalline phase for the C-terminal domain of calmodulin are in excellent agreement with its 1.68-A crystal structure, but agree considerably less with the 2.2-A structure.
Chou
J J
JJ
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Delaglio
F
F
Bax
A
A
D000818:Animals; D002147:Calmodulin / Q000737:chemistry*; D002247:Carbon Isotopes; D018360:Crystallography, X-Ray; D008322:Mammals; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results
D002147:Calmodulin; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins