A simple method to quantitatively measure polypeptide J_HNHα coupling constants from TOCSY or NOESY spectra

A simple linear relationship between the J_HN_Hα coupling constant and the linewidth (Δν_1/2) of in-phase NMR peaks has been identified. This relationship permits the rapid and accurate determination of polypeptide J_HN_Hα coupling constants from a simple inspection of amide cross peaks in homonuclear ¹H TOCSY or ¹H NOESY spectra. By using the appropriate set of processing parameters we show that J_HN_Hα 0.5(Δν_1/2) - MW/5000 + 1.8 for TOCSY spectra and J_HN_Hα 0.6(Δν_1/2) - MW/5000 - 0.9 for NOESY spectra, where Δν_1/2 is the half-height linewidth in Hz and MW is the molecular weight of the protein in Da. The simplicity of this relationship, combined with the ease with which Δν_1/2 measurements can be made, means that J_HN_Hα coupling constants can now be rapidly determined (up to 100 measurements in less than 30 min) without the need for any complex curve-fitting algorithms. Tests on 11 different polypeptides involving more than 650 separate J_HN_Hα measurements have shown that this method yields coupling constants with an rmsd error (relative to X-ray data) of less than 0.9 Hz. Furthermore, the correlation coefficient between the predicted NMR coupling constants and those derived from high-resolution X-ray crystal structures is typically better than 0.89. These simple linear relationships have been found to be valid for peptides as small as 1 kDa to proteins as large as 20 kDa. Despite the method's simplicity, these results are comparable to the accuracy and precision of the best techniques published to date