Abstract
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 ∼1/(21 J C′N) (reference intensity) and ∼1/1 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-Å crystal structure, but agree considerably less with the 2.2-Å structure.
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Chou, J.J., Delaglio, F. & Bax, A. Measurement of one-bond 15N-13C′ dipolar couplings in medium sized proteins. J Biomol NMR 18, 101–105 (2000). https://doi.org/10.1023/A:1008358318863
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DOI: https://doi.org/10.1023/A:1008358318863