Facile measurement of polypeptide JHNH alpha coupling constants from HMQC-J spectra.

A method, based on linewidth measurements, is described which permits the rapid and facile determination of JHNH alpha coupling constants from 15N labeled proteins. Using appropriately processed HMQC-J data, we have found that a simple linear relationship exists between the half-height linewidth (delta nu1/2) of 15N-1H cross peaks and their corresponding JHNH alpha coupling constants. Tests indicate that this technique permits the accurate measurements of up to 100 JHNH alpha coupling constants in less than 30 min. Furthermore, the JHNH alpha measurements can be done manually--without the need of any computer-based curve-fitting or minimization. Comparisons between JHNH alpha values predicted from high resolution X-ray structures and those determined using this technique indicate that the method is both accurate and precise (correlation coefficient = 0.90, rmsd = 0.75 Hz).