Measurement of one-bond 15N-13C′ dipolar couplings in medium sized proteins

A simple and accurate method is described for measurement of (1)J(C'N) splittings in isotopically enriched proteins. The method is of the quantitative J correlation type, and the (1)J(C'N) splitting is derived from the relative intensity in two 3D TROSY-HNCO spectra with (1)J(C'N) dephasing intervals of similar to1/(2(1)J(C'N)) (reference intensity) and similar to1/(1)J(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 (1)J(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 (1)J(C'N) value. Dipolar couplings obtained from the difference in (1)J(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-Angstrom crystal structure, but agree considerably less with the 2.2-Angstrom structure.