A SIMPLE METHOD TO QUANTITATIVELY MEASURE POLYPEPTIDE J-(HH-ALPHA)-H-N COUPLING-CONSTANTS FROM TOCSY OR NOESY SPECTRA

A simple linear relationship between the J(H)N(H) alpha coupling const ant and the linewidth (Delta nu(1/2)) of in-phase NMR peaks has been i dentified. This relationship permits the rapid and accurate determinat ion of polypeptide J(H)N(H) alpha coupling constants from a simple ins pection of amide cross peaks in homonuclear H-1 TOCSY or H-1 NOESY spe ctra. By using the appropriate set of processing parameters we show th at J(H)N(H) alpha = 0.5(Delta nu(1/2))-MW/5000 + 1.8 for TOCSY spectra and J(H)N(H) alpha=0.6(Delta nu(1/2))-MW/5000-0.9 for NOESY spectra, where Delta nu(1/2) is the half-height linewidth in Hz and MW is the m olecular weight of the protein in Da. The simplicity of this relations hip, combined with the ease with which Delta nu(1/2) measurements can be made, means that J(H)N(H) alpha coupling constants can now be rapid ly 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(H)N(H) alpha measureme nts 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 a nd those derived from high-resolution X-ray crystal structures is typi cally better than 0.89. These simple linear relationships have been fo und 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 dat e.